ORCID Profile
0000-0002-9675-1444
Current Organisation
University of Melbourne
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Analytical Spectrometry | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Analytical Biochemistry | Analytical Chemistry | Biochemistry and cell biology | Systems biology | Characterisation of Biological Macromolecules | Instrumental Methods (excl. Immunological and Bioassay Methods) | Medicinal and Biomolecular Chemistry | Structural biology (incl. macromolecular modelling) | Macromolecular and Materials Chemistry | Synthesis of Materials | Microbiology not elsewhere classified | Receptors and Membrane Biology | Signal Transduction | Synthetic Biology | Sociology and Social Studies of Science and Technology | Systems Biology | Biological physics | Plant Cell and Molecular Biology | Medical Biotechnology | Analytical Biochemistry | Proteomics and intermolecular interactions (excl. medical proteomics) | Medical Biochemistry: Lipids | Medical Biotechnology Diagnostics (incl. Biosensors) | Biological mathematics | Applied mathematics
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Chemical Sciences | Workforce Transition and Employment | Organic Industrial Chemicals (excl. Resins, Rubber and Plastics) | Biological sciences | Production of Biofuels (Biomass) | Chemical sciences | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in Technology | Human Pharmaceutical Treatments (e.g. Antibiotics) | Expanding Knowledge in the Agricultural and Veterinary Sciences |
Related Links
Publications
Experimental and theoretical proton affinities of methionine, methionine sulfoxide and their N- and C-terminal derivatives
Publisher: Elsevier BV
Date: 11-2007
Identification and characterization of recombinant murine interleukin-6 with a C-terminal pentapeptide extension using capillary reversed phase HPLC-MS and Edman degradation
Publisher: Wiley
Date: 11-1997
DOI: 10.1002/(SICI)1099-0801(199711)11:6<337::AID-BMC687>3.0.CO;2-E
Abstract: The rising cost of clinical development, license submissions, commercial product launches, and affiliate management in all countries around the world, coupled with the ethical obligation to ensure that eligible patients have access to new treatments, has led some pharmaceutical and biopharmaceutical companies to review their approach to access to medicine. The traditional US first launch, followed by European Union approval and then a strategic launch process, can eventually ensure access in the key markets with developed healthcare systems. For many other countries, providing access via the current legislation available for unlicensed medicine supply can provide a solution for increasing access. This option can be considered for broadening access to a greater number of eligible patients in more countries where unlicensed supply may be the only option, for ex le, if no clinical trials or commercial product supplies are available. This article looks specifically at the key financial and reimbursement considerations for unlicensed medicines and how some companies are adopting a "charged for" early access model that can be sustainable and affordable from their perspective. It is also important to consider how sustainable a charged program would be for the patient and the relevant payer, as they may expect an unlicensed treatment is provided free of charge. However, if the sponsor or manufacturer simply cannot afford to run a free supply program, the patient is faced with a more serious problem, that of no access at all, either charged or free. The objective of this article is to raise awareness amongst interested stakeholders from different perspectives, including the patients. Unlicensed medicines are usually only prescribed when there is a serious or life-threatening unmet need, and the implications for the company, physician, patient, and payer should be clear if access to treatment depends on the ability to pay.
Analysis of Retina and Erythrocyte Glycerophospholipid Alterations in a Rat Model of Type 1 Diabetes
Publisher: SAGE Publications
Date: 12-2009
DOI: 10.1016/J.JALA.2009.07.003
Abstract: An automated tandem mass spectrometry—based analysis using precursor ion and neutral loss scans in a triple quadrupole (QqQ) mass spectrometer has been used to identify and quantify changes in the abundances of glycerophospholipids extracted from retina and erythrocytes in a rat streptozotocin model of type I diabetes, 6 and 36 weeks after the induction of diabetes, compared with age-matched nondiabetic controls. The utility of an “internal standard” method compared with an “internal standard free” method for quantification of differences in the abundances of specific lipid ions was evaluated in both retina and erythrocyte lipid extracts. In retina, equivalent results were obtained by using the internal standard and internal standard free methods for quantification. In erythrocytes, the two methods of analysis yielded significantly different results, suggesting that factors intrinsic to particular s le types may influence the outcome of label-free lipidome quantification approaches. Overall increases (∼25–35%) in the abundances of major retina glycerophospholipid classes were demonstrated in rats at 6 weeks of diabetes, relative to control animals. However, at 36 weeks of diabetes, subsequent overall decreases in retina glycerophosphatidylcholine (GPCho) and glycerophosphatidylethanolamine (GPEtn) abundances of 16% and 33%, respectively, were observed. Additionally, retina and erythrocyte GPCho lipids at both 6 and 36 weeks of diabetes exhibited increased incorporation of linoleic acid (18:2n6) and a decrease in docosahexaenoic acid (DHA (22:6n3) ) content. Finally, an approximately fivefold increase in the abundances of specific glycated GPEtn (Amadori-GPEtn) lipids were observed in the retina of 36-week diabetic rats, with a corresponding 1.6-fold increase of Amadori-GPEtn lipids in diabetic erythrocytes.
The origin and control ofex vivooxidative peptide modifications prior to mass spectrometry analysis
Publisher: Wiley
Date: 27-02-2008
Abstract: The origin and control of ex vivo s le handling related oxidative modifications of methionine-, S-alkyl cysteine-, and tryptophan-containing peptides obtained from typical "in-solution" or "in-gel" proteolytic digestion strategies, have been examined by capillary HPLC and MS/MS. The origin of increased oxidation levels were found to be predominantly associated with the extensive ex vivo s le handling steps required for gel electrophoresis and/or in-gel proteolytic digestion of proteins prior to analysis by MS. Conditions for deliberately controlling the oxidation state (both oxidation and reduction) of these peptides, as well as for those containing cysteine, have been evaluated using a series of model synthetic peptides and standard tryptic protein digests. Essentially complete oxidation of methionine- and S-alkyl cysteine-containing peptides was achieved by reaction with 30% hydrogen peroxide/5% acetic acid at room temperature for 30 min. Under these conditions, cysteine was also converted to cysteic acid, while only limited oxidation of tryptophan to oxindolylalanine, and methionine and S-alkyl cysteine sulfoxides to their respective sulfones, were observed. Efficient reduction of methionine- and S-alkyl cysteine sulfoxide-containing peptides was achieved by reaction in 1 M dimethylsulfide/10 M hydrochloric acid at room temperature for 10 and 45 min, respectively. None of the reduction conditions evaluated were found to result in the reduction of oxindolylalanine, cysteic acid, or methionine sulfone.
Identification of truncated E. coli-expressed proteins with a novel C-terminal 10Sa RNA decapeptide extension
Publisher: Elsevier
Date: 1996
N-3 Polyunsaturated Fatty Acids Prevent Diabetic Retinopathy by Inhibition of Retinal Vascular Damage and Enhanced Endothelial Progenitor Cell Reparative Function
Publisher: Public Library of Science (PLoS)
Date: 29-01-2013
Dissociation reactions of gaseous ferro-, ferri-, and apo-cytochrome c ions
Publisher: American Chemical Society (ACS)
Date: 07-2001
Hybrid 213 nm photodissociation of cationized Sterol lipid ions yield [M]+. Radical products for improved structural characterization using multistage tandem mass spectrometry
Publisher: Elsevier BV
Date: 2021
2015 ASMS “Emerging Investigators” Focus Section
Publisher: American Chemical Society (ACS)
Date: 03-03-2015
The developmental lipidome of Haemonchus contortus
Publisher: Elsevier BV
Date: 10-2018
DOI: 10.1016/J.IJPARA.2018.06.002
Abstract: Lipids play crucial roles in the biology of organisms, particularly relating to cellular membranes, energy storage, and intra- or inter-cellular signalling. Despite the recent expansion of the lipidomics field, very little is known about the biology of lipids in metzoan pathogens, and, to date, there has been no global lipidomic study of a parasitic nematode. Using Haemonchus contortus (barber's pole worm) as a model, we describe the first known global lipidome for a parasitic nematode via high throughput LC-MS/MS-based lipidomics. We identified a total of 554 lipid species across four lipid categories, and 18 lipid classes exhibited alterations among six developmental stages (eggs L3 and exsheathed L3 (xL3) and L4 larval stages female and male adults) of H. contortus. The lipid composition and abundance of H. contortus changed significantly during the transition from free-living (egg, L3 and xL3) to parasitic (L4 and adult) stages. The three main changes observed were: (i) decreased synthesis of triradylglycerols (ii) increased glycerophospholipids (predominantly glycerophosphoethanolamines and glycerophosphocholines) and (iii) a 'cooperative' modulation of ether-linked lipids and saturated fatty acids. These changes suggest specific adaptations, in terms of nutrient acquisition, metabolism and development, as the nematode makes its transition to the parasitic stage inside the host animal. This lipidomic data set serves as a stimulus for studies to understand lipid biology in parasitic worms, and their roles in parasite-host interactions and disease processes.
n-3 PUFAs enhance the frequency of murine B-cell subsets and restore the impairment of antibody production to a T-independent antigen in obesity
Publisher: Elsevier BV
Date: 11-2013
DOI: 10.1194/JLR.M042457
The search for stable gas phase b1 ions derived from aliphatic amino acids: a combined experimental andab initio study
Publisher: Wiley
Date: 2000
DOI: 10.1002/1097-0231(20000730)14:14<1220::AID-RCM13>3.0.CO;2-3
An Early Warning Monitoring System for Illicit Drug Use at Large Public Events: ‘Close to Real Time’ Trace Residue Analysis of Discarded Drug Packaging Samples
Publisher: American Chemical Society (ACS)
Date: 02-06-2021
DOI: 10.26434/CHEMRXIV.14693016.V1
Abstract: Inspired by the exchange principle espoused by Edmond Locard (1877-1966), which states “every contact leaves a trace”, we report here the development and application of a strategy for trace residue s ling and analysis of discarded ‘Drug Packaging S les’ (DPS), as part of an early warning monitoring system for illicit drug use at large public events. Using Direct Analysis in Real Time (DART) - mass spectrometry (MS) and -tandem mass spectrometry (MS/MS), rapid and high-throughput identification and characterisation of a wide range of illicit drugs and adulterant substances was achieved, including those present in complex poly-drug mixtures and at low relative abundances, and with analysis times of less than one minute per s le. 1362 DPS were analysed either ‘off-site’ using laboratory-based instrumentation or in ‘on-site’ in ‘close to real time’ using a transportable mass spectrometer housed within a customised mobile analytical laboratory. 92.2% of DPS yielded positive results for at least one of 15 different pharmacologically active drugs and/or adulterants, including cocaine, MDMA, and ketamine, as well as numerous ‘novel psychoactive substances’ (NPS). Notably, polydrug mixtures were more common than single drugs, with 52.6% of positive DPS found to contain more than one substance, and with 42 different drug and polydrug combinations observed throughout the study. For analyses performed ‘on-site’, reports to key stakeholders including event organisers, first aid and medical personnel, and peer-based harm reduction workers could be provided in as little as 5 minutes after s le collection. Then, following risk assessment of the potential harms associated with their use, drug advisories or alerts were then disseminated to event staff and patrons, and subsequently to the general public, when substances with particularly toxic properties were identified.
Specific covalent modification of the tryptophan residues in murine interleukin‐6
Publisher: Wiley
Date: 10-1993
DOI: 10.1111/J.1432-1033.1993.TB18217.X
Abstract: Modification of recombinant murine interleukin-6 (mIL-6) with the tryptophan-specific reagent 2-nitrophenylsulfenyl chloride under mild acidic conditions, 0.1 M sodium acetate, pH 3.5, yielded a derivative containing 2.02 mol 2-nitrophenylsulfenyl tryptophan/mol protein. The sites of modification were identified as Trp36 and Trp160. No detectable side reactions occurred on other amino acids in the molecule, as indicated by the combination of endoproteinase Asp-N peptide mapping, Edman degradation and electrospray mass spectrometry. Sulfenylation of the two tryptophan residues in mIL-6 caused a 50% reduction in both the biological activity in the murine-hybridoma-growth-factor assay using 7TD1 cells and receptor-binding affinity to mIL-6 receptors. Sulfenylation of mIL-6 did not significantly affect the overall conformation of the protein as measured by farultraviolet circular dichroism and binding to the neutralizing anti-mIL-6 mAb 6B4. The sulfenylated protein was, however, significantly less stable [delta delta G(H2O) = 3.98 kJ/mol] than unmodified mIL-6 as measured by urea-gradient gel electrophoresis.
Complete amino acid sequence of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone Actinia tenebrosa
Publisher: Wiley
Date: 06-1990
DOI: 10.1111/J.1432-1033.1990.TB15579.X
Abstract: The complete amino acid sequence of the cardiac stimulatory and haemolytic protein tenebrosin-C, from the Australian sea anemone Actinia tenebrosa, has been determined by Edman degradation of the intact molecule and fragments produced by treatment of the polypeptide chain with cyanogen bromide and enzymatic cleavage with endoproteinase Asp-N, thermolysin and trypsin. The molecule is a single-chain polypeptide consisting of 179 amino acid residues with a calculated molecular mass of 19,797 Da. Tenebrosin-C shows a high degree of amino acid sequence similarity (63%) with Stoichactis helianthus cytolysin III [Blumenthal, K. M. and Kem, W. R. (1983) J. Biol. Chem. 258, 5574-5581] and is identical to a partial sequence (90 residues) reported for equinatoxin, a cardiostimulatory and haemolytic protein isolated from the European sea anemone Actinia equina [Ferlan, I. and Jackson, K. (1983) Toxicon Suppl. 3, 141-144]. No amino acid sequence similarity was detected between tenebrosin-C and other protein sequences stored in available databases. The predicted secondary structure of tenebrosin-C suggests that it is a compact, highly structured molecule.
Tandem Mass Spectrometry of Ribonuclease A and B: N-Linked Glycosylation Site Analysis of Whole Protein Ions
Publisher: American Chemical Society (ACS)
Date: 12-12-2001
DOI: 10.1021/AC015618L
Abstract: Recently, an approach for the "top down" sequence analysis of whole protein ions has been developed, employing electrospray ionization, collision-induced dissociation, and ion/ion proton-transfer reactions in a quadrupole ion trap mass spectrometer. This approach has now been extended to an analysis of the [M + 12H]12+ to [M + 5H]5+ ions of ribonuclease A and its N-linked glycosylated analogue, ribonuclease B, to determine the influence of the posttranslational modification on protein fragmentation. In agreement with previous studies on the fragmentation of a range of protein ions, facile gas-phase fragmentation was observed to occur along the protein backbone at the C-terminal of aspartic acid residues, and at the N-terminal of proline, depending on the precursor ion charge state. Interestingly, no evidence was found for gas-phase deglycosylation of the N-linked sugar in ribonuclease B, presumably due to effective competition from the facile amide bond cleavage channels that "protect" the N-linked glycosidic bond from cleavage. Thus, localization of the posttranslational modification site may be determined by analysis of the "protein fragment ion mass fingerprint".
A Census of Hsp70-Mediated Proteome Solubility Changes upon Recovery from Heat Stress
Publisher: American Chemical Society (ACS)
Date: 07-04-2022
DOI: 10.1021/ACS.JPROTEOME.1C00920
Abstract: Eukaryotic cells respond to heat shock through several regulatory processes including upregulation of stress responsive chaperones and reversible shutdown of cellular activities through formation of protein assemblies. However, the underlying regulatory mechanisms of the recovery of these heat-induced protein assemblies remain largely elusive. Here, we measured the proteome abundance and solubility changes during recovery from heat shock in the mouse Neuro2a cell line. We found that prefoldins and translation machinery are rapidly down-regulated as the first step in the heat shock response. Analysis of proteome solubility reveals that a rapid mobilization of protein quality control machineries, along with changes in cellular energy metabolism, translational activity, and actin cytoskeleton are fundamental to the early stress responses. In contrast, longer term adaptation to stress involves renewal of core cellular components. Inhibition of the Hsp70 family, pivotal for the heat shock response, selectively and negatively affects the ribosomal machinery and delays the solubility recovery of many nuclear proteins. ProteomeXchange: PXD030069.
A thiol probe for measuring unfolded protein load and proteostasis in cells
Publisher: Springer Science and Business Media LLC
Date: 07-09-2017
DOI: 10.1038/S41467-017-00203-5
Abstract: When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum . TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.
Emerging Investigators Special Issue
Publisher: Royal Society of Chemistry (RSC)
Date: 2006
DOI: 10.1039/B600451M
Inhibition of Cytokine Signaling in Human Retinal Endothelial Cells through Downregulation of Sphingomyelinases by Docosahexaenoic Acid
Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 06-2010
DOI: 10.1167/IOVS.09-4731
The potential of extracellular vesicle lipid profiling in Alzheimer's disease.
Publisher: Authorea, Inc.
