ORCID Profile
0000-0001-5988-2632
Current Organisations
Københavns Universitet
,
IT University of Copenhagen
,
Polytechnique Montréal
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Publications
Identification of Novel Protein Functions and Signaling Mechanisms by Genetics and Quantitative Phosphoproteomics in Caenorhabditis elegans
Publisher: Springer New York
Date: 2014
DOI: 10.1007/978-1-4939-1142-4_9
Abstract: Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology for measuring relative changes in protein and phosphorylation levels at a global level. We have applied this method to the model organism Caenorhabditis elegans in combination with RNAi-mediated gene knockdown by feeding the nematode on pre-labeled lysine auxotroph Escherichia coli. In this chapter, we describe in details the generation of the E. coli strain, incorporation of heavy isotope-labeled lysine in C. elegans, and the procedure for a comprehensive global phosphoproteomic experiment.
Quantitative proteomics by amino acid labeling in C. elegans
Publisher: Springer Science and Business Media LLC
Date: 28-08-2002
DOI: 10.1038/NMETH.1675
Abstract: We demonstrate labeling of Caenorhabditis elegans with heavy isotope-labeled lysine by feeding them with heavy isotope-labeled Escherichia coli. Using heavy isotope-labeled worms and quantitative proteomics methods, we identified several proteins that are regulated in response to loss or RNAi-mediated knockdown of the nuclear hormone receptor 49 in C. elegans. The combined use of quantitative proteomics and selective gene knockdown is a powerful tool for C. elegans biology.
TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation
Publisher: eLife Sciences Publications, Ltd
Date: 08-02-2021
DOI: 10.7554/ELIFE.62873
Abstract: Protein interaction is critical molecular regulatory activity underlining cellular functions and precise cell fate choices. Using TWIST1 BioID-proximity-labeling and network propagation analyses, we discovered and characterized a TWIST-chromatin regulatory module (TWIST1-CRM) in the neural crest cells (NCC). Combinatorial perturbation of core members of TWIST1-CRM: TWIST1, CHD7, CHD8, and WHSC1 in cell models and mouse embryos revealed that loss of the function of the regulatory module resulted in abnormal differentiation of NCCs and compromised craniofacial tissue patterning. Following NCC delamination, low level of TWIST1-CRM activity is instrumental to stabilize the early NCC signatures and migratory potential by repressing the neural stem cell programs. High level of TWIST1 module activity at later phases commits the cells to the ectomesenchyme. Our study further revealed the functional interdependency of TWIST1 and potential neurocristopathy factors in NCC development.
Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture
Publisher: American Chemical Society (ACS)
Date: 25-06-2013
DOI: 10.1021/PR400099J
Abstract: Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151.
Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment
Publisher: Elsevier BV
Date: 08-2011
Affinity proteomics for interactome and phosphoproteome screening in synaptosomes
Publisher: Springer New York
Date: 2018
Centering inclusivity in the design of online conferences—An OHBM–Open Science perspective
Publisher: Oxford University Press (OUP)
Date: 08-2021
DOI: 10.1093/GIGASCIENCE/GIAB051
Abstract: As the global health crisis unfolded, many academic conferences moved online in 2020. This move has been hailed as a positive step towards inclusivity in its attenuation of economic, physical, and legal barriers and effectively enabled many in iduals from groups that have traditionally been underrepresented to join and participate. A number of studies have outlined how moving online made it possible to gather a more global community and has increased opportunities for in iduals with various constraints, e.g., caregiving responsibilities. Yet, the mere existence of online conferences is no guarantee that everyone can attend and participate meaningfully. In fact, many elements of an online conference are still significant barriers to truly erse participation: the tools used can be inaccessible for some in iduals the scheduling choices can favour some geographical locations the set-up of the conference can provide more visibility to well-established researchers and reduce opportunities for early-career researchers. While acknowledging the benefits of an online setting, especially for in iduals who have traditionally been underrepresented or excluded, we recognize that fostering social justice requires inclusivity to actively be centered in every aspect of online conference design. Here, we draw from the literature and from our own experiences to identify practices that purposefully encourage a erse community to attend, participate in, and lead online conferences. Reflecting on how to design more inclusive online events is especially important as multiple scientific organizations have announced that they will continue offering an online version of their event when in-person conferences can resume.