Date: 12-05-2023
DOI: 10.22541/AU.168388562.25020996/V1
Abstract: Over the past two decades, there has been increasing research into the molecular composition and function of small extracellular vesicles in the central nervous system. This is due in part to the recognition that small extracellular vesicles likely contribute to the pathogenesis of neurological diseases such as Alzheimer’s disease, but also an understanding that small extracellular vesicles are a source of potential biomarkers. Small extracellular vesicles carry specific cargo that reflects their biogenesis and cellular origins, including protein, RNA and lipid. While the protein and RNA content of small extracellular vesicles in the central nervous system diseases and have been studied extensively, our understanding of the lipidome of small extracellular vesicles in the central nervous system is still in its infancy. Lipids play a significant role in maintaining central nervous system structure and function, and the dysregulation of lipid metabolism is known to occur in many neurological disorders, including Alzheimer’s disease. Here we review what is currently known about lipid dyshomeostasis in Alzheimer’s disease. We propose that small extracellular vesicle lipids may provide insight into the pathophysiology and progression of Alzheimer’s disease and other neurological disorders, and, in the future perhaps, aid in disease monitoring and detection.
Chemical Derivatization and Ultrahigh Resolution and Accurate Mass Spectrometry Strategies for “Shotgun” Lipidome Analysis
Publisher: American Chemical Society (ACS)
Date: 30-08-2016
DOI: 10.1021/ACS.ACCOUNTS.6B00030
Abstract: Lipids play critical structural and functional roles in the regulation of cellular homeostasis, and it is increasingly recognized that the disruption of lipid metabolism or signaling or both is associated with the onset and progression of certain metabolically linked diseases. As a result, the field of lipidomics has emerged to comprehensively identify and structurally characterize the erse range of lipid species within a s le of interest and to quantitatively monitor their abundances under different physiological or pathological conditions. Mass spectrometry (MS) has become a critical enabling platform technology for lipidomic researchers. However, the presence of isobaric (i.e., same nominal mass) and isomeric (i.e., same exact mass) lipids within complex lipid extracts means that MS-based identification and quantification of in idual lipid species remains a significant analytical challenge. Ultrahigh resolution and accurate mass spectrometry (UHRAMS) offers a convenient solution to the isobaric mass overlap problem, while a range of chromatographic separation, differential extraction, intrasource separation and selective ionization methods, or tandem mass spectrometry (MS/MS) strategies may be used to address some types of isomeric mass lipid overlaps. Alternatively, chemical derivatization strategies represent a more recent approach for the separation of lipids within complex mixtures, including for isomeric lipids. In this Account, we highlight the key components of a lipidomics workflow developed in our laboratory, whereby certain lipid classes or subclasses, namely, aminophospholipids and O-alk-1'-enyl (i.e., plasmalogen) ether-containing lipids, are shifted in mass following sequential functional group selective chemical derivatization reactions prior to "shotgun" nano-ESI-UHRAMS analysis, "targeted" MS/MS, and automated database searching. This combined derivatization and UHRAMS approach resolves both isobaric mass lipids and certain categories of isomeric mass lipids within crude lipid extracts, with no requirement for extensive s le handling prior to analysis, with additional potential for enhanced ionization efficiencies, improved molecular level structural characterization, and multiplexed relative quantification. When integrated with a monophasic method for the simultaneous global extraction of both highly polar and nonpolar lipids, this workflow has been shown to enable the sum composition level identification and relative quantification of 500-600 in idual lipid species across four lipid categories and from 36 lipid classes and subclasses, in only 1-2 min data acquisition time and with minimal s le consumption. Thus, while some analytical challenges remain to be addressed, shotgun lipidomics workflows encompassing chemical derivatization strategies have particular promise for the analysis of s les with limited availability that require rapid and unbiased assessment of global lipid metabolism.
Solution structure of a polypeptide containing four heptad repeat units from a merozoite surface antigen of Plasmodium falciparum
Publisher: American Chemical Society (ACS)
Date: 03-1995
DOI: 10.1021/BI00011A001
Abstract: The Plasmodium falciparum antigen SPAM (secreted polymorphic antigen associated with merozoites) contains an unusual set of heptad repeat units with alanine at the a and d positions. Twelve heptads with the consensus sequence AXXAXXX occur in three blocks of four, linked by short nonrepetitive sequences. A 38-residue polypeptide comprising the first block of four heptad units and five flanking residues at either end, SPAM-H1, has been synthesized and its structure in aqueous solution determined from 1H NMR data. Sedimentation equilibrium showed the peptide to be monomeric in aqueous solution. Its structure was determined from 1H NMR-derived distance and dihedral angle constraints by using distance geometry calculations, restrained simulated annealing, and conjugate gradient energy minimization in the CHARMm force field. The polypeptide contains an alpha-helix extending from Ser10 (position e of the first heptad) to at least Lys32 (position f of the fourth heptad) and possibly as far as Val35. The helix is bent, partly as result of a kink around residues 19-20. The conformations of the nine N-terminal residues and the six C-terminal residues are not well defined by the NMR data. The rms deviation from the average of the 20 best structures over the well-defined region (residues 11-31, which have backbone angular order parameters > 0.8) was 1.56 A for backbone heavy atoms (N, C alpha, and C) and 2.12 A for all heavy atoms. 2H2O exchange experiments identified slowly exchanging amide protons near the C-terminus and the last two turns of the helix. The unusual stability of the C-terminus reflects the presence of a new C-capping motif, which may involve the side chain of an asparagine in a position external to the C-cap residue. Possible interactions of the H1 sequence with the other two heptad repeat units in the intact merozoite antigen are discussed.
Automated Neutral Loss and Data Dependent Energy Resolved “Pseudo MS3” for the Targeted Identification, Characterization and Quantitative Analysis of Methionine-Containing Peptides
Publisher: SAGE Publications
Date: 04-2008
DOI: 10.1255/EJMS.931
Abstract: A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary high-performance liquid chromatography and automated data dependent neutral loss scan mode tandem mass spectrometry (MS/MS) and “pseudo multiple mass spectrometry (MS 3 )” product ion scans in a triple quadrupole mass spectrometer has been developed for the “targeted” gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. Selective gas-phase “enrichment” and identification is performed via neutral loss scan mode MS/MS, by low energy collision-induced dissociation of the derivatized methionine side chain, resulting in the formation of a single characteristic product ion. Structural characterization of identified peptides is then achieved by automatically subjecting the characteristic neutral loss product ion to further dissociation by data dependent product ion scan mode pseudo MS 3 under higher collision energy conditions. Quantitative analysis is achieved by measurement of the abundances of characteristic product ions formed by sequential neutral loss scan mode MS/MS experiments from “light” ( 12 C) and “heavy” ( 13 C) stable isotope encoded fixed-charge derivatized peptides. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method employed here was able to achieve accurate quantification for in idual peptides at levels as low as 100fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest.
Role of the sulfhydryl group on the gas phase fragmentation reactions of protonated cysteine and cysteine containing peptides
Publisher: American Chemical Society (ACS)
Date: 12-1998
‘Top down’ protein characterization via tandem mass spectrometry
Publisher: Wiley
Date: 07-2002
DOI: 10.1002/JMS.346
Abstract: Technological and scientific advances over the past decade have enabled protein identification and characterization strategies to be developed that are based on subjecting intact protein ions and large protein fragments directly to tandem mass spectrometry. These approaches are referred to collectively as 'top down' to contrast them with 'bottom up' approaches whereby protein identification is based on mass spectrometric analysis of peptides derived from proteolytic digestion, usually with trypsin. A key step in enabling top down approaches has been the ability to assign tandem mass spectrometer product ion identities, which can be done either via high resolving power or through product ion charge state manipulation. The ability to determine product ion charge states has permitted studies of the reactions, including dissociation, ion-molecule reactions, ion-electron reactions and ion-ion reactions of high-mass, multiply charged protein ions. Electrospray ionization combined with high magnetic field strength Fourier transform ion cyclotron resonance has proven to be particularly powerful for detailed protein characterization owing to its high mass resolution and mass accuracy and its ability to effect electron capture-induced dissociation. Other types of tandem mass spectrometers are also beginning to find increasing use in top down protein identification/characterization studies. Charge state manipulation via ion-ion reactions in electrodynamic ion traps, for ex le, enables top down strategies to be considered using instruments with relatively modest mass resolution capabilities. Precursor ion charge state manipulation techniques have also recently been demonstrated to be capable of concentrating and charge-state purifying proteins in the gas phase. Advances in technologies applied to the structural analysis of whole protein ions and in understanding their reactions, such as those described here, are providing new options for the study of complex protein mixtures.
Mass spectrometric characterization and activity of zinc-activated proinsulin C-peptide and C-peptide mutants
Publisher: Royal Society of Chemistry (RSC)
Date: 2010
DOI: 10.1039/B917600D
Abstract: Numerous reports have demonstrated an active role for proinsulin C-peptide in ameliorating chronic complications associated with diabetes mellitus. It has been recently reported that some of these activities are dependent upon activation of C-peptide with certain metal ions, such as Fe(II), Cr(III) or Zn(II). In an effort to gain a greater understanding of the structure/function dependence of the peptide-metal interactions responsible for this activity, a series of experiments involving the use of electrospray ionization (ESI), matrix assisted laser desorption/ionization (MALDI) and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of C-peptide in the presence or absence of Zn(II) have been carried out. Additionally, various C-peptide mutants with alanine substitution at in idual aspartic acid or glutamic acid residues throughout the C-peptide sequence were analyzed. CID-MS/MS of wild type C-peptide in the presence of Zn(II) indicated multiple sites for metal binding, localized at acidic residues within the peptide sequence. Mutations of in idual acidic residues did not significantly affect this fragmentation behavior, suggesting that no single acidic residue is critical for binding. However, ESI-MS analysis revealed an approximately 50% decrease in relative Zn(II) binding for each of the mutants compared to the wild type sequence. Furthermore, a significant decrease in activity was observed for each of the Zn(II)-activated mutant peptides compared to the wild type C-peptide, indicated by measurement of ATP released from erythrocytes, with a 75% decrease observed for the Glu27 mutant. Additional studies on the C-terminal pentapeptide of C-peptide EGSLQ, as well as a mutant C-terminal pentapeptide sequence AGSLQ, revealed that substitution of the glutamic acid residue resulted in a complete loss of activity, implicating a central role for Glu27 in Zn(II)-mediated C-peptide activity.
Do all b2 ions have oxazolone structures? Multistage mass spectrometry and ab initio studies on protonated N-acyl amino acid methyl ester model systems
Publisher: Elsevier BV
Date: 09-2001
Evaluation of ion activation strategies and mechanisms for the gas-phase fragmentation of sulfoquinovosyldiacylglycerol lipids from Rhodobacter sphaeroides
Publisher: Elsevier BV
Date: 04-2012
Somatic proteome of Haemonchus contortus
Publisher: Elsevier BV
Date: 03-2019
DOI: 10.1016/J.IJPARA.2018.12.003
Abstract: Currently, there is a dearth of proteomic data to underpin fundamental investigations of parasites and parasitism at the molecular level. Here, using a high throughput LC-MS/MS-based approach, we undertook the first reported comprehensive, large-scale proteomic investigation of the barber's pole worm (Haemonchus contortus) - one of the most important parasitic nematodes of livestock animals worldwide. In total, 2487 unique H. contortus proteins representing different developmental stages/sexes (i.e. eggs, L3s and L4s, female (Af) and male (Am) adults) were identified and quantified with high confidence. Bioinformatic analyses of this proteome revealed substantial alterations in protein profiles during the life cycle, particularly in the transition from the free-living to the parasitic phase, and key groups of proteins involved specifically in feeding, digestion, metabolism, development, parasite-host interactions (including immunomodulation), structural remodelling of the body wall and adaptive processes during parasitism. This proteomic data set will facilitate future molecular, biochemical and physiological investigations of H. contortus and related nematodes, and the discovery of novel intervention targets against haemonchosis.
Gas-phase reactions of protonated tryptophan
Publisher: American Chemical Society (ACS)
Date: 2004
DOI: 10.1016/J.JASMS.2003.09.011
Abstract: The gas phase reactions of protonated tryptophan have been examined in a quadrupole ion trap using a combination of collision induced dissociation, hydrogen-deuterium exchange, regiospecific deuterium labeling and molecular orbital calculations (at the B3LYP/6-31G* level of theory). The loss of ammonia from protonated tryptophan is observed as the primary fragmentation pathway, with concomitant formation of a [M + H - NH(3)](+) ion by nucleophilic attack from the C3 position of the indole side chain. Hydrogen-deuterium exchange and regiospecific deuterium labeling reveals that scrambling of protons in the C2 and C4 positions of the indole ring, via intramolecular proton transfer from the thermodynamically preferred site of protonation at the amino nitrogen, precedes ammonia loss. Molecular orbital calculations have been employed to demonstrate that the activation barriers to intramolecular proton transfer are lower than that for NH(3) loss.
Leaving group and gas phase neighboring group effects in the side chain losses from protonated serine and its derivatives
Publisher: American Chemical Society (ACS)
Date: 12-2000
Statistical and Mechanistic Approaches to Understanding the Gas-Phase Fragmentation Behavior of Methionine Sulfoxide Containing Peptides
Publisher: American Chemical Society (ACS)
Date: 29-04-2004
DOI: 10.1021/PR0499646
Abstract: Recently, we carried out a statistical analysis of a 'tryptic' peptide tandem mass spectrometry database in order to identify sequence-dependent patterns for the gas-phase fragmentation behavior of protonated peptide ions, and to improve the models for peptide fragmentation currently incorporated into peptide sequencing and database search algorithms [Kapp, E. A., Schutz, F., Reid, G. E., Eddes, J. S., Moritz, R. L., O'Hair, R. A. J., Speed, T. P. and Simpson, R. J. Anal. Chem. 2003, 75, 6251-6264.]. Here, we have reexamined this database in order to determine the effect of a common post-translational or process induced modification, methionine oxidation, on the appearance and relative abundances of the product ions formed by low energy collision induced dissociation of peptide ions containing this modification. The results from this study indicate that the structurally diagnostic neutral loss of methane sulfenic acid (CH3SOH, 64Da) from the side chain of methionine sulfoxide residues is the dominant fragmentation process for methionine sulfoxide containing peptide ions under conditions of low proton mobility, i.e., when ionizing proton(s) are sequestered at strongly basic amino acids such as arginine, lysine or histidine. The product ion abundances resulting from this neutral loss were found to be approximately 2-fold greater than those resulting from the cleavage C-terminal to aspartic acid, which has previously been shown to be enhanced under the same conditions. In close agreement with these statistical trends, experimental and theoretical studies, employing synthetic "tryptic" peptides and model methionine sulfoxide containing peptide ions, have determined that the mechanism for enhanced methionine sulfoxide side chain cleavage proceeds primarily via a 'charge remote' process. However, the mechanism for dissociation of the side chain for these ions was observed to change as a function of proton mobility. Finally, the transition state barrier for the charge remote side chain cleavage mechanism is predicted to be energetically more favorable than that for charge remote cleavage C-terminal to aspartic acid.
Photoinduced intermolecular cross-linking of gas phase triacylglycerol lipid ions
Publisher: SAGE Publications
Date: 06-2015
DOI: 10.1255/EJMS.1344
Abstract: Complex mixtures of plant-derived triglycerol (TG) lipids are commonly used as feedstock components for the production of industrial polymers. However, there remains a need for the development of analytical strategies to investigate the intrinsic intermolecular cross-linking reactivity of in idual TG molecules within these mixtures as a function of their structures and physicochemical properties, and for the characterization of the resultant products. Here, to address this need, we describe a novel multistage tandem mass spectrometry based method for intermolecular cross-linking and subsequent structural characterization of TG lipid ions in the gas phase. Cross-linking reactions were initiated using 266 nm ultraviolet photodissociation tandem mass spectrometry (UVPD-MS/MS) of saturated or unsaturated TG dimers introduced via electrospray ionization into a linear ion trap mass spectrometer as noncovalent complexes with protonated 3,4-, 2,4- or 3,5-diiodoaniline (diIA). UVPD resulted in the initial formation of an anilinyl biradical via the sequential loss of two iodine radicals, which underwent further reaction to yield multiple cross-linked TG products along with competing noncross-linking processes. These chemistries are proposed to occur via sequential combinations of hydrogen abstraction (H-abstraction), radical addition, and radical recombination. Multistage collision-induced dissociation tandem mass spectrometry (CID-MS n ) was used to obtain evidence for the structures and mechanisms of formation for these products, as a function of both the TG lipid and diIA ion structures. The efficiency of the UVPD reaction was shown to be dependent on the number of unsaturation sites present within the TG lipids. However, when unsaturation sites were present, the formation of the cross-linked and noncross-linked product ions via H-abstraction and radical addition mechanisms was found to be competitive. Finally, the identity of the anilinyl biradical (e.g., 3,4- versus 2,4-substituted) was found to significantly affect the distribution of these two types of product ions. Importantly, owing to the observed propensity for cross-linking to occur via H-abstraction-initiated processes, this novel gas-phase cross-linking reaction provides a convenient method to link two molecules covalently without the requirement of any specific functional group, and therefore could be applied to examine the gas-phase intermolecular interactions and cross-linking of a wide range of biomolecular classes.
Capillary column chromatography improves sample preparation for mass spectrometric analysis: Complete characterization of human α‐enolase from two‐dimensional gels following in situ proteolytic
Publisher: Wiley
Date: 05-1998
Abstract: Two-dimensional polyacrylamide gel electrophoresis (2-DE) in combination with mass spectrometry is an extremely powerful tool for characterizing complex mixtures of proteins. In many cases, the success of this approach relies upon the ability to recover peptides at high concentrations and free of interfering artifacts from in-gel and/or on-membrane enzymatic digests. In previous studies, we demonstrated that capillary or microcolumn (< 350 microm ID) reversed-phase high performance liquid chromatography (RP-HPLC) is a powerful microseparation technique for proteins and peptides (Moritz, R. L. and Simpson, R. J., J. Chromatogr. 1992, 599, 119-130). Here we evaluate various capillary column RP-HPLC/mass spectrometric approaches for identifying and characterizing 2-DE resolved proteins. For these studies, stable and efficient 0.20 mm and 0.32 mm internal diameter (ID) fused-silica columns with hydrophilic polyvinylidene difluoride (PVDF) frits were fabricated and slurry packed with 7 microm spherical, 300 A pore size, C8 bonded phase silica particles. We show that capillary column chromatography is a rapid and efficient desalting/concentrating (ON/OFF) technique for s le cleanup prior to protein identification by peptide-mass fingerprinting using matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry. While marginally more peptide mass information can be obtained by stepped elution of the peptide mixture with increasing concentrations of organic solvent, best results were obtained by fractionation of the peptide mixture using a linear 60 min gradient. One salient feature of this study was the observation that, in contrast to the stepped elution and gradient approaches, the ionization of peptide T1 (m/z 2402.2 SGETEDTFIADLVV(PeCys)TGQIK) was almost completely suppressed using the ON/OFF approach. Maximal amino acid sequence coverage, a necessary prerequisite for complete characterization of a protein, was accomplished using a capillary column (0.2 mm ID) directly coupled with an electrospray ionization (ESI) ion-trap tandem mass spectrometer. For ex le, from an in situ tryptic digest of alpha-enolase isolated by 2-DE from the human breast carcinoma cell line MDA-MB231, 71% of the amino acid sequence was obtained. In addition to identifying two possible N-terminal acetylated alpha-enolase variants, Asn153Asp and Ile152Asp/Asn153Ile, the tandem mass spectrometric data revealed the presence of a number of process-induced modifications of alpha-enolase such as methionine oxidation and cysteine amidoethylation.
Characterization of a Novel GDP-mannose:Serine-protein Mannose-1-phosphotransferase from Leishmania mexicana
Publisher: Elsevier BV
Date: 03-1999
Two‐dimensional electrophoretic analysis of proteins expressed by normal and cancerous human crypts: Application of mass spectrometry to peptide‐mass fingerprinting
Publisher: Wiley
Date: 1994
Abstract: Protein patterns of normal human colonic crypts, isolated from different regions of the large intestine, and several colorectal cancer cell lines were compared using two-dimensional electrophoresis gels (2-DE). As detected by intrinsic radiolabeling and Coomassie Brilliant Blue staining, the protein patterns for normal crypts isolated from the ascending, and descending, regions of the colon and the rectum, were almost (> 95%) identical. While 75-80% of the protein spots from normal crypts and the colorectal cancer cell line (LIM 1863), a cell line that grows as organoids and differentiates spontaneously into crypt-like structures in vitro, can be matched, the relative expression levels of a large number of proteins differ. At least two protein spots (undetectable in the protein pattern from normal cells), proteins a (M(r) approximately 18,000, pI 6.7-6.9) and b (M(r) approximately 24,000, pI 5.9-6.0), were detected in the 2-DE gel protein pattern in the three cell lines LIM 1863, LIM 1215 and LIM 1899. The identity of these proteins is not yet known and further studies are required before they can be considered as potential colon tumor markers. Approximately 60% of the cellular proteins from LIM 1215 cells, a colon carcinoma cell line that exhibits many properties associated with columnar cells, can be matched with LIM 1863 cells. The results presented here represent an initial phase in our efforts to develop a comprehensive protein database for normal human colon cells and several colorectal cancer cell lines. While our initial protein identification relied on microsequencing methodologies, we are presently evaluating peptide-mass fingerprinting, utilizing capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry, as a means for rapid identification of proteins at subpicomole levels. Using this approach, protein #3 (M(r) approximately 66,000, pI 6.2) was identified as heat shock protein 60 from as few as seven tryptic peptide masses when they were screened against the molecular weight search (MOWSE) peptide-mass database.
Rapid and comprehensive ‘shotgun’ lipidome profiling of colorectal cancer cell derived exosomes
Publisher: Elsevier BV
Date: 10-2015
A Non-canonical Pathway with Potential for Safer Modulation of Transforming Growth Factor-β1 in Steroid-Resistant Airway Diseases
Publisher: Elsevier BV
Date: 02-2019
Synthesis, Structural Characterization, and Gas-Phase Unimolecular Reactivity of Bis(diphenylphosphino)amino Copper Hydride Nanoclusters [Cu3 (X)(μ3 -H)((PPh2 )2 NH)3 ](BF4 ), Where X = μ2 -Cl and μ3 -BH4
Publisher: American Chemical Society (ACS)
Date: 19-09-2016
DOI: 10.1021/ACS.INORGCHEM.6B01696
Abstract: An electrospray ionization mass spectrometry (ESI-MS) survey of the types of cationic copper clusters formed from an acetonitrile solution containing a 1:1:20 mixture of tetrakis(acetonitrile)copper(I) tetrafluoroborate [Cu(MeCN)
N-Terminal Derivatization and Fragmentation of Neutral Peptides via Ion−Molecule Reactions with Acylium Ions: Toward Gas-Phase Edman Degradation?
Publisher: American Chemical Society (ACS)
Date: 18-01-2001
DOI: 10.1021/JA003070E
Abstract: The gas-phase ion-molecule reactions of neutral alanylglycine have been examined with various mass-selected acylium ions RCO(+) (R= CH(3), CD(3), C(6)H(5), C(6)F(5) and (CH(3))( 2)N), as well as the transacylation reagent O-benzoylbenzophenone in a Fourier transform ion cyclotron resonance mass spectrometer. Reactions of the gaseous dipeptide with acylium ions trapped in the ICR cell result in the formation of energized [M + RCO](+) adduct ions that fragment to yield N-terminal b-type and C-terminal y-type product ions, including a modified b(1) ion which is typically not observed in the fragmentation of protonated peptides. Judicious choice of the acylium ion employed allows some control over the product ion types that are observed (i.e., b versus y ions). The product ion distributions from these ion--molecule reactions are similar to those obtained by collision-activated dissociation in a triple quadrupole mass spectrometer of the authentic N-acylated alanylglycine derivatives. These data indicate that derivatization of the peptide in the gas-phase occurs at the N-terminal amine. Ab initio molecular orbital calculations, performed to estimate the thermochemistry of the steps associated with adduct formation as well as product ion formation, indicate that (i) the initially formed adduct is energized and hence likely to rapidly undergo fragmentation, and (ii) the likelihood for the formation of modified b(1) ions in preference to y(1) ions is dependent on the R substituent of the acylium ion. The reaction of the tetrapeptide valine--alanine--alanine--phenylalanine with the benzoyl cation was also found to yield a number of product ions, including a modified b(1) ion. This result suggests that the new experimental approach described here may provide a tool to address one of the major limitations associated with traditional mass spectrometric peptide sequencing approaches, that is, determination of the identity and order of the two N-terminal amino acids. Analogies are made between the reactions observed here and the derivatization and N-terminal cleavage reactions employed in the condensed-phase Edman degradation method.
Femtosecond Laser-Induced Ionization/Dissociation of Protonated Peptides
Publisher: American Chemical Society (ACS)
Date: 07-01-2009
DOI: 10.1021/JA8089119
Abstract: Although tandem mass spectrometry has revolutionized the identification and structural characterization of peptides and proteins, future advances in comprehensive proteome analysis will depend on the development of improved methods for ion activation that yield greater sequence information, and with selective control over the fragmentation chemistry. This report presents initial findings that demonstrate the utility of a novel ion activation method using ultrashort (approximately 30 fs) laser pulses as a means to overcome the limitations of current technologies, while opening the door to solving significant challenges in protein and peptide analysis.
Capillary HPLC: A Method for Protein Isolation and Peptide Mapping
Publisher: Elsevier BV
Date: 09-1994
Mechanistic Insights into the Multistage Gas-Phase Fragmentation Behavior of Phosphoserine- and Phosphothreonine-Containing Peptides
Publisher: American Chemical Society (ACS)
Date: 09-01-2018
DOI: 10.1021/PR0705136
Abstract: The increasing use of multistage tandem mass spectrometry (MS/MS and MS (3)) methods for comprehensive phosphoproteome analysis studies, as well as the emerging application of in silico spectral intensity prediction algorithms in enhanced database search analysis strategies, necessitate the development of an improved understanding of the mechanisms and other factors that affect the gas-phase fragmentation reactions of phosphorylated peptide ions. To address this need, we have examined the multistage collision-induced dissociation (CID) behavior of a set of singly and doubly charged phosphoserine- and phosphothreonine-containing peptide ions, as well as their regioselectively or uniformly deuterated derivatives, in a quadrupole ion trap mass spectrometer. Consistent with previous reports, the neutral loss of phosphoric acid (H 3PO 4) was observed as a dominant reaction pathway upon MS/MS. The magnitude of this loss was found to be highly dependent on the proton mobility of the precursor ion for both phosphoserine- and phosphothreonine-containing peptides. In contrast to that currently accepted in the literature, however, the results obtained in this study unequivocally demonstrate that the loss of H 3PO 4 does not predominantly occur via a "charge-remote" beta-elimination reaction. The observation of product ions corresponding to the loss of formaldehyde (CH 2O, 30 Da, or CD 2O, 32 Da) or acetaldehyde (CH 3CHO, 44 Da) upon MS (3) dissociation of the [M+ nH-H 3PO 4] ( n+ ) product ions from phosphoserine- and phosphothreonine-containing peptide ions, respectively, provide experimental evidence for a "charge-directed" mechanism involving an S N2 neighboring group participation reaction, resulting in the formation of a cyclic product ion. Potentially, these "diagnostic" MS (3) product ions may provide additional information to facilitate the characterization of phosphopeptides containing multiple potential phosphorylation sites.
Structural Characterization and Absolute Quantification of Microcystin Peptides Using Collision-Induced and Ultraviolet Photo-Dissociation Tandem Mass Spectrometry
Publisher: American Chemical Society (ACS)
Date: 29-05-2018
Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1194/JLR.M082495
Automated solid-phase peptide synthesis: Use of 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium tetrafluoroborate for coupling of ert- butyloxycarbonyl amino acids
Publisher: Elsevier BV
Date: 02-1992
DOI: 10.1016/0003-2697(92)90470-R
Abstract: 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) has been adapted for use as a coupling reagent for tert-butyloxycarbonyl (Boc) amino acids in automated solid-phase peptide synthesis. When compared to the existing preformed symmetrical anhydride procedure employing dicyclohexyl-carbodiimide (DCC), the use of TBTU in the presence of 1-hydroxybenzotriazole (HOBt) provides a more efficient coupling procedure for Boc-amino acid derivatives. Overall cycle times using TBTU/HOBt coupling reagents (30 min) compare favorably to those of the DCC-mediated procedure (approx 65 min). Dimethylformamide can be used as the sole solvent for both activation and coupling reactions. Implementation of TBTU/HOBt coupling conditions does not require replumbing of any lines of the Applied Biosystems Model 430A instrument and necessitates changes to only three reagent bottle positions. The variable coupling efficiencies of Boc-asparagine following activation with TBTU/HOBt (as low as 89%) can be overcome by protection of the amide function of Boc-asparagine with the 9-xanthyl group. Ex les of the synthesis and characterization of a number of peptides ranging in length from 13 to 29 residues are given.
Relationship between the cytolysins tenebrosin-C from Actinia tenebrosa and equinatoxin II from Actinia equina
Publisher: Elsevier BV
Date: 10
DOI: 10.1016/0041-0101(92)90497-S
Abstract: The chemical, physical and biological properties of the cytolysin tenebrosin-C from Actinia tenebrosa have been compared with those of equinatoxin II from Actinia equina. The two proteins are indistinguishable by reverse-phase and cation-exchange HPLC and capillary zone electrophoresis, and give similar peptide fragments upon cyanogen bromide cleavage (as judged by the chromatographic behaviour, ultraviolet absorption spectra, amino acid composition and N-terminal amino acid sequences of the peptides). Their cardiac stimulatory activities are identical, and their haemolytic activities are similar, with equinatoxin II having slightly greater activity. These data indicate that the two molecules are either identical in all 179 amino acid positions, or differ by no more than one or two residues. These findings are discussed in the context of the taxonomic relationship between the two species of sea anemone.
Mechanisms for the proton mobility-dependent gas-phase fragmentation reactions of S-alkyl cysteine sulfoxide-containing peptide ions
Publisher: American Chemical Society (ACS)
Date: 09-2007
DOI: 10.1016/J.JASMS.2007.06.014
Abstract: Mechanisms for the gas-phase fragmentation reactions of singly and multiply protonated precursor ions of the model S-alkyl cysteine sulfoxide-containing peptides GAILCGAILK, GAILCGAILR, and VTMGHFCNFGK prepared by reaction with iodomethane, iodoacetamide, iodoacetic acid, acrylamide, or 4-vinylpyridine, followed by oxidation with hydrogen peroxide, as well as peptides obtained from an S-carboxyamidomethylated and oxidized tryptic digest of bovine serum albumin, have been examined using multistage tandem mass spectrometry, hydrogen/deuterium exchange and molecular orbital calculations (at the B3LYP/6-31 + G(d,p) level of theory). Consistent with previous reports, CID-MS/MS of the S-alkyl cysteine sulfoxide-containing peptide ions resulted in the dominant "non-sequence" neutral loss of an alkyl sulfenic acid (XSOH) from the modified cysteine side chains under conditions of low proton mobility, irrespective of the alkylating reagent employed. Dissociation of uniformly deuterated precursor ions of these model peptides determined that the loss of alkyl sulfenic acid in each case occurred via a "charge-remote" five-centered cis-1,2 elimination reaction to yield a dehydroalanine-containing product ion. Similarly, the charge state dependence to the mechanisms and product ion structures for the losses of CO(2), CO(2) + H(2)O and CO(2) + CH(2)O from S-carboxymethyl cysteine sulfoxide-containing peptides, and for the losses of CH(2)CHCONH(2) and CH(2)CHC(5)H(4)N, respectively, from S-amidoethyl and S-pyridylethyl cysteine sulfoxide-containing peptide ions have also been determined. The results from these studies indicate that both the proton mobility of the peptide precursor ion and the nature of the S-alkyl substituent have a significant influence on the abundances and charge states of the product ions resulting from the various competing fragmentation pathways.
Altered Lipid Composition and Enhanced Nutritional Value of Arabidopsis Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2
Publisher: Oxford University Press (OUP)
Date: 02-2013
Abstract: Enhancement of acyl-CoA–dependent triacylglycerol (TAG) synthesis in vegetative tissues is widely discussed as a potential avenue to increase the energy density of crops. Here, we report the identification and characterization of Chlamydomonas reinhardtii diacylglycerol acyltransferase type two (DGTT) enzymes and use DGTT2 to alter acyl carbon partitioning in plant vegetative tissues. This enzyme can accept a broad range of acyl-CoA substrates, allowing us to interrogate different acyl pools in transgenic plants. Expression of DGTT2 in Arabidopsis thaliana increased leaf TAG content, with some molecular species containing very-long-chain fatty acids. The acyl compositions of sphingolipids and surface waxes were altered, and cutin was decreased. The increased carbon partitioning into TAGs in the leaves of DGTT2-expressing lines had little effect on transcripts of the sphingolipid/wax/cutin pathway, suggesting that the supply of acyl groups for the assembly of these lipids is not transcriptionally adjusted. Caterpillars of the generalist herbivore Spodoptera exigua reared on transgenic plants gained more weight. Thus, the nutritional value and/or energy density of the transgenic lines was increased by ectopic expression of DGTT2 and acyl groups were erted from different pools into TAGs, demonstrating the interconnectivity of acyl metabolism in leaves.
Affecting Proton Mobility in Activated Peptide and Whole Protein Ions via Lysine Guanidination
Publisher: American Chemical Society (ACS)
Date: 16-10-2003
DOI: 10.1021/PR034054U
Abstract: We have evaluated the effect of lysine guanidination in peptides and proteins on the dissociation of protonated ions in the gas phase. The dissociation of guanidinated model peptide ions compared to their unmodified forms showed behavior consistent with concepts of proton mobility as a major factor in determining favored fragmentation channels. Reduction of proton mobility associated with lysine guanidination was reflected by a relative increase in cleavages occurring C-terminal to aspartic acid residues as well as increases in small molecule losses. To evaluate the effect of guanidination on the dissociation behavior of whole protein ions, bovine ubiquitin was selected as a model. Essentially, all of the amide bond cleavages associated with the +10 charge state of fully guanidinated ubiquitin were observed to occur C-terminal to aspartic acid residues, unlike the dissociation behavior of the +10 ion of the unmodified protein, where competing cleavage N-terminal to proline and nonspecific amide bond cleavages were also observed. The +8 and lower charge states of the guanidinated protein showed prominent losses of small neutral molecules. This overall fragmentation behavior is consistent with current hypotheses regarding whole protein dissociation that consider proton mobility and intramolecular charge solvation as important factors in determining favored dissociation channels, and are also consistent with the fragmentation behaviors observed for the guanidinated model peptide ions. Further evaluation of the utility of condensed phase guanidination of whole proteins is necessary but the results described here confirm that guanidination can be an effective strategy for enhancing C-terminal aspartic acid cleavages. Gas phase dissociation exclusively at aspartic acid residues, especially for whole protein ions, could be useful in identifying and characterizing proteins via tandem mass spectrometry of whole protein ions.