An analysis-ready and quality controlled resource for pediatric brain white-matter research
Publisher: Springer Science and Business Media LLC
Date: 12-10-2022
DOI: 10.1038/S41597-022-01695-7
Abstract: We created a set of resources to enable research based on openly-available diffusion MRI (dMRI) data from the Healthy Brain Network (HBN) study. First, we curated the HBN dMRI data (N = 2747) into the Brain Imaging Data Structure and preprocessed it according to best-practices, including denoising and correcting for motion effects, susceptibility-related distortions, and eddy currents. Preprocessed, analysis-ready data was made openly available. Data quality plays a key role in the analysis of dMRI. To optimize QC and scale it to this large dataset, we trained a neural network through the combination of a small data subset scored by experts and a larger set scored by community scientists. The network performs QC highly concordant with that of experts on a held out set (ROC-AUC = 0.947). A further analysis of the neural network demonstrates that it relies on image features with relevance to QC. Altogether, this work both delivers resources to advance transdiagnostic research in brain connectivity and pediatric mental health, and establishes a novel paradigm for automated QC of large datasets.
Reactive oxygen species (ROS)-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large-scale phosphoproteomics screen
Publisher: Elsevier BV
Date: 03-2016
Publisher Correction: An analysis-ready and quality controlled resource for pediatric brain white-matter research
Publisher: Springer Science and Business Media LLC
Date: 31-10-2022
Reactivity and mechanism of the reactions of 4-methylbenzoquinone with amino acid residues in β-lactoglobulin
Publisher: Elsevier BV
Date: 02-2024
A Presynaptic Phosphosignaling Hub for Lasting Homeostatic Plasticity
Publisher: Elsevier BV
Date: 2020
DOI: 10.2139/SSRN.3728948
Oxidation of Whey Proteins during Thermal Treatment Characterized by a Site-Specific LC–MS/MS-Based Proteomic Approach
Publisher: American Chemical Society (ACS)
Date: 05-04-2022
Abstract: Thermal treatment is often employed in food processing to tailor product properties by manipulating the ingredient functionality, but these elevated temperatures may accelerate oxidation and nutrient loss. Here, oxidation of different whey protein systems [α-lactalbumin (α-LA), β-lactoglobulin (β-LG), a mix of α-LA and β-LG (whey model), and a commercial whey protein isolate (WPI)] was investigated during heat treatment at 60-90 °C and a UHT-like treatment by LC-MS-based proteomic analysis. The relative modification levels of each oxidation site were calculated and compared among different heat treatments and s le systems. Oxidation increased significantly in protein systems after heating at ≥90 °C but decreased in systems with higher complexity [pure protein (α-LA > β-LG) > whey model > WPI]. In α-LA, Cys, Met, and Trp residues were found to be most prone to oxidation. In β-LG-containing protein systems, Cys residues were suggested to scavenge most of the reactive oxidants and undergo an oxidation-mediated disulfide rearrangement. The rearranged disulfide bonds contributed to protein aggregation, which was suggested to provide physical protection against oxidation. Overall, limited loss of amino acid residues was detected after acidic hydrolysis followed by UHPLC analysis, which showed only a minor effect of heat treatment on protein oxidation in these protein systems.
Multidimensional Strategy for Sensitive Phosphoproteomics Incorporating Protein Prefractionation Combined with SIMAC, HILIC, and TiO2 Chromatography Applied to Proximal EGF Signaling
Publisher: American Chemical Society (ACS)
Date: 15-10-2011
DOI: 10.1021/PR200641X
Abstract: Comprehensive enrichment and fractionation is essential to obtain a broad coverage of the phosphoproteome. This inevitably leads to s le loss, and thus, phosphoproteomics studies are usually only performed on highly abundant s les. Here, we present a comprehensive phosphoproteomics strategy applied to 400 μg of protein from EGF-stimulated HeLa cells. The proteins are separated into membrane and cytoplasmic fractions using sodium carbonate combined with ultracentrifugation. The phosphopeptides were separated into monophosphorylated and multiphosphorylated pools using sequential elution from IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and nonphosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4700 unique phosphopeptides, while 636 phosphosites were changing following short-term EGF stimulation, many of which were not previously known to be involved in EGFR signaling. We further compared three different data processing programs and found large differences in their peptide identification rates due to different implementations of recalibration and filtering. Manually validating a subset of low-scoring peptides exclusively identified using the MaxQuant software revealed a large percentage of false positive identifications. This indicates that, despite having highly accurate precursor mass determination, peptides with low fragment ion scores should not automatically be reported in phosphoproteomics studies.