Zinc-activated C-peptide resistance to the type 2 diabetic erythrocyte is associated with hyperglycemia-induced phosphatidylserine externalization and reversed by metformin.
Publisher: Royal Society of Chemistry (RSC)
Date: 2009
DOI: 10.1039/B908241G
S‐Pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: A rapid mass spectrometric method for identifying cysteine‐containing p
Publisher: Wiley
Date: 1996
Abstract: In-gel proteolytic digestion of acrylamide-gel separated proteins is a method widely used for generating peptide fragments for the purpose of identifying proteins by Edman degratation, tandem mass spectrometry, and peptide-mass fingerprinting. However, it is well recognised for disulfide-bonded proteins electrophoresed under reducing conditions that if no precautions are taken to minimise disulfide bond formation during protein digestion or peptide isolation, complex peptide maps can result. Here, we describe an improved method for in-gel protein digestion. It consists of first reducing and S-pyridylethylating Coomassie Brilliant Blue R-250-stained proteins immobilised in the whole gel slab with dithiothreitol and 4-vinylpyridine, excising the in idual stained and alkylated proteins, and then digesting them in situ in the gel matrix with trypsin or Achromobacter lyticus protease I. Peptide fragments generated in this manner are extracted from the gel piece and purified to homogeneity by a rapid (< or = 12 min) reversed-phase high performance liquid chromatography (HPLC) procedure, based upon conventional silica supports. Recoveries of peptides are increased by S-pyridylethylation of acrylamide-immobilised proteins prior to in-gel digestion. Further, the levels of gel-related contaminants, which otherwise result in suppression of s le signals during electrosprayionisation mass spectrometry, are greatly reduced by the reduction/alkylation step. Additionally, we demonstrate that S-beta-(4-pyridylethyl)-cysteine containing peptides can be readily identified during reversed-phase HPLC by absorbance at 254 nm, and during electrospray ionisation tandem mass spectrometry by the appearance of a characteristic-pyridylethyl fragment ion of 106 Da. The position of cysteine residues in a sequence can be determined as phenylthiohydantoin S-beta-(4-pyridylethyl)-cysteine during Edman degradation, and by tandem mass spectrometry.
Reprogrammed Lipid Metabolism and the Lipid-Associated Hallmarks of Colorectal Cancer
Publisher: MDPI AG
Date: 29-07-2022
Abstract: Lipids have erse structures, with multifarious regulatory functions in membrane homeostasis and bioenergetic metabolism, in mediating functional protein–lipid and protein–protein interactions, as in cell signalling and proliferation. An increasing body of evidence supports the notion that aberrant lipid metabolism involving remodelling of cellular membrane structure and changes in energy homeostasis and signalling within cancer-associated pathways play a pivotal role in the onset, progression, and maintenance of colorectal cancer (CRC) and their tumorigenic properties. Recent advances in analytical lipidome analysis technologies have enabled the comprehensive identification and structural characterization of lipids and, consequently, our understanding of the role they play in tumour progression. However, despite progress in our understanding of cancer cell metabolism and lipidomics, the key lipid-associated changes in CRC have yet not been explicitly associated with the well-established ‘hallmarks of cancer’ defined by Hanahan and Weinberg. In this review, we summarize recent findings that highlight the role of reprogrammed lipid metabolism in CRC and use this growing body of evidence to propose eight lipid metabolism-associated hallmarks of colorectal cancer, and to emphasize their importance and linkages to the established cancer hallmarks.
The Proteome and Lipidome of Extracellular Vesicles from Haemonchus contortus to Underpin Explorations of Host–Parasite Cross–Talk
Publisher: MDPI AG
Date: 30-06-2023
Abstract: Many parasitic worms have a major adverse impact on human and animal populations worldwide due to the chronicity of their infections. There is a growing body of evidence indicating that extracellular vesicles (EVs) are intimately involved in modulating (suppressing) inflammatory/immune host responses and parasitism. As one of the most pathogenic nematodes of livestock animals, Haemonchus contortus is an ideal model system for EV exploration. Here, employing a multi-step enrichment process (in vitro culture, followed by ultracentrifugation, size exclusion and filtration), we enriched EVs from H. contortus and undertook the first comprehensive (qualitative and quantitative) multi-omic investigation of EV proteins and lipids using advanced liquid chromatography–mass spectrometry and informatics methods. We identified and quantified 561 proteins and 446 lipids in EVs and compared these molecules with those of adult worms. We identified unique molecules in EVs, such as proteins linked to lipid transportation and lipid species (i.e., sphingolipids) associated with signalling, indicating the involvement of these molecules in parasite-host cross-talk. This work provides a solid starting point to explore the functional roles of EV-specific proteins and lipids in modulating parasite-host cross-talk, and the prospect of finding ways of disrupting or interrupting this relationship to suppress or eliminate parasite infection.
Metal-activated C-peptide facilitates glucose clearance and the release of a nitric oxide stimulus via the GLUT1 transporter
Publisher: Springer Science and Business Media LLC
Date: 27-10-2007
Limited Proteolysis via Millisecond Digestions in Protease-Modified Membranes
Publisher: American Chemical Society (ACS)
Date: 21-09-2012
DOI: 10.1021/AC3019153
Editorial: 2021 ASMS “Emerging Investigators” Focus
Publisher: American Chemical Society (ACS)
Date: 09-2021
Early Warning System for Illicit Drug Use at Large Public Events: Trace Residue Analysis of Discarded Drug Packaging Samples
Publisher: American Chemical Society (ACS)
Date: 30-08-2021
Abstract: Inspired by Locard's exchange principle, which states "every contact leaves a trace", a trace residue s ling strategy has been developed for the analysis of discarded drug packaging s les (DPS), as part of an early warning system for illicit drug use at large public events including music/dance festivals. Using direct analysis in real time/mass spectrometry and tandem mass spectrometry, rapid and high-throughput identification and characterization of a wide range of illicit drugs and adulterant substances was achieved, including in complex polydrug mixtures and at low relative ion abundances. A total of 1362 DPS were analyzed either off-site using laboratory-based instrumentation or on-site and in close to real time using a transportable mass spectrometer housed within a mobile analytical laboratory, with each analysis requiring less than 1 min per s le. Of the DPS analyzed, 92.2% yielded positive results for at least one of 15 different drugs and/or adulterants, including cocaine, MDMA, and ketamine, as well as numerous novel psychoactive substances (NPS). Also, 52.6% of positive DPS were found to contain polydrug mixtures, and a total of 42 different drug and polydrug combinations were observed throughout the study. For analyses performed on-site, reports to key stakeholders including event organizers, first aid and medical personnel, and peer-based harm reduction workers could be provided in as little as 5 min after s le collection. Following risk assessment of the potential harms associated with their use, drug advisories or alerts were then disseminated to event staff and patrons and subsequently to the general public when substances with particularly toxic properties were identified.
Quantitative lipidomic analysis of Ascaris suum
Publisher: Public Library of Science (PLoS)
Date: 02-12-2020
DOI: 10.1371/JOURNAL.PNTD.0008848
Abstract: Ascaris is a soil-transmitted nematode that causes ascariasis, a neglected tropical disease affecting predominantly children and adolescents in the tropics and subtropics. Approximately 0.8 billion people are affected worldwide, equating to 0.86 million disability-adjusted life-years (DALYs). Exploring the molecular biology of Ascaris is important to gain a better understanding of the host-parasite interactions and disease processes, and supports the development of novel interventions. Although advances have been made in the genomics, transcriptomics and proteomics of Ascaris , its lipidome has received very limited attention. Lipidomics is an important sub-discipline of systems biology, focused on exploring lipids profiles in tissues and cells, and elucidating their biological and metabolic roles. Here, we characterised the lipidomes of key developmental stages and organ systems of Ascaris of porcine origin via high throughput LC-MS/MS. In total, 500 lipid species belonging to 18 lipid classes within three lipid categories were identified and quantified–in precise molar amounts in relation to the dry weight of worm material–in different developmental stages/sexes and organ systems. The results showed substantial differences in the composition and abundance of lipids with key roles in cellular processes and functions (e.g. energy storage regulation and membrane structure) among distinct stages and among organ systems, likely reflecting differing demands for lipids, depending on stage of growth and development as well as the need to adapt to constantly changing environments within and outside of the host animal. This work provides the first step toward understanding the biology of lipids in Ascaris , with possibilities to work toward designing new interventions against ascariasis.
Mechanisms for the selective gas-phase fragmentation reactions of methionine side chain fixed charge sulfonium ion containing peptides
Publisher: American Chemical Society (ACS)
Date: 12-2006
DOI: 10.1016/J.JASMS.2006.07.013
Abstract: To enable the development of improved tandem mass spectrometry based methods for selective proteome analysis, the mechanisms, product ion structures, and other factors influencing the gas-phase fragmentation reactions of methionine side-chain derivatized "fixed-charge" phenacylsulfonium ion containing peptide ions have been examined. Dissociation of these peptide ions results in the exclusive characteristic loss of the derivatized side chain, thereby enabling their selective identification. The resultant product ion(s) are then subjected to further dissociation to obtain sequence information for subsequent protein identification. Molecular orbital calculations (at the B3LYP/6-31 + G (d,p) level of theory) performed on a simple peptide model, together with experimental evidence obtained by multistage dissociation of a regioselectively deuterated methionine derivatized sulfonium ion containing tryptic peptide, indicate that fragmentation of the fixed charge containing peptide ions occurs via SN2 reactions involving the N- and C-terminal amide bonds adjacent to the methionine side chain, resulting in the formation of stable cyclic five- and six-membered iminohydrofuran and oxazine product ions, respectively. These studies further indicate that the rings formed via these neighboring group reactions are stable to further dissociation by MS3. As a consequence, the formation of b- or y-type sequence ions are "skipped" at the site of cyclization. Despite this, complete sequence information is still obtained because of the presence of both cyclic products.
Jack Throck Watson, May 2, 1939–September 3, 2016
Publisher: American Chemical Society (ACS)
Date: 13-02-2017
Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis
Publisher: Elsevier BV
Date: 05-2017
DOI: 10.1016/J.CELREP.2017.04.029
Abstract: Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.
The Effect of Post-Translational and Process-Induced Modifications on the Multistage Gas-Phase Fragmentation Reactions of Protonated Peptides
Publisher: Bentham Science Publishers Ltd.
Date: 02-2009
DOI: 10.2174/138620709787315445
Abstract: Significant effort has been extended in recent years toward the development and application of 'targeted' approaches for the identification, characterization and quantitative analysis of post-translational or process-induced protein modifications, based on the multistage tandem mass spectrometry (MS/MS and MS(3)) fragmentation reactions of their proteolytically derived peptide ions. Although these approaches have been successfully employed to date, the development of an improved understanding of the mechanisms and other factors (e.g., proton mobility, peptide conformation, product ion structures, etc.) that influence the multistage fragmentation reactions of modified peptide ions would facilitate further advances in the field. In this review, the important role of such mechanistic studies for rationalizing the effect of post-translational (e.g., phosphoserine- and phosphothreonine-containing peptides) and process-induced (e.g., oxidative modifications of methionine- and S-alkyl cysteine-containing peptides) protein modifications on the multistage collision induced dissociation gas-phase fragmentation reactions of proteolytically derived peptide ions are highlighted. Furthermore, recent efforts toward the development of chemical derivatization strategies for controlling and directing the gas-phase fragmentation reactions of protonated peptides toward the formation of analytically useful fragmentation pathways will be discussed, as well as the use of alternative dissociation techniques including electron capture dissociation (ECD) and electron transfer dissociation (ETD).
Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry
Publisher: American Chemical Society (ACS)
Date: 07-2005
DOI: 10.1016/J.JASMS.2005.03.015
Abstract: To enable the development of a tandem mass spectrometry (MS/MS) based methodology for selective protein identification and differential quantitative analysis, a novel derivatization strategy is proposed, based on the formation of a "fixed-charge" sulfonium ion on the side-chain of a methionine amino acid residue contained within a protein or peptide of interest. The gas-phase fragmentation behavior of these side chain fixed charge sulfonium ion containing peptides is observed to result in exclusive loss of the derivatized side chain and the formation of a single characteristic product ion, independently of charge state or amino acid composition. Thus, fixed charge containing peptide ions may be selectively identified from complex mixtures, for ex le, by selective neutral loss scan mode MS/MS methods. Further structural interrogation of identified peptide ions may be achieved by subjecting the characteristic MS/MS product ion to multistage MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by energy resolved "pseudo" MS3 in a triple quadrupole mass spectrometer. The general principles underlying this fixed charge derivatization approach are demonstrated here by MS/MS, MS3 and "pseudo" MS3 analysis of side chain fixed-charge sulfonium ion derivatives of peptides containing methionine formed by reaction with phenacylbromide. Incorporation of "light" and "heavy" isotopically encoded labels into the fixed-charge derivatives facilitates the application of this method to the quantitative analysis of differential protein expression, via measurement of the relative abundances of the neutral loss product ions generated by dissociation of the light and heavy labeled peptide ions. This approach, termed "selective extraction of labeled entities by charge derivatization and tandem mass spectrometry" (SELECT), thereby offers the potential for significantly improved sensitivity and selectivity for the identification and quantitative analysis of peptides or proteins containing selected structural features, without requirement for extensive fractionation or otherwise enrichment from a complex mixture prior to analysis.
Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry
Publisher: Wiley
Date: 04-03-2009
DOI: 10.1002/MAS.20219
Abstract: Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of phosphopeptides on TiO(2) seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex s les. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purification of small (microL) s les. On-plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid s le-handling steps, but this technique is likely limited to relatively simple s les such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways.
Charge-State-Dependent Sequence Analysis of Protonated Ubiquitin Ions via Ion Trap Tandem Mass Spectrometry
Publisher: American Chemical Society (ACS)
Date: 31-05-2001
DOI: 10.1021/AC0101095
Abstract: One of the major factors governing the "top-down" sequence analysis of intact multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions. To more fully understand this effect, electrospray ionization coupled to a quadrupole ion trap mass spectrometer, collision-induced dissociation, and gas-phase ion/ion reactions have been employed to examine the fragmentation of the [M + 12H]12+ to [M + H]+ ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed cleavage at aspartic acid residues was observed. At intermediate charge states, (+7, +8, and +9), extensive nonspecific fragmentation of the protein backbone was observed, with 50% sequence coverage obtained from the [M + 8H]8+ ion alone. At high charge states, (+10, +11, +12), the single dominant channel that was observed was the preferential fragmentation of a single proline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton model", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquitin in the gas phase, examined by ion mobility and hydrogen/deuterium exchange measurements.
Phosphopeptide Enrichment with TiO2-Modified Membranes and Investigation of Tau Protein Phosphorylation
Publisher: American Chemical Society (ACS)
Date: 28-05-2013
DOI: 10.1021/AC400198N
Gaseous apomyoglobin ion dissociation in a quadrupole ion trap: [M + 2H] 2+ -[M + 21H] 21+ 1 1Prepared for submission to the International Journal of Mass Spectrometry in honor of Prof. R. Graham Cook
Publisher: Elsevier BV
Date: 11-2001
Leaving group effects on the selectivity of the gas‐phase fragmentation reactions of side chain fixed‐charge‐containing peptide ions
Publisher: Wiley
Date: 11-12-2006
DOI: 10.1002/JMS.1150
Abstract: The effect of trialkylsulfonium versus quaternaryalkylammonium ions on the multistage gas-phase fragmentation reactions of side chain, fixed-charge, cysteine-containing peptides has been examined in a quadrupole linear ion trap. These tandem mass spectrometry experiments reveal that selective loss of dialkylsulfide from fixed-charge sulfonium ion derivatives is the dominant fragmentation pathway regardless of the degree of proton mobility, indicating that it is the most energetically favored fragmentation pathway. In contrast, the loss of trimethylamine from the side chain of fixed-charge ammonium-ion-containing cysteine derivatives appears to be less energetically favored, and as a result extensive charge-remote fragmentation is observed under low proton mobility conditions, while under conditions of high proton mobility, amide bond fragmentation reactions dominate. These findings are supported by molecular orbital calculations at the B3LYP/6-31 + G(d, p) level of theory, which showed that the neutral loss of dimethylsulfide is both thermodynamically and kinetically preferred over the loss of trimethylamine.
The Disulfide Bond Structure of Plasmodium Apical Membrane Antigen-1
Publisher: Elsevier BV
Date: 11-1996
Characterization of ornithine and glutamine lipids extracted from cell membranes of Rhodobacter sphaeroides
Publisher: American Chemical Society (ACS)
Date: 02-2009
A mass spectrometric and ab initio study of the pathways for dehydration of simple glycine and cysteine-containing peptide [M+H]+ ions
Publisher: American Chemical Society (ACS)
Date: 09-1998
Disulfide bond structure and N-glycosylation sites of the extracellular domain of the human interleukin-6 receptor
Publisher: Elsevier BV
Date: 03-1999
Response to Comment on: Tikhonenko et al. (2010) Remodeling of Retinal Fatty Acids in an Animal Model of Diabetes: A Decrease in Long-Chain Polyunsaturated Fatty Acids Is Associated with a Decrease in
Publisher: American Diabetes Association
Date: 07-2010
DOI: 10.2337/DB10-0623
Examining the role of lipid mediators in diabetic retinopathy
Publisher: Informa UK Limited
Date: 12-2012
DOI: 10.2217/CLP.12.68
A biosensor-based framework to measure latent proteostasis capacity
Publisher: Springer Science and Business Media LLC
Date: 18-01-2018
DOI: 10.1038/S41467-017-02562-5
Abstract: The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.
Helminth lipidomics: Technical aspects and future prospects
Publisher: Elsevier BV
Date: 2021
Strategies for internal amino acid sequence analysis of proteins separated by polyacrylamide gel electrophoresis
Publisher: Elsevier BV
Date: 10-1990
DOI: 10.1016/0021-9673(90)85148-O
Abstract: An evaluation has been made of various strategies for obtaining internal amino acid sequence data from electrophoretically separated proteins. Electroblotting, in situ proteolysis and extraction, and direct electroelution are compared. Electroblotting of protein or peptides from gels resulted in poor yields (typically, 1-7%). However, higher yields (3-67%) were achieved by in situ enzymatic cleavage followed by acid extraction of the peptides from the gel. Peptides extracted from the gel were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), on short, small-bore columns (100 x 2.1 mm I.D.), to enable recovery of peptides in small volumes (ca. 50 microliters) suitable for microsequence analysis. Capillary zone electrophoresis under acidic conditions (pH 2.5) was used to assess peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivative during sequence analysis. This derivative does not co-chromatograph with any known PTH-amino acid. Direct electrophoretic elution of protein from gels yielded between 45-50% of applied protein. Proteins recovered from gels by electrophoretic elution required further purification by inverse-gradient RP-HPLC [R. J. Simpson, R. L. Moritz, E. C. Nice and B. Grego, Eur. J. Biochem., 165 (1987) 21] to remove sodium dodecylsulphate and acrylamide-related contaminants for sequence analysis.
Detailed Structural Characterization of Sphingolipids via 193 nm Ultraviolet Photodissociation and Ultra High Resolution Tandem Mass Spectrometry
Publisher: American Chemical Society (ACS)
Date: 28-04-2017
DOI: 10.1007/S13361-017-1668-1
Abstract: Sphingolipids serve not only as components of cellular membranes but also as bioactive mediators of numerous cellular functions. As the biological activities of these lipids are dependent on their structures, and due to the limitations of conventional ion activation methods employed during tandem mass spectrometry (MS/MS), there is a recognized need for the development of improved structure-specific methods for their comprehensive identification and characterization. Here, positive-ionization mode 193 nm ultraviolet photodissociation (UVPD)-MS/MS has been implemented for the detailed structural characterization of lipid species from a range of sphingolipid classes introduced to the mass spectrometer via electrospray ionization as their lithiated or protonated adducts. These include sphingosine d18:1(4E), dihydrosphingosine (sphinganine) d18:0, sphingadiene d18:2(4E,11Z), the isomeric sphingolipids ceramide d18:1(4E)/18:0 and dihydroceramide d18:0/18:1(9Z), ceramide-1-phosphate d18:1(4Z)/16:0, sphingomyelin d18:1(4E)/18:1(9Z) the glycosphingolipids galactosyl ceramide d18:1(4E)/24:1(15Z) and lactosyl ceramide d18:1(4E)/24:0, and several endogenous lipids present within a porcine brain total lipid extract. In addition to the product ions formed by higher energy collision dissociation (HCD), UVPD is shown to yield a series of novel structurally diagnostic product ions resulting from cleavage of both sphingosine carbon-carbon and acyl chain carbon-carbon double bonds for direct localization of site(s) of unsaturation, as well as via diagnostic cleavages of the sphingosine backbone and N-C amide bond linkages. With activation timescales and dissociation efficiencies similar to those found in conventional MS/MS strategies, this approach is therefore a promising new tool in the arsenal of ion activation techniques toward providing complete structural elucidation in automated, high-throughput lipid analysis workflows. Graphical Abstract ᅟ.
Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization
Publisher: American Chemical Society (ACS)
Date: 06-07-2011
Oxidation-Induced Conformational Changes in Calcineurin Determined by Covalent Labeling and Tandem Mass Spectrometry
Publisher: American Chemical Society (ACS)
Date: 20-10-2014
DOI: 10.1021/BI5009744
Shedding light on isomeric FAHFA lipid structures using 213 nm ultraviolet photodissociation mass spectrometry
Publisher: SAGE Publications
Date: 22-09-2020
Abstract: Fatty Acid Esters of Hydroxy Fatty Acids (FAHFAs) are a recently discovered class of biological active lipids with anti-diabetic and anti-inflammatory functions. Given that structure and function are intimately related, we report here the use of direct infusion multi-stage hybrid tandem mass spectrometry involving sequential Collisional Activated Dissociation (CAD) and 213 nm UltraViolet PhotoDissociation (UVPD), as a novel technique for the unambiguous denovo identification and detailed structural characterisation of FAHFA lipid ions, including determination of the esterified fatty acid identity, the hydroxy fatty acid identity and position of esterification, and localization of the site(s) of endogenous unsaturations, without need for chromatographic separation or authentic reference standards. The utility of this approach is demonstrated for the identification of in idual FAHFA lipids introduced to the mass spectrometer in positive ionization mode as their lithiated adducts, as well as from mixtures containing isomeric FAHFA species with differing esterification sites, including those that are not resolved by current liquid chromatography methods.
‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility
Publisher: American Chemical Society (ACS)
Date: 08-2010
DOI: 10.1016/J.JASMS.2010.03.047
Abstract: Protein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. However, a common problem associated with the use of selective chemical labeling methods for mapping protein solvent accessible residues is that when a complicated peptide mixture resulting from a large protein or protein complex is analyzed, the modified peptides may be difficult to identify and characterize amongst the largely unmodified peptide population (i.e., the 'needle in a haystack' problem). To address this challenge, we describe here the development of a strategy involving the synthesis and application of a novel 'fixed charge' sulfonium ion containing lysine-specific protein modification reagent, S,S'-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), coupled with capillary HPLC-ESI-MS, automated CID-MS/MS, and data-dependant neutral loss mode MS(3) in an ion trap mass spectrometer, to map the surface accessible lysine residues in a small model protein, cellular retinoic acid binding protein II (CRABP II). After reaction with different reagent:protein ratios and digestion with Glu-C, modified peptides are selectively identified and the number of modifications within each peptide are determined by CID-MS/MS, via the exclusive neutral loss(es) of dimethylsulfide, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility) of the peptide. The observation of these characteristic neutral losses are then used to automatically 'trigger' the acquisition of an MS(3) spectrum to allow the peptide sequence and the site(s) of modification to be characterized. Using this approach, the experimentally determined relative solvent accessibilities of the lysine residues were found to show good agreement with the known solution structure of CRABP II.
A two‐dimensional gel database of human colon carcinoma proteins
Publisher: Wiley
Date: 1997
Abstract: The master two‐dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11 , 883–891 Ji et al., Electrophoresis 1994, 15 , 391–405). Updated two‐dimensional electrophoretic (2‐DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2‐DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their M r I loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2‐DE resolved proteins. This procedure relies on a combination of capillary column (0.10–0.32 mm internal diameter) reversed‐phase HPLC peptide mapping of in‐gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision‐induced dissociation or post‐source‐decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro‐techniques employed in this laboratory for identifying 2‐DE resolved proteins can be accessed via the internet URL: www.ludwig.edu.au .
Characterization of the Protease Processing Sites in a Multidomain Proteinase Inhibitor Precursor from Nicotiana Alata
Publisher: Wiley
Date: 05-1995
DOI: 10.1111/J.1432-1033.1995.TB20558.X
Abstract: A gene encoding a 40.3-kDa serine proteinase inhibitor (PI) precursor is expressed at high levels in the stigma of the ornamental tobacco, Nicotiana alata. The precursor is processed proteolytically in vivo to release five homologous proteinase inhibitors of approximately 6 kDa, as well as two flanking peptides. The five PIs have been purified from stigmas and identified by N-terminal sequencing, electrospray mass spectrometry and inhibition activity against chymotrypsin or trypsin. One of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeated six times in the precursor molecule. In the plant, the initial cleavage probably occurs between asparagine and the aspartate residues and ragged ends are formed by subsequent trimming. In vitro, the protease-sensitive linker region is selectively cleaved by the endoproteinases Asp-N, Glu-C and Lys-C to release fully active approximately 6-kDa PIs that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effectively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 precursor molecule indicates that processing is required to activate or expose all of the four trypsin inhibitory sites.
Special Focus: Honoring John Yates for Receiving the 2019 John B. Fenn Award for a Distinguished Contribution in Mass Spectrometry
Publisher: American Chemical Society (ACS)
Date: 07-2020
Multistage tandem mass spectrometry of anionic phosphatidylcholine lipid adducts reveals novel dissociation pathways
Publisher: Elsevier BV
Date: 06-2006
Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33
Publisher: Wiley
Date: 1997
Abstract: The murine monoclonal antibody A33 (mAbA33) recognises a human cell membrane‐associated antigen selectively expressed in epithelial cells of the lower gastrointestinal tract and 90% of colonic cancers, but is not detected in a wide range of other normal tissues by immunohistochemical analysis. In phase I/II clinical triasl, mAbA33 has been shown to target advanced colon cancers and the humanised version is currently being evaluated in therapy studies. Although the mAbA33 has been well characterised by immunohistochemical and clinical studies, until recently, the target antigen has remained poorly defined. This was largely attributable to the antigenic determinant recognised by mAbA33 being dependent on the native spatial conformation of the A33 antigen which impeded its identification by conventional two‐dimensional electrophoresis (2‐DE) and immunoblot analysis. We have developed an immunoblot method, based on nonreducing/non‐urea precast 2‐DE gels, that has facilitated the purification of the detergent (0.3% Triton X‐100) solubilised A33 antigen from the human colon cancer cell lines LIM1215 and SW1222. Under these 2‐DE conditions, the A33 antigen electrophoreses with an apparent M r ˜ 41000 and p I 5.0–6.0. Attempts to isolate the A33 antigen from 2‐DE gels for direct structural analysis were unsuccessful, due to its co‐electrophoresis with actin and cytokeratin proteins. However, using Western blot and biosensor detection the A33 antigen has been purified chromatographically and N ‐terminal sequence analysis was possible. Using polyclonal antibodies raised against a synthetic peptide corresponding to the N ‐terminal region of the A33 antigen we have used Western blot analysis to localise the molecule in our master 2‐DE protein database for normal human colon crypts and several colon carcinoma cell lines (URL address: www.ludwig.edu.au ). Under reducing 2‐DE conditions, the A33 antigen electrophoresis as 6 differentially charged isoforms (p I 4.6–4.8) with a single molecular weight species at M r ˜ 55000.
Molecular Evidence for the Involvement of Alpha Crystallin in the Colouration/Crosslinking of Crystallins in Age-related Nuclear Cataract
Publisher: Elsevier BV
Date: 12-1997
Abstract: The proteins of the lens which become insoluble, crosslinked and coloured as a result of the onset of human nuclear cataract have been studied using a combination of enzymatic digestion and HPLC/mass spectrometry (MS). The objective was to determine if such an approach could provide information on the identities of the polypeptide components involved in the colouration and crosslinking and to discover whether any crystallins predominate in this characteristic post-translational modification process. Initially, coloured high molecular weight peptides were isolated from a tryptic/chymotryptic digest of the 6 M guanidine hydrochloride-insoluble lens protein fraction. These tryptic/chymotryptic peptides were then incubated with pronase and the small peptides released, purified by gel filtration. All but one of the peptides analysed by HPLC/MS/MS were found to contain proline. Peptides derived from alpha-crystallin were found to comprise the great majority of the peptides characterised. No gamma-crystallin peptides were observed. Both alpha A-crystallin and alpha B-crystallin were represented. Further, all but one of these peptides were derived from the N-terminal region of the alpha-crystallin subunits: a region recently implicated in the chaperone activity of alpha-crystallin. This finding suggests that the putative N-terminal domain of alpha-crystallin may be involved at the molecular level in the process of crosslinking and colouration which is known to be characteristic of age-related nuclear cataract. It is, therefore, conceivable that an early stage of these cataractous modifications may involve alpha-crystallin acting as a molecular chaperone.
Two‐dimensional electrophoretic analysis of human breast carcinoma proteins: Mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2
Publisher: Wiley
Date: 1997
Abstract: MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (Eur. J. Biochem. 1993, 213 , 701–710), comprises several distinct structural domains including an src homology‐3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif. Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction. To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N ‐terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23–77), fused to glutathione S‐transferase. This fusion protein was expressed in Escherichia coli , purified using gluthathione‐Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2‐SH3 domain binding proteins from lysates of 35 S‐labelled MDA‐MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low‐abundance proteins with a molecular weights ( M r ) of ˜ 31 500 and ˜ 34 000, bound consistently to the MLK2N protein. To establish accurately the M r / isoelectric point (p I ) loci of these MLK2‐SH3 domain binding proteins, a number of abundant proteins in a two‐dimensional electrophoresis (2‐DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in‐gel proteolysis and fractionation by rapid (˜ 12 min)˜ microbore column (2.1 mm ID) reversed‐phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide‐mass fingerprinting and electrospray (ESI) – mass spectrometry (MS)‐MS utilizing capillary (0.2–0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2‐DE protein database for the human breast carcinoma cell line MDA‐MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: www.ludwig.edu.au/www/jpsl/jpslhome.html ).
Ion/ion chemistry as a top-down approach for protein analysis
Publisher: Elsevier BV
Date: 02-2002
DOI: 10.1016/S0958-1669(02)00285-9
Abstract: Developing methodology for analyzing complex protein mixtures in a rapid fashion is one of the most challenging problems facing analytical biochemists today. Recent advances in mass spectrometry for the analysis of intact proteins (i.e. the top-down approach) show great promise for rapid protein identification. The ion/ion chemistry approach for the detection and identification of target proteins in complex matrices, determination of fragmentation channels as a function of precursor ion charge state, and post-translational modification characterization are discussed with particular emphasis on tandem mass spectrometry of intact proteins.
Synthesis and X‐Ray Crystallographic Characterisation of Frustum‐Shaped Ligated [Cu18H16(DPPE)6]2+ and [Cu16H14(DPPA)6]
Publisher:
Wiley
Date:
16-01-2018
Abstract: We report new structural motifs for Cu nanoclusters that conceptually represent seed crystals for large face-centred cubic (FCC) crystal growth. Kinetically controlled syntheses, high resolution mass spectrometry experiments for determination of the dication formulae and crystallographic characterisation were carried out for [Cu
Publisher: Wiley
Date: 16-01-2018
Abstract: We report new structural motifs for Cu nanoclusters that conceptually represent seed crystals for large face-centred cubic (FCC) crystal growth. Kinetically controlled syntheses, high resolution mass spectrometry experiments for determination of the dication formulae and crystallographic characterisation were carried out for [Cu
Mutation detection using mass spectrometric separation of tiny oligonucleotide fragments
Publisher: Cold Spring Harbor Laboratory
Date: 12-06-2002
DOI: 10.1101/GR.GR-1578R
Synthesis of diazo functionalized solid supports and their application towards the enrichment of phosphorylated peptides
Publisher: Royal Society of Chemistry (RSC)
Date: 2009
DOI: 10.1039/B906577F
Abstract: We describe herein the synthesis and application of a range of diazo-functionalized materials as platforms for the solid phase enrichment of phosphorylated peptides from protein digests. Diazo-functionalized solid phase resins are employed to bind covalently and specifically with the phosphate moiety of a peptide substrate, allowing for non-phosphorylated peptides to be efficiently removed by filtration. The technique described herein is compatible with enrichment of phospho-serine, threonine and tyrosine residues and was used to successfully enrich phosphorylated substrates from beta-casein and ovalbumin digests in low-picomolar quantities.