Detection of protein oxidation products by fluorescence spectroscopy and trilinear data decomposition
Publisher: Elsevier BV
Date: 12-2011
DOI: 10.1016/J.FOODCHEM.2022.133732
Abstract: Current analytical methods studying protein oxidation modifications require laborious s le preparation and chromatographic methods. Fluorescence spectroscopy is an alternative, as many protein oxidation products are fluorescent. However, the complexity of the signal causes misinterpretation and quantification errors if single emission spectra are used. Here, we analyzed the entire fluorescence excitation-emission matrix using the trilinear decomposition method parallel factor analysis (PARAFAC). Two s le sets were used: a calibration set based on known mixtures of tryptophan, tyrosine, and four oxidation products, and a second s le set of oxidized protein solutions containing UV-illuminated β-lactoglobulin. The PARAFAC model succeeded in resolving the signals of the model systems into the pure fluorophore components and estimating their concentrations. The estimated concentrations for the illuminated β-lactoglobulin s les were validated by liquid chromatography-mass spectrometry. Our approach is a promising tool for reliable identification and quantification of fluorescent protein oxidation products, even in a complex protein system.
Covalent bonding of 4-methylcatechol to β-lactoglobulin results in the release of cysteine-4-methylcatechol adducts after in vitro digestion
Publisher: Elsevier BV
Date: 12-2022
DOI: 10.1016/J.FOODCHEM.2022.133775
Abstract: Protein-polyphenol adducts are formed upon covalent bonding between oxidized polyphenols and proteins. 4-Methylcatechol (4MC) is a polyphenol with origin in coffee and is oxidized to 4-methylbenzoquinone (4MBQ) under conditions used during food processing. The present study characterizes 4MBQ-induced covalent modifications on β-lactoglobulin (β-LG) from bovine milk, (henceforth β-LQ) and the effect on protein digestibility. Significant thiol and amine loss was found in β-LQ compared to β-LG. Site-specific 4MBQ-induced modifications were identified on Cys, Lys, Arg, His and Trp in β-LQ. No significant differences between β-LG and β-LQ on in vitro digestibility were observed by assessment with SDS-PAGE, degree of hydrolysis and LC-MS/MS unmodified peptide intensities. Cys-4MC adduct (1.7 ± 0.1 µmol/g) was released from β-LQ after in vitro digestion. Thus, it is relevant to investigate how released Cys-4MC adducts are absorbed in vivo in future studies.
TiSH — a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.JPROT.2012.08.007
Abstract: Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO(2) phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated "TiSH"). The advantages of the strategy include a high specificity and s le preparation workload reduction due to the TiO(2) pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~6600 unique phosphopeptides from 300 μg of peptides/condition (22 unique phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity>94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of s le with high sensitivity and specificity.
Chemical Deamidation: A Common Pitfall in Large-Scale N-Linked Glycoproteomic Mass Spectrometry-Based Analyses
Publisher: American Chemical Society (ACS)
Date: 22-02-2012
DOI: 10.1021/PR2011268
Abstract: N-Linked glycoproteins are involved in several diseases and are important as potential diagnostic molecules for biomarker discovery. Therefore, it is important to provide sensitive and reliable analytical methods to identify not only the glycoproteins but also the sites of glycosylation. Recently, numerous strategies to identify N-linked glycosylation sites have been described. These strategies have been applied to cell lines and several tissues with the aim of identifying many hundreds (or thousands) of glycosylation events. With high-throughput strategies however, there is always the potential for false positives. The confusion arises since the protein N-glycosidase F (PNGase F) reaction used to separate N-glycans from formerly glycosylated peptides catalyzes the cleavage and deamidates the asparagine residue. This is typically viewed as beneficial since it acts to highlight the modification site. We have evaluated this common large-scale N-linked glycoproteomic strategy and proved potential pitfalls using Escherichia coli as a model organism, since it lacks the N-glycosylation machinery found in mammalian systems and some pathogenic microbes. After isolation and proteolytic digestion of E. coli membrane proteins, we investigated the presence of deamidated asparagines. The results show the presence of deamidated asparagines especially with close proximity to a glycine residue or other small amino acid, as previously described for spontaneous in vivo deamidation. Moreover, we have identified deamidated peptides with incorporation of (18)O, showing the pitfalls of glycosylation site assignment based on deamidation of asparagine induced by PNGase F in (18)O-water in large-scale analyses. These data experimentally prove the need for more caution in assigning glycosylation sites and "new" N-linked consensus sites based on common N-linked glycoproteomics strategies without proper control experiments. Besides showing the spontaneous deamidation, we provide alternative methods for validation that should be used in such experiments.