Femtosecond laser scalpel technology for proteomic mass spectrometry
Publisher: SPIE
Date: 12-02-2009
DOI: 10.1117/12.808934
Analytical Challenges and Recent Advances in Mass Spectrometry Based Lipidomics
Publisher: American Chemical Society (ACS)
Date: 08-12-2017
Characterization of rat brain stathmin isoforms by two‐dimensional gel electrophoresis‐matrix assisted laser desorption/ionization and electrospray ionizationion trap mass spectrometry
Publisher: Wiley
Date: 05-1998
Abstract: Stathmin is a regulatory phosphoprotein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. There are at least 14 isoforms of stathmin that migrate on two-dimensional electrophoresis (2-DE): two unphosphorylated, and 12 increasingly phosphorylated proteins. Following extracellular stimuli, stathmin is phosphorylated on four serines (Ser16, Ser25, Ser38, and Ser63) by several kinases, such as mitogen-activated protein (MAP), cdc2 kinase, protein kinase A, and Ca2+/calmodulin-dependent kinase-Gr. While all forms of stathmin are derived from the same protein encoded by a single mRNA, the precise nature of the post-translational modifications has not been clear. In this study we have characterized three rat brain stathmin isoforms, #1, #3 and #4, which electrophorese on 2-DE with apparent molecular weight (Mr)/isoelectric point (pI) values of 15,500/6.2, 15,000/6.1, and 15,000/6.0, respectively. The phosphorylation status of these isoforms was determined using a combination of peptide mapping, matrix-assisted laser desorption/ionization mass spectrometry and electrospray-ionization ion trap mass spectrometry. Stathmin isoform #1 was not phosphorylated, stathmin isoform #3 was phosphorylated on Ser38 only, and stathmin isoform #4 was phosphorylated on Ser38 however, the phosphorylation status of Ser63 could not be determined. In addition, three proteins which electrophorese near stathmin were identified in order to more accurately define the Mr I locus of this region of the 2-DE gel map. These include: phosphatidyl ethanolamine binding protein (Mr approximately 18,000 I 6.0), synuclein forms 2 and 3 (Mr approximately 14,000 I 5.4), and synuclein form 2 (Mr approximately 15,000 I 5.0).
Characterization and multiplexed quantification of derivatized aminophospholipids
Publisher: Elsevier BV
Date: 11-2015
Two‐dimensional gel database of human breast carcinoma cell expressed proteins: An update
Publisher: Wiley
Date: 05-1998
Abstract: Previously, we reported a two-dimensional gel map and database with molecular weight/isoelectric point (Mr I) loci for 22 proteins expressed in the breast carcinoma cell line, MDA-MB231 (Rasmussen et al., Electrophoresis 1997, 18, 588-598). Here we update this database with Mr I loci for a further nine cytoplasmic proteins and three Triton X-114 solubilised membrane proteins from MDA-MB231 cells. In addition, a novel protein, previously represented only in expressed sequence tag (EST) databases, has been identified as a Triton X-114 soluble protein and assigned an Mr I locus. During the course of isolating proteins from the Triton X-114 fraction, we compared recoveries of proteins in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels after isoelectric focusing (IEF) using either immobilised pH gradients or carrier holytes. In these experiments, a significantly higher proportion of membrane proteins were visible in SDS-polyacrylamide gels after the use of carrier holytes for the first dimension. We also report our mass spectrometric-based procedure for identifying two-dimensional electrophoresis (2-DE) gel-resolved proteins, combining in-gel enzymatic digestion, 0.2 mm internal diameter (ID) capillary column reversed-phase high-performance liquid chromatography (RP-HPLC) peptide mapping and electrospray ionisation--ion trap--mass spectrometry.
Altered Lipid Metabolism in Residual White Adipose Tissues of Bscl2 Deficient Mice
Publisher: Public Library of Science (PLoS)
Date: 16-12-2013
Performance of a quadrupole ion trap mass spectrometer adapted for ion/ion reaction studies
Publisher: Elsevier BV
Date: 2003
Charge state dependent fragmentation of gaseous protein ions in a quadrupole ion trap: bovine ferri-, ferro-, and apo-cytochrome c
Publisher: Elsevier BV
Date: 08-2002
Hidden information on protein function in censuses of proteome foldedness
Publisher: Cold Spring Harbor Laboratory
Date: 25-02-2021
DOI: 10.1101/2021.02.24.432609
Abstract: Methods that assay protein foldedness with proteomics have generated censuses of protein folding stabilities in biological milieu. Surprisingly, different censuses poorly correlate with each other. Here, we show that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding. About one quarter of cysteine or methionine sidechains in proteins in mammalian cell lysate increase in reactivity upon chemical denaturant titration consistent with two-state unfolding. Paradoxically, up to one third decreased reactivity, which were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings. We show that proteome folding stability censuses are ill-defined because they earmark hidden information on conformation and ligand binding.
A Point Mutation Converts Dihydroneopterin Aldolase to a Cofactor-Independent Oxygenase
Publisher: American Chemical Society (ACS)
Date: 19-09-2006
DOI: 10.1021/JA063455I
Abstract: Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (1) to 6-hydroxymethyl-7,8-dihydropterin (4) in the folate biosynthetic pathway. Substitution of a conserved tyrosine residue at the active site of DHNA by phenylalanine converts the enzyme to a cofactor-independent oxygenase, which generates mainly 7,8-dihydroxanthopterin (6) rather than 4. 6 is generated via the same enol intermediate as in the wild-type enzyme-catalyzed reaction, but this species undergoes an oxygenation reaction to form 6. The conserved tyrosine residue plays only a minor role in the formation of the enol reaction intermediate but a critical role in the protonation of the enol intermediate to form 4.
Characterization of smokeless powders using nanoelectrospray ionization mass spectrometry (nESI-MS)
Publisher: Springer Science and Business Media LLC
Date: 26-02-2009
DOI: 10.1007/S00216-009-2689-9
Abstract: Smokeless powder is one of the most common types of explosives used in civilian ammunition and, hence, its detection and identification is of great forensic value. Based on comparison of physical properties, extraction yield in methanol, and the spectra obtained using nanoelectrospray ionization and multistage tandem mass spectrometry (MS/MS) in a quadrupole ion trap mass spectrometer, a method was developed to identify and differentiate unburned smokeless powders from different brands of ammunition. The mass spectrometry method was optimized for the simultaneous detection of the organic stabilizers commonly present in smokeless powders: methyl centralite, ethyl centralite, and diphenylamine. All but two of the powders were differentiated however, the two that were not differentiated were produced by the same manufacturer. Gunshot residue from the cartridges was deposited on cotton cloth and collision-induced dissociation MS/MS was used to identify low levels of ethyl centralite in the residue, despite the presence of contaminants.
Quantification of Competing H3PO4 Versus HPO3 + H2O Neutral Losses from Regioselective 18O-Labeled Phosphopeptides
Publisher: American Chemical Society (ACS)
Date: 19-11-2013
DOI: 10.1007/S13361-013-0744-4
Abstract: Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of protonated phosphopeptide ions. Two competing fragmentation pathways are involved in this process, namely, the direct loss of H3PO4 from the phosphorylated residue and the combined losses of HPO3 and H2O from the phosphorylation site and from an additional site within the peptide, respectively. These competing pathways produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS(3) for phosphorylation site localization. To quantify the relative contributions of these pathways and to determine the conditions under which each pathway predominates, we have examined the ion trap CID-MS/MS fragmentation of a series of regioselective (18)O-phosphate ester labeled phosphopeptides prepared using novel solution-phase amino acid synthesis and solid-phase peptide synthesis methodologies. By comparing the intensity of the -100 Da (-H3PO3 (18)O) versus -98 Da (-[HPO3 + H2O]) neutral loss product ions formed upon MS/MS, quantification of the two pathways was achieved. Factors that affect the extent of formation of the competing neutral losses were investigated, with the combined loss pathway predominantly occurring under conditions of limited proton mobility, and with increased combined losses observed for phosphothreonine compared with phosphoserine-containing peptides. The combined loss pathway was found to be less dominant under ion activation conditions associated with HCD-MS/MS. Finally, the contribution of carboxylic acid functional groups and backbone amide bonds to the water loss in the combined loss fragmentation pathway was determined via methyl esterification and by examination of a phosphopeptide lacking side-chain hydroxyl groups.
Trace Residue Identification, Characterization and Longitudinal Monitoring of the Novel Synthetic Opioid β-U10, from Discarded Drug Paraphernalia
Publisher: American Chemical Society (ACS)
Date: 09-03-2022
DOI: 10.26434/CHEMRXIV-2022-2BR4R
Abstract: Empirical data regarding dynamic alterations in illicit drug supply markets in response to the COVID-19 pandemic, including the potential for introduction of novel drug substances and/or increased poly-drug combinations at the ‘street’ level (i.e., directly proximal to the point of consumption), is currently lacking. Here, a high-throughput strategy employing ambient ionization-mass spectrometry is described for the trace residue identification, characterization and longitudinal monitoring of illicit drug substances found within ,600 discarded drug paraphernalia (DDP) s les collected during a pilot study of an early warning system for illicit drug use in Melbourne, Australia from August 2020-February 2021, while significant COVID-19 lockdown conditions were imposed. The utility of this approach is demonstrated for the de novo identification and structural characterization of β-U10, a previously unreported naphthamide analogue within the ‘U-series’ of synthetic opioid drugs, including differentiation from its α-U10 isomer without need for s le preparation or chromatographic separation prior to analysis. Notably, β-U10 was observed with 23 other drug substances, most commonly in temporally distinct clusters with heroin, etizolam and diphenhydramine, and in a total of 182 different poly-drug combinations. Finally, longitudinal monitoring of the number and weekly ‘average signal intensity’ (ASI) values of identified substances, developed here as a semi-quantitative proxy indicator of changes in availability, relative purity and compositions of street level drug s les, revealed that increases in the number of identifications and ASI for β-U10 and etizolam coincided with a 50% decrease in the number of positive detections and an order of magnitude decrease in the ASI for heroin.
Fragmentation reactions of all 64 protonated trimer oligodeoxynucleotides and 16 mixed base tetramer oligodeoxynucleotides via tandem mass spectrometry in an ion trap
Publisher: Elsevier BV
Date: 2000
Evaluation of Gas-Phase Rearrangement and Competing Fragmentation Reactions on Protein Phosphorylation Site Assignment Using Collision Induced Dissociation-MS/MS and MS3
Publisher: American Chemical Society (ACS)
Date: 14-11-2008
DOI: 10.1021/AC801768S
Abstract: The development of strategies directed toward comprehensive analysis of the phosphoproteome have undoubtedly been facilitated by recent advances in the application of ion trap tandem mass spectrometry-based techniques for routine phosphopeptide identification. However, when multiple potential sites of phosphorylation exist within a phosphorylated peptide sequence, unambiguous characterization of the site of phosphorylation remains a significant challenge. Here, the gas-phase fragmentation reactions of a series of 33 synthetic phospho-serine, -threonine, and -tyrosine peptides containing multiple potential phosphorylation sites have been examined using collision induced dissociation (CID) and multistage tandem mass spectrometry (MS/MS and MS(3)) in a linear quadrupole ion trap. From this study, 15 of the peptides (45%) gave rise to product ions that were formed following initial transfer of a phosphate group from the phosphorylated residue to an unmodified hydroxyl-containing amino acid residue upon CID-MS/MS. The propensity for this rearrangement was found to be highly dependent on the precursor ion charge state and amino acid composition (i.e, proton mobility) of the peptide and was observed predominantly for peptides under "nonmobile" or "partially mobile" protonation conditions. The observation of these rearrangement reactions and/or the lack of product ions that provided definitive evidence for the correct site of phosphorylation, limited the ability to unambiguously assign the correct site of phosphorylation to only 12 of the 33 peptides (36%). Furthermore, the observation of competing fragmentation reactions for the neutral loss of 98 Da from these precursor ions (i.e., the loss of H(3)PO(4) versus the combined losses of HPO(3) and H(2)O) indicates that CID-MS(3) of [M + nH - 98](n+) ions may not be used for unambiguous phosphorylation site localization.
Combined Genetic and Metabolic Manipulation of Lipids in Rhodobacter sphaeroides Reveals Non-Phospholipid Substitutions in Fully Active Cytochrome c Oxidase
Publisher: American Chemical Society (ACS)
Date: 22-04-2011
DOI: 10.1021/BI1017039
Examining factors that influence erroneous phosphorylation site localization via competing fragmentation and rearrangement reactions during ion trap CID ‐MS /MS and ‐
Publisher:
Wiley
Date:
03-2013
Abstract: Factors influencing the magnitude of competing fragmentation and intramolecular phosphate group rearrangement reactions during CID-MS/MS and CID-MS(3) of protonated phosphopeptide ions in ion trap mass spectrometers, and their effect on phosphorylation site localization using automated search algorithms, have been examined by systematically varying the peptide composition, the identity, number, and position of the phosphorylated "donor" and nonphosphorylated "acceptor" residues, and the proton mobility of the precursor ion charge states for a synthetic phosphopeptide library. CID-MS(3) of product ions formed via combined neutral losses of HPO3 and H2 O, rather than direct loss of H3 PO4 from phosphotyrosine containing peptides yielded incorrect phosphorylation site assignments, while correct phosphorylation site assignments for phosphothreonine and phosphoserine containing peptides were highly dependant on the relative abundance of these competing fragmentation pathways. Abundant phosphate group rearrangement product ions were observed from CID-MS/MS of multiply protonated phosphopeptide ions, with increased rearrangement under nonmobile or partially mobile protonation conditions, and as a function of the identity and number of the donor and acceptor residues. A clear inverse trend was observed between the litude of these rearrangement reactions and the confidence for phosphorylation site localization, and rearrangement played a contributing role in erroneous phosphorylation site assignment for several peptides.
Publisher: Wiley
Date: 03-2013
Abstract: Factors influencing the magnitude of competing fragmentation and intramolecular phosphate group rearrangement reactions during CID-MS/MS and CID-MS(3) of protonated phosphopeptide ions in ion trap mass spectrometers, and their effect on phosphorylation site localization using automated search algorithms, have been examined by systematically varying the peptide composition, the identity, number, and position of the phosphorylated "donor" and nonphosphorylated "acceptor" residues, and the proton mobility of the precursor ion charge states for a synthetic phosphopeptide library. CID-MS(3) of product ions formed via combined neutral losses of HPO3 and H2 O, rather than direct loss of H3 PO4 from phosphotyrosine containing peptides yielded incorrect phosphorylation site assignments, while correct phosphorylation site assignments for phosphothreonine and phosphoserine containing peptides were highly dependant on the relative abundance of these competing fragmentation pathways. Abundant phosphate group rearrangement product ions were observed from CID-MS/MS of multiply protonated phosphopeptide ions, with increased rearrangement under nonmobile or partially mobile protonation conditions, and as a function of the identity and number of the donor and acceptor residues. A clear inverse trend was observed between the litude of these rearrangement reactions and the confidence for phosphorylation site localization, and rearrangement played a contributing role in erroneous phosphorylation site assignment for several peptides.
Identification of p65-Associated Phosphoproteins by Mass Spectrometry after On-Plate Phosphopeptide Enrichment Using Polymer-oxotitanium Films
Publisher: American Chemical Society (ACS)
Date: 04-05-2010
DOI: 10.1021/PR901200M
Abstract: Stimuli-induced protein phosphorylation plays a vital role in signal transduction and transcriptional activities in eukaryotic cells. This work aims to develop analysis techniques that rapidly detect stimulus-specific intracellular protein phosphorylation and association, with specific emphasis on identifying phosphoproteins associated with p65, a nuclear regulatory factor. The analytical strategy includes immunoprecipitation of the target protein along with its associated proteins, tryptic digestion directly on the antibody beads, on-plate phosphopeptide enrichment for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) to identify phosphopeptides and phosphorylation sites. Enrichment of the phosphopeptides from the tryptic digest occurs on a polymer-oxotitanium-modified gold wafer (Au-P-oxoTi) and consumes as little as 1 microL of digest solution. The Au-P-oxoTi wafers can capture both mono- and multiphosphorylated peptides from model protein digests containing high concentrations of nonphosphopeptides, urea, and salts. When combined with MALDI-MS/MS, the enrichment technique reveals nine phosphopeptides from p65-associated proteins immunoprecipitated from human acute monocytic leukemia (THP-1) cell nuclear extracts. Semiquantitative MALDI-MS shows that the levels of these proteins increase dramatically after treatment with tumor necrosis factor (TNF)-alpha. Overall, these techniques facilitated the identification of five p65-associated proteins, two of which were not previously reported to interact with p65.
Peptide mapping and internal sequencing of proteins electroblotted from two-dimensional gels onto polyvinylidene difluoride membranes
Publisher: Elsevier BV
Date: 1989
DOI: 10.1016/S0021-9673(01)93881-6
Abstract: Direct sequence analysis of proteins electroblotted from two-dimensional polyacrylamide gels onto immobilizing matrices provides an efficient technique for obtaining N-terminal sequence data for proteins not amenable to purification by reversed-phase high-performance liquid chromatography (RP-HPLC). We present in this paper a procedure for obtaining peptide fragments from electroblotted proteins for internal amino acid sequence analysis. First, Coomassie Blue-stained proteins are extracted from poly inylidene difluoride membranes, using a detergent mixture of sodium dodecylsulfate and Triton X-100. Proteins are then separated from the detergent mixture by a chromatographic procedure which relies on the ability of proteins to interact with certain reversed-phase sorbents at high organic solvent concentrations. Under these conditions, detergents and Coomassie Blue are not retained and pass through the column. Proteins are recovered by simultaneously: (i) introducing trifluoroacetic acid into the mobile phase and (ii) decreasing the organic solvent concentration. After proteolytic fragmentation, peptides are purified by microbore-column (1-2 mm I.D.) RP-HPLC for microsequence analysis.
Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation
Publisher: Public Library of Science (PLoS)
Date: 18-12-2012
Does side chain water loss from protonated threonine yield N-protonated dehydroamino-2-butyric acid?
Publisher: Wiley
Date: 15-08-1998
DOI: 10.1002/(SICI)1097-0231(19980815)12:15<999::AID-RCM274>3.0.CO;2-K
Evaluation of ultraviolet photodissociation tandem mass spectrometry for the structural assignment of unsaturated fatty acid double bond positional isomers
Publisher: Springer Science and Business Media LLC
Date: 31-01-2020
Gas-Phase Concentration, Purification, and Identification of Whole Proteins from Complex Mixtures
Publisher: American Chemical Society (ACS)
Date: 30-05-2002
DOI: 10.1021/JA025966K
Abstract: Five proteins present in a relatively complex mixture derived from a whole cell lysate fraction of E. coli have been concentrated, purified, and dissociated in the gas phase, using a quadrupole ion trap mass spectrometer. Concentration of intact protein ions was effected using gas-phase ion/ion proton-transfer reactions in conjunction with mass-to-charge dependent ion "parking" to accumulate protein ions initially dispersed over a range of charge states into a single lower charge state. Sequential ion isolation events interspersed with additional ion parking ion/ion reaction periods were used to "charge-state purify" the protein ion of interest. Five of the most abundant protein components present in the mixture were subjected to this concentration urification procedure and then dissociated by collisional activation of their intact multiply charged precursor ions. Four of the five proteins were subsequently identified by matching the uninterpreted product ion spectra against a partially annotated protein sequence database, coupled with a novel scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential cleavage sites. The identification of these proteins illustrates the potential of this "top-down" protein identification approach to reduce the reliance on condensed-phase chemistries and extensive separations for complex protein mixture analysis.
Nonreducing two‐dimensional polyacrylamide gel electrophoretic analysis of human colonic proteins
Publisher: Wiley
Date: 1995
Abstract: Immunochemical detection of proteins with antigenic determinants that are dependent on the native spatial conformation of the protein can often pose problems with conventional two-dimensional polyacrylamide gel electrophoresis (2-DE). For ex le, many antigenic determinants are readily destroyed by reducing agents and/or urea, reagents which are critical components of many of the conventional isoelectric focusing and immobilized-pH-gradient (IPG) protocols used in the first electrophoretic dimension. Here we describe the use of commercially available precast 2-DE gels for performing nonreducing/non-urea 2-DE of proteins extracted from the human colon cancer cell line LIM 1215 with 0.3% Triton X-100 that permit the identification of antigens with conformational determinants by immunoblot analysis. Previous, related studies demonstrated the usefulness of peptide-mass fingerprinting for identifying 2-DE resolved proteins. Here we show how partial protein sequence data obtained by rapid peptide mapping, using capillary column liquid chromatography directly coupled with electrospray ionization tandem mass spectrometric methodologies, enhances the usefulness of this approach for identifying incompletely resolved proteins. The nonreducing 2-DE gel images reported in this study, along with our master 2-DE gel protein database for both normal human colonic crypts and several colon-cancer-derived cell lines, and information regarding microtechniques employed in this laboratory for obtaining structural data on 2-DE resolved proteins can be accessed over the Internet using World Wide Web (URL address: http:@www.ludwig.edu.au).
Exploring the significance of lipids in Alzheimer's disease and the potential of extracellular vesicles
Publisher: Wiley
Date: 31-08-2023
Proteomic analysis of the human colon carcinoma cell line (LIM 1215): Development of a membrane protein database
Publisher: Wiley
Date: 05-2000
DOI: 10.1002/(SICI)1522-2683(20000501)21:9<1707::AID-ELPS1707>3.0.CO;2-Q
Ion Parking during Ion/Ion Reactions in Electrodynamic Ion Traps
Publisher: American Chemical Society (ACS)
Date: 08-12-2001
DOI: 10.1021/AC0109671
Abstract: Under appropriate ion density conditions, it is possible to selectively inhibit rates of ion/ion reactions in a quadrupole ion trap via the application of oscillatory voltages to one or more electrodes of the ion trap. The phenomenon is demonstrated using dipolar resonance excitation applied to the end-cap electrodes of a three-dimensional quadrupole ion trap. The application of a resonance excitation voltage tuned to inhibit the ion/ion reaction rate of a specific range of ion mass-to-charge ratios is referred to as "ion parking". The bases for rate inhibition are (i) an increase in the relative velocity of the ion/ion reaction pair, which reduces the cross section for ion/ion capture and, at least in some cases, (ii) reduction in the time of physical overlap of positively charged and negatively charged ion clouds. The efficiency and specificity of the ion parking experiment is highly dependent upon ion densities, trapping conditions, ion charge states, and resonance excitation conditions. The ion parking experiment is illustrated herein along with applications to the concentration of ions originally present over a range of charge states into a selected charge state and in the selection of a particular ion from a set of ions derived from a simple protein mixture.
Mining a Tandem Mass Spectrometry Database To Determine the Trends and Global Factors Influencing Peptide Fragmentation
Publisher: American Chemical Society (ACS)
Date: 03-10-2003
DOI: 10.1021/AC034616T
Abstract: A database of 5500 unique peptide tandem mass spectra acquired in an ion trap mass spectrometer was assembled for peptides derived from proteins digested with trypsin. Peptides were identified initially from their tandem mass spectra by the SEQUEST algorithm and subsequently validated manually. Two different statistical methods were used to identify sequence-dependent fragmentation patterns that could be used to improve fragmentation models incorporated into current peptide sequencing and database search algorithms. The currently accepted "mobile proton" model was expanded to derive a new classification scheme for peptide mass spectra, the "relative proton mobility" scale, which considers peptide ion charge state and amino acid composition to categorize peptide mass spectra into peptide ions containing "nonmobile", "partially mobile", or "mobile" protons. Quantitation of amide bond fragmentation, both N- and C-terminal to any given amino acid, as well as the positional effect of an amino acid in a peptide and peptide length on such fragmentation, has been determined. Peptide bond cleavage propensities, both positive (i.e., enhanced) and negative (i.e., suppressed), were determined and ranked in order of their cleavage preferences as primary, secondary, or tertiary cleavage effects. For ex le, primary positive cleavage effects were observed for Xaa-Pro and Asp-Xaa bond cleavage for mobile and nonmobile peptide ion categories, respectively. We also report specific pairwise interactions (e.g., Asn-Gly) that result in enhanced amide bond cleavages analogous to those observed in solution-phase chemistry. Peptides classified as nonmobile gave low or insignificant scores, below reported MS/MS score thresholds (cutoff filters), indicating that incorporation of the relative proton mobility scale classification would lead to improvements in current MS/MS scoring functions.
Two‐dimensional electrophoretic analysis of mixed lineage kinase 2 N‐terminal domain binding proteins
Publisher: Wiley
Date: 05-1998
Abstract: The mixed lineage kinase 2 (MLK2) protein contains several structurally distinct domains including an src homology (SH) 3 domain, a kinase catalytic domain, two leucine zippers, a basic motif and a cdc42/rac interactive binding motif. These domains have been recognized mainly for their involvement in protein-protein interactions in signal transduction networks. The SH3 domain in particular has been implicated in control of signaling events. To identify proteins that interact with MLK2, the N-terminal 100 amino acids, including the SH3 domain, were expressed as a glutathione S-transferase (GST) fusion protein. This fusion protein (MLK2N) was used as an affinity ligand to isolate binding proteins from lysates of 35S-radiolabeled MDA-MB231 breast carcinoma cells. When the radiolabeled binding proteins were subjected to 2-DE, proteins of Mr 55,000, 31,500 and 34,000 bound consistently to the MLK2N domain fusion protein, but not to the GST control. Two of the binding proteins were isolated from whole cell lysates by preparative 2-DE and subjected to in-gel digestion and capillary or microbore reverse-phase high performance liquid chromatography (RP-HPLC). Resultant peptides were analyzed by peptide mass fingerprinting, N-terminal Edman degradation or tandem mass spectrometry. The 55,000 protein was identified as the cytoskeletal protein, beta-tubulin, and this was verified by immunoblotting of proteins in the MLK2N binding fraction with anti-tubulin antibodies. The 31,500 protein has been identified as prohibitin, a protein that has been implicated in both signal transduction and cell cycle arrest.
Purification and characterization of a recombinant murine interleukin‐6
Publisher: Wiley
Date: 08-1992
DOI: 10.1111/J.1432-1033.1992.TB17123.X
Abstract: Murine interleukin-6 (IL-6), when expressed in Escherichia coli using the pUC9 vector, accumulated as insoluble aggregates or 'inclusion bodies'. After selective urea washing of the inclusion bodies, to remove extraneous proteins, murine IL-6 was solubilized with 8 M guanidine hydrochloride and then rapidly purified to homogeneity by gel-permeation chromatography followed by reversed-phase HPLC. It was demonstrated that complete disulfide bond formation in murine IL-6 occurred during the early urea washing/guanidine hydrochloride extraction steps, so no refolding step was required. When fully reduced murine IL-6 was dissolved in 8 M guanidine hydrochloride and allowed to air-oxidize, complete disulfide bond formation, monitored by analytical reversed-phase HPLC, was shown to occur within 13 h at 6 degrees C. About 25 mg pure protein was obtained from 37 g wet cells. This recombinant murine IL-6 had a specific activity in the hybridoma growth factor assay of 2 x 10(8) U/mg, which is equivalent to that of native murine IL-6. During the purification procedure, a number of variant forms of murine IL-6 were isolated and partially characterized. Two of these forms, T1 and T3, were C-terminal deletants of murine IL-6 lacking about 60 and 20 amino acids from the C-terminus, respectively, while the other form, T2, was an N-terminal deletant lacking 37 amino acids from the N-terminus. None of these variant forms of murine IL-6 bound to the murine IL-6 receptor and, consequently, all were inactive in the hybridoma growth factor assay.
Loss of charged versus neutral heme from gaseous holomyoglobin ions
Publisher: Wiley
Date: 09-11-2001
DOI: 10.1002/RCM.512
Abstract: The dissociation of holomyoglobin ions ranging in charge state from +10 to +2 has been studied using collisional activation in a quadrupole ion trap. Collisional activation times and litudes were varied to investigate the effects of these variables on dissociation of the heme group from the holoprotein. The onset of neutral heme loss occurs at a lower activation litude than loss of charged heme. For solutions of ferri-myoglobin, charged heme loss was prominent for +10 to +4 holomyoglobin ions, while neutral heme loss product was found to be dominant for charge states +3 and +2. For any given charge state, activation of holomyoglobin ions from a solution containing primarily ferro-myoglobin yielded significantly more abundant neutral heme loss products than was observed for activation of ions from solutions containing primarily ferri-myoglobin. The relative concentrations of the two oxidation states were shown to be affected by redox chemistry within the nano-electrospray emitter used in this work. Results from a double activation experiment revealed that the precursor ions of a given charge state contained a mixture of two populations, with ferro-myoglobin giving rise to neutral heme loss upon dissociation and ferri-myoglobin yielding charged heme. No evidence for electron transfer upon collisional activation of ferri-myoglobin ions was observed. Furthermore, little or no evidence for electron transfer associated with ion/ion reactions with anions derived from perfluoro-1,3-dimethylcyclohexane was observed. Definitive results could not be drawn for the lowest precursor ion charge states (+3 and +2) due to low dissociation efficiencies.
Complementary precursor ion and neutral loss scan mode tandem mass spectrometry for the analysis of glycerophosphatidylethanolamine lipids from whole rat retina
Publisher: Springer Science and Business Media LLC
Date: 11-03-2009
Establishing Signature Fragments for Identification and Sequencing of Dityrosine Cross-Linked Peptides Using Ultraviolet Photodissociation Mass Spectrometry
Publisher: American Chemical Society (ACS)
Date: 06-09-2019
DOI: 10.1021/ACS.ANALCHEM.9B02986
Abstract: Dityrosine cross-linking of Aβ peptides and α-synuclein is increasingly becoming recognized as a biomarker of neuropathological diseases. However, there remains a need for the development of analytical methods that enable the specific and selective identification of dityrosine cross-linked proteins and peptides in complex biological s les. Here, we report that the gas-phase fragmentation of protonated dityrosine cross-linked peptides under ultraviolet photodissociation (UVPD) tandem mass spectrometry (MS/MS) conditions results in the cleavage across C
Synthesis, structure, and condensed-phase reactivity of [Ag3(μ3-H)(μ3-BH4)LPh3](BF4) (LPh = bis(diphenylphosphin
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8DT02437E
Abstract: Electrospray ionisation mass spectrometry (ESI-MS) was used to monitor the reaction of AgBF 4 , bis(diphenylphosphino)amine (dppa = (Ph 2 P) 2 NH = L Ph ) and NaBH 4 in acetonitrile.
Gas phase ion-molecule reactions in a modified ion trap: H/D exchange of non-covalent complexes and coordinatively unsaturated platinum complexes
Publisher: Wiley
Date: 30-11-1998
DOI: 10.1002/(SICI)1097-0231(19981130)12:22<1701::AID-RCM392>3.0.CO;2-S
Cardiolipin Deficiency in Rhodobacter sphaeroides Alters the Lipid Profile of Membranes and of Crystallized Cytochrome Oxidase, but Structure and Function Are Maintained
Publisher: American Chemical Society (ACS)
Date: 22-04-2011
DOI: 10.1021/BI101702C
ExoCarta 2012: database of exosomal proteins, RNA and lipids
Publisher: Oxford University Press (OUP)
Date: 11-10-2011
DOI: 10.1093/NAR/GKR828
‘Top-down’ characterization of site-directed mutagenesis products of Staphylococcus aureus dihydroneopterin aldolase by multistage tandem mass spectrometry in a linear quadrupole ion trap
Publisher: Royal Society of Chemistry (RSC)
Date: 2006
DOI: 10.1039/B512012H
Abstract: The gas-phase fragmentation reactions of a series of site-directed mutagenesis products of Staphylococcus aureus dihydroneopterin aldolase have been examined by multistage tandem mass spectrometry (MS/MS and MS(3)) in a linear quadrupole ion trap in order to explore the utility of this instrumentation for routine 'top-down' recombinant protein characterization. Following a rapid low resolution survey of the fragmentation behavior of the precursor ions from the wild type (WT) protein, selected charge states were subjected to detailed structural characterization by using high resolution 'zoom' and 'ultrazoom' resonance ejection MS/MS product ion scans. Dissociation of the [M + 18H](18+) charge state yielded a range of product ions from which extensive sequence information could be derived. In contrast, dissociation of the [M + 20H](20+) charge state resulted in a single dominant y(96) product ion formed by fragmentation between adjacent Ile/Gly residues, with only limited sequence coverage. Further extensive sequence information was readily obtained however, by MS(3) dissociation of this initial product. From the combined MS/MS and MS(3) spectra an overall sequence coverage of 66.9%, with fragmentation of 85 of the 127 amide bonds within the WT protein, was obtained. MS/MS and MS(3) of three of the four site-directed mutagenesis products (E29A), (Y61F) and (E81A) were found to yield essentially identical product ion spectra to the WT protein, indicating that these modifications had no significant influence on the fragmentation behavior. The specific site of modification could be unambiguously determined in each case by characterization of product ions resulting from fragmentation of amide bonds on either side of the mutation site. In contrast, MS/MS and MS(3) of the K107A mutant led to significantly different product ion spectra dominated by cleavages occurring N-terminal to proline, which restricted the ability to localize the modification site to within only an 8 amino acid region of the sequence. This work highlights the need for further studies to characterize the charge state, sequence and structural dependence to the low energy collision induced dissociation reactions of multiply protonated intact protein ions.
Dissociation of Multiple Protein Ion Charge States Following a Single Gas-Phase Purification and Concentration Procedure
Publisher: American Chemical Society (ACS)
Date: 16-08-2002
DOI: 10.1021/AC025587+
Abstract: The formation of a range of precursor ion charge states from a single concentrated and purified charge state, followed by activation of each charge state, is introduced as a means to obtain more protein structural information than is available from dissociation of a single charge state alone. This approach is illustrated using off-resonance collisional activation of the [M + 8H]8+ to [M + 6H]6+ precursor ions of the bacteriophage MS2 viral coat protein following concentration and purification of the [M + 8H]8+ charge state. This range of charge states was selected on the basis of an ion trap collisional activation study of the effects of precursor ion charge state on the dissociation of the [M + 12H]12+ to [M + 5H]5+ ions. Gas-phase ion/ion proton-transfer reactions and the ion parking technique were applied to purify and concentrate selected precursor ion charge states as well as to simplify the product ion spectra. The high-charge-state ions fragment preferentially at the N-terminal side of proline residues while the product ion spectra of the lowest charge states investigated are dominated by C-terminal aspartic acid cleavages. Maximum structural information is obtained by fragmentation of the intermediate-charge states.
2015 ASMS Fall Workshop: Lipids and Lipidomics
Publisher: American Chemical Society (ACS)
Date: 14-03-2016
Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis
Publisher: American Chemical Society (ACS)
Date: 22-10-2015
C-terminal Extension of Truncated Recombinant Proteins in Escherichia coli with a 10Sa RNA Decapeptide
Publisher: Elsevier BV
Date: 04-1995
Ionic Reagent for Controlling the Gas-Phase Fragmentation Reactions of Cross-Linked Peptides
Publisher: American Chemical Society (ACS)
Date: 11-2008
DOI: 10.1021/AC801625E
Abstract: Chemical cross-linking combined with proteolytic digestion and mass spectrometry (MS) is a promising approach to provide inter- and intramolecular distance constraints for the structural characterization of protein topologies and functional multiprotein complexes. Despite the relative straightforwardness of these methodologies, the identification and characterization of cross-linked proteins presents a significant analytical challenge, due to the complexity of the resultant peptide mixtures, as well as the array of inter-, intra-, or "dead-end"-cross-linked peptides that may be generated from a single cross-linking experiment. To address these issues, we describe here the synthesis, characterization, and initial evaluation of a novel "fixed charge" sulfonium ion-containing crosslinking reagent, S-methyl 5,5'-thiodipentanoylhydroxysuc-cinimide. The peptide products obtained by reaction with this reagent are all shown to fragment exclusively via facile cleavage of the C-S bond directly adjacent to the fixed charge during CID-MS/MS, resulting in the formation of characteristic product ions that enable the presence and type (i.e., inter, intra, or dead-end) of the cross-linked products to be readily determined, independently of the "proton mobility" of the precursor ion. Subsequent isolation and dissociation of these products by MS3 provides additional structural information required for identification of the peptide sequences involved in the cross-linking reactions, as well as for characterization of the specific site(s) at which cross-linking has occurred. The specificity of these gas-phase fragmentation reactions, as well as the solubility and stability of the cross-linking reagent under aqueous conditions, suggests that this strategy holds great promise for use in future studies aimed at the structural analysis of large proteins or multiprotein assemblies.
Emerging methods in proteomics: top-down protein characterization by multistage tandem mass spectrometry
Publisher: Royal Society of Chemistry (RSC)
Date: 2007
DOI: 10.1039/B618499P
Abstract: "Top-down" mass spectrometry methods have emerged as an attractive alternative to conventional "bottom-up" approaches for the comprehensive characterization of co- and post-translational protein modifications. Here we present a brief overview of current strategies employed for top-down protein characterization and discuss the key technical challenges and solutions associated with their implementation on a range of mass spectrometry instrument platforms. For more specific details regarding the in idual strategies described herein, interested readers are referred to the references cited at the end of this article.
Derivatization of protonated peptides via gas phase ion—molecule reactions with acetone
Publisher: American Chemical Society (ACS)
Date: 03-2000
Do amines react with protonated peptides in the gas phase via transacylation reactions to induce peptide bond cleavage?
Publisher: Wiley
Date: 2000
DOI: 10.1002/1097-0231(20000930)14:18<1707::AID-RCM83>3.0.CO;2-9
Energetics and Dynamics of the Fragmentation Reactions of Protonated Peptides Containing Methionine Sulfoxide or Aspartic Acid via Energy- and Time-Resolved Surface Induced Dissociation
Publisher: American Chemical Society (ACS)
Date: 10-2007
DOI: 10.1021/JP073040Z
Abstract: The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64 Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility in these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (nonmobile proton conditions) to lysine (partially mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFECs) reveal that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1-2 orders of magnitude lower than nonselective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to nonselective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these processes are much slower compared to amide bond cleavage, explaining why these selective bond cleavages are not observed if fragmentation is performed under mobile proton conditions. This study further affirms that fragmentation of peptide ions in the gas phase are predominantly governed by entropic effects.
Comparative bioinformatic analysis suggests that specific dauer-like signalling pathway components regulate Toxocara canis development and migration in the mammalian host
Publisher: Springer Science and Business Media LLC
Date: 14-01-2019
The Unconventional Role of Acid Sphingomyelinase in Regulation of Retinal Microangiopathy in Diabetic Human and Animal Models
Publisher: American Diabetes Association
Date: 20-08-2011
DOI: 10.2337/DB10-0550
Abstract: Acid sphingomyelinase (ASM) is an important early responder in inflammatory cytokine signaling. The role of ASM in retinal vascular inflammation and vessel loss associated with diabetic retinopathy is not known and represents the goal of this study. Protein and gene expression profiles were determined by quantitative RT-PCR and Western blot. ASM activity was determined using Amplex Red sphingomyelinase assay. Caveolar lipid composition was analyzed by nano-electrospray ionization tandem mass spectrometry. Streptozotocin-induced diabetes and retinal ischemia-reperfusion models were used in in vivo studies. We identify endothelial caveolae-associated ASM as an essential component in mediating inflammation and vascular pathology in in vivo and in vitro models of diabetic retinopathy. Human retinal endothelial cells (HREC), in contrast with glial and epithelial cells, express the plasma membrane form of ASM that overlaps with caveolin-1. Treatment of HREC with docosahexaenoic acid (DHA) specifically reduces expression of the caveolae-associated ASM, prevents a tumor necrosis factor-α–induced increase in the ceramide-to-sphingomyelin ratio in the caveolae, and inhibits cytokine-induced inflammatory signaling. ASM is expressed in both vascular and neuroretina however, only vascular ASM is specifically increased in the retinas of animal models at the vasodegenerative phase of diabetic retinopathy. The absence of ASM in ASM−/− mice or inhibition of ASM activity by DHA prevents acellular capillary formation. This is the first study demonstrating activation of ASM in the retinal vasculature of diabetic retinopathy animal models. Inhibition of ASM could be further explored as a potential therapeutic strategy in treating diabetic retinopathy.
Effect of Reduced Retinal VLC-PUFA on Rod and Cone Photoreceptors
Publisher: Association for Research in Vision and Ophthalmology (ARVO)
Date: 19-05-2014
A monophasic extraction strategy for the simultaneous lipidome analysis of polar and nonpolar retina lipids
Publisher: Elsevier BV
Date: 08-2014
DOI: 10.1194/JLR.D050302
Trace residue identification, characterization, and longitudinal monitoring of the novel synthetic opioid β‐U10, from discarded drug paraphernalia
Publisher: Wiley
Date: 23-05-2022
DOI: 10.1002/DTA.3284
Abstract: Empirical data regarding dynamic alterations in illicit drug supply markets in response to the COVID‐19 pandemic, including the potential for introduction of novel drug substances and/or increased poly‐drug combination use at the “street” level, that is, directly proximal to the point of consumption, are currently lacking. Here, a high‐throughput strategy employing ambient ionization‐mass spectrometry is described for the trace residue identification, characterization, and longitudinal monitoring of illicit drug substances found within ,600 discarded drug paraphernalia (DDP) s les collected during a pilot study of an early warning system for illicit drug use in Melbourne, Australia from August 2020 to February 2021, while significant COVID‐19 lockdown conditions were imposed. The utility of this approach is demonstrated for the de novo identification and structural characterization of β‐U10, a previously unreported naphthamide analog within the “U‐series” of synthetic opioid drugs, including differentiation from its α‐U10 isomer without need for s le preparation or chromatographic separation prior to analysis. Notably, β‐U10 was observed with 23 other drug substances, most commonly in temporally distinct clusters with heroin, etizolam, and diphenhydramine, and in a total of 182 different poly‐drug combinations. Longitudinal monitoring of the number and weekly “average signal intensity” (ASI) values of identified substances, developed here as a semi‐quantitative proxy indicator of changes in availability, relative purity and compositions of street level drug s les, revealed that increases in the number of identifications and ASI for β‐U10 and etizolam coincided with a 50% decrease in the number of positive detections and an order of magnitude decrease in the ASI for heroin.
Remodeling of Retinal Fatty Acids in an Animal Model of Diabetes
Publisher: American Diabetes Association
Date: 29-10-2009
DOI: 10.2337/DB09-0728
Abstract: The results of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort study revealed a strong association between dyslipidemia and the development of diabetic retinopathy. However, there are no experimental data on retinal fatty acid metabolism in diabetes. This study determined retinal-specific fatty acid metabolism in control and diabetic animals. Tissue gene and protein expression profiles were determined by quantitative RT-PCR and Western blot in control and streptozotocin-induced diabetic rats at 3–6 weeks of diabetes. Fatty acid profiles were assessed by reverse-phase high-performance liquid chromatography, and phospholipid analysis was performed by nano-electrospray ionization tandem mass spectrometry. We found a dramatic difference between retinal and liver elongase and desaturase profiles with high elongase and low desaturase gene expression in the retina compared with liver. Elovl4, an elongase expressed in the retina but not in the liver, showed the greatest expression level among retinal elongases, followed by Elovl2, Elovl1, and Elovl6. Importantly, early-stage diabetes induced a marked decrease in retinal expression levels of Elovl4, Elovl2, and Elovl6. Diabetes-induced downregulation of retinal elongases translated into a significant decrease in total retinal docosahexaenoic acid, as well as decreased incorporation of very-long-chain polyunsaturated fatty acids (PUFAs), particularly 32:6n3, into retinal phosphatidylcholine. This decrease in n3 PUFAs was coupled with inflammatory status in diabetic retina, reflected by an increase in gene expression of proinflammatory markers interleukin-6, vascular endothelial growth factor, and intercellular adhesion molecule-1. This is the first comprehensive study demonstrating diabetes-induced changes in retinal fatty acid metabolism. Normalization of retinal fatty acid levels by dietary means or/and modulating expression of elongases could represent a potential therapeutic target for diabetes-induced retinal inflammation.
Global Analysis of Retina Lipids by Complementary Precursor Ion and Neutral Loss Mode Tandem Mass Spectrometry
Publisher: Humana Press
Date: 2009
Rapid separation of proteins and peptides using conventional silica-based supports: Identification of 2-D gel proteins following in-gel proteolysis
Publisher: Elsevier
Date: 1995
Protein painting reveals pervasive remodeling of conserved proteostasis machinery in response to pharmacological stimuli
Publisher: Cold Spring Harbor Laboratory
Date: 15-05-2022
DOI: 10.1101/2022.05.14.491969
Abstract: Accurate spatio-temporal organization of the proteome is essential for cellular homeostasis. However, a detailed mechanistic understanding of this organization and how it is altered in response to external stimuli in the intact cellular environment is as-yet unrealized. To address this need, ‘protein painting’ methods have emerged as a way to gain insight into the conformational status of proteins within cells at the proteome-wide scale. For ex le, tetraphenylethene maleimide (TPE-MI) has previously been used to quantify the engagement of quality control machinery with client proteins in cell lysates. Here, we showcase the ability of TPE-MI to additionally reveal proteome network remodeling in whole cells in response to a cohort of commonly used pharmacological stimuli of varying specificity. We report specific, albeit heterogeneous, responses to in idual stimuli that coalesce on a conserved set of core cellular machineries. This work expands our understanding of proteome conformational remodeling in response to cellular stimuli, and provides a blueprint for assessing how these conformational changes may contribute to disorders characterized by proteostasis imbalance.
Comprehensive Lipidome Profiling of Isogenic Primary and Metastatic Colon Adenocarcinoma Cell Lines
Publisher: American Chemical Society (ACS)
Date: 10-10-2012
DOI: 10.1021/AC302154G
Quantification of the Compositional Information Provided by Immonium Ions on a Quadrupole-Time-of-Flight Mass Spectrometer
Publisher: American Chemical Society (ACS)
Date: 20-06-2008
DOI: 10.1021/AC8006076
Phosphopeptide Enrichment Using MALDI Plates Modified with High-Capacity Polymer Brushes
Publisher: American Chemical Society (ACS)
Date: 26-06-2008
DOI: 10.1021/AC702472J
Abstract: Matrix-assisted laser desorption/ionization plates coated with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes that are derivatized with Fe(III)-nitrilotriacetate (NTA) complexes allow selective, efficient phosphopeptide enrichment prior to analysis by mass spectrometry (MS). Fe(III)-NTA-PHEMA brushes (60 nm thick) have a phosphopeptide binding capacity of 0.6 microg/cm(2) and exhibit phosphopeptide recoveries of over 70%, whereas much thinner polymer films containing Fe(III)-NTA afford a recovery of only 20%, and a monolayer of Fe(III)-NTA shows a recovery of just 10%. Recoveries are determined by comparing signals from enriched unlabeled phosphopeptides with those of their deuterium-labeled analogues that were added to the plate just prior to addition of matrix. Mass spectra of phosphopeptide-containing s les enriched using Fe(III)-NTA-PHEMA-modified plates also demonstrate higher recoveries or fewer interfering peaks than corresponding spectra obtained with enrichment using several commercially available Fe(III)-containing films and resins or metal oxide materials. When analyzing tryptic digests of beta-casein, the Fe(III)-NTA-PHEMA brushes allow detection of as little as 15 fmol of phosphopeptide. Moreover, with both ovalbumin and beta-casein digests, phosphopeptide signals dominate the mass spectra obtained using these modified plates.
Impact of alogliptin and pioglitazone on lipid metabolism in islets of prediabetic and diabetic Zucker Diabetic Fatty rats
Publisher: Elsevier BV
Date: 05-2015
DOI: 10.1016/J.BCP.2015.03.010
Abstract: Prolonged exposure of pancreatic beta (β) cells to elevated glucose and free fatty acids (FFA) as occurs in type 2 diabetes results in loss of β cell function and survival. In Zucker Diabetic Fatty (ZDF) rats, β cell failure is associated with increased triacylglyceride (TAG) synthesis and disruption of the glycerolipid/FFA (GL/FFA) cycle, a critical arm of glucose-stimulated insulin secretion (GSIS). The aim of this study was to determine the impact of activation of PPARγ and increased incretin action via dipeptidyl-peptidase inhibition using pioglitazone and/or alogliptin, respectively, on islet lipid metabolism in prediabetic and diabetic ZDF rats. Transition of control prediabetic ZDF rats to diabetes was associated with reduced plasma insulin levels, reduced islet insulin content and GSIS, reduced stearoyl-CoA desaturase 2 (SCD 2) expression, and increased islet TAG, diacylglyceride (DAG) and ceramides species containing saturated FA. Treatment of prediabetic ZDF rats with a combination of pioglitazone and alogliptin, but not in idually, prevented the transition to diabetes and was associated with marked lowering of islet TAG and DAG levels. Pioglitazone and alogliptin, however, did not restore SCD2 expression, the degree of FA saturation in TAG, DAG or ceramides, islet insulin content, or lower ceramide levels. These findings are consistent with activation of PPARγ and increased incretin action working in concert to restore GL/FFA cycle in β cells of ZDF rats. Restoration of the GL/FFA cycle without correcting islet FA desaturation, production of islet ceramides, and/or insulin sensitivity, however, may place these islets at risk for β cell failure.
2017 ASMS ‘Emerging Investigators’ Focus
Publisher: American Chemical Society (ACS)
Date: 28-02-2017
Internal amino acid sequecing of proteins by in situ cyanogen bromide cleavage in polyacrylamide gels
Publisher: Elsevier BV
Date: 1990
DOI: 10.1016/0006-291X(90)91922-F
Abstract: A new method was developed for generating peptide fragments for amino acid sequence analysis from polyacrylamide-gel separated proteins. This method involves in situ CNBr treatment of proteins in the polyacrylamide gel after their separation by electrophoresis. Pure CNBr peptides were recovered either by solvent extraction followed by microbore column reversed-phase HPLC or, alternatively, by a second electrophoretic separation step (SDS-PAGE) followed by electrotransfer of the peptides onto polyvinylidene difluoride (PVDF) membranes. These approaches yielded sequence data at subnanomole levels for a wide range of CNBr fragments recovered from gel-separated proteins.
Fabrication of Stable Packed Capillary Reversed-Phase Columns for Protein Structural Analysis
Publisher: Springer Science and Business Media LLC
Date: 07-1997
Abstract: Capillary column ( 3400 cm/hr i.e., approximately 40 microliters/min for 200-micron ID columns) and the loading of large s le volumes (up to 500 microliters). The accurate low flow rates (0.4-4.0 microliters/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr. 599, 119-130]. Column performance was evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots.
Related Organisations
Related Funding Activities
Discovery Projects - Grant ID: DP0342771
Start Date: 2003
End Date: 06-2004
Amount: $193,035.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP170103093
Start Date: 2017
End Date: 12-2020
Amount: $466,500.00
Funder: Australian Research Council
View Funded ActivityARC Centres Of Excellence - Grant ID: CE230100001
Start Date: 2023
End Date: 12-2029
Amount: $35,000,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP190102464
Start Date: 02-2019
End Date: 12-2022
Amount: $410,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP230101050
Start Date: 2023
End Date: 12-2025
Amount: $543,594.00
Funder: Australian Research Council
View Funded ActivityIndustrial Transformation Training Centres - Grant ID: IC170100020
Start Date: 07-2018
End Date: 12-2024
Amount: $3,279,502.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP130100535
Start Date: 2013
End Date: 12-2015
Amount: $260,000.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE200100117
Start Date: 2020
End Date: 12-2020
Amount: $1,275,000.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE160100015
Start Date: 2016
End Date: 12-2016
Amount: $500,000.00
Funder: Australian Research Council
View Funded Activity