Centering inclusivity in the design of online conferences - An OHBM - Open Science perspective
Publisher: Center for Open Science
Date: 23-03-2021
Abstract: As the global health crisis unfolded throughout the world, many academic conferences moved online in 2020. This move has been hailed as a positive step towards inclusivity in its attenuation of economic, physical and legal barriers and effectively enabled many in iduals who have traditionally been underrepresented to join and participate. A number of studies have outlined how moving online made it possible to gather a more global community and has increased opportunities for in iduals with various constraints, e.g. caregiving responsibilities. Yet, the mere existence of online conferences is unfortunately no guarantee that everyone can attend and participate meaningfully. In fact, many elements of an online conference are still significant barriers to truly erse participation: the tools used can be inaccessible for some in iduals the scheduling choices can favour some geographical locations the setup of the conference can provide more visibility to well-established researchers and reduce opportunities for early career researchers. While acknowledging the benefits of an online setting, especially for in iduals who have traditionally been underrepresented or excluded, we recognize that fostering social justice requires inclusivity to actively be centered in every aspect of online conference design.Here, we draw from the literature and from our own experiences to identify practices that purposefully encourage a erse community to: attend, participate in, and lead online conferences. Reflecting on how to design more inclusive online events is especially important as multiple scientific organizations have announced that they will continue offering an online version of their event when in-person conferences can resume.
Comprehensive Quantitative Comparison of the Membrane Proteome, Phosphoproteome, and Sialiome of Human Embryonic and Neural Stem Cells
Publisher: Elsevier BV
Date: 2014
Erratum: Structural basis for phosphorylation and lysine acetylation cross-talk in a kinase motif associated with myocardial ischemia and cardioprotection (The Journal of Biological Chemistry (2014) 289 (25890-25906))
Publisher: Elsevier BV
Date: 12-2014
A Systems-level Characterization of the Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells.
Publisher: Elsevier BV
Date: 10-2019
Author Correction: An analysis-ready and quality controlled resource for pediatric brain white-matter research
Publisher: Springer Science and Business Media LLC
Date: 17-11-2022
Site-Specific Characterization of Heat-Induced Disulfide Rearrangement in Beta-Lactoglobulin by Liquid Chromatography–Mass Spectrometry
Publisher: American Chemical Society (ACS)
Date: 13-01-2022
Abstract: Disulfides are important for maintaining the protein native structure, but they may undergo rearrangement in the presence of free Cys residues, especially under elevated temperatures. Disulfide rearrangement may result in protein aggregation, which is associated with
Improving the phosphoproteome coverage for limited sample amounts using TiO2 -SIMAC-HILIC (TiSH) phosphopeptide enrichment and fractionation
Publisher: Springer New York
Date: 2016
DOI: 10.1007/978-1-4939-3049-4_11
Abstract: Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological s les, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the s le loss resulting from fractionation, this procedure is mainly performed when large quantities of s le are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.
TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation
Publisher: Cold Spring Harbor Laboratory
Date: 07-09-2020
DOI: 10.1101/2020.09.06.285387
Abstract: Protein interaction is critical molecular regulatory activity underlining cellular functions and precise cell fate choices. Using TWIST1 BioID-proximity-labelling and network propagation analyses, we discovered and characterized a TWIST-chromatin regulatory module (TWIST1-CRM) in the neural crest cell (NCC). Combinatorial perturbation of core members of TWIST1-CRM: TWIST1, CHD7, CHD8, and WHSC1 in cell models and mouse embryos revealed that loss of the function of the regulatory module resulted in abnormal specification of NCCs and compromised craniofacial tissue patterning. Our results showed that in the course of cranial neural crest differentiation, phasic activity of TWIST1 and the interacting chromatin regulators promote the choice of NCC fate while suppressing neural stem cell fates, and subsequently enhance ectomesenchyme potential and cell motility. We have revealed the connections between TWIST1 and potential neurocristopathy factors which are functionally interdependent in NCC specification. Moreover, the NCC module participate in the genetic circuit delineating dorsal-ventral patterning of neural progenitors in the neuroepithelium.
Technologies and challenges in large‐scale phosphoproteomics
Publisher: Wiley
Date: 03-2013
Abstract: Phosphorylation, the reversible addition of a phosphate group to amino acid side chains of proteins, is a fundamental regulator of protein activity, stability, and molecular interactions. Most cellular processes, such as inter- and intracellular signaling, protein synthesis, degradation, and apoptosis, rely on phosphorylation. This PTM is thus involved in many diseases, rendering localization and assessment of extent of phosphorylation of major scientific interest. MS-based phosphoproteomics, which aims at describing all phosphorylation sites in a specific type of cell, tissue, or organism, has become the main technique for discovery and characterization of phosphoproteins in a nonhypothesis driven fashion. In this review, we describe methods for state-of-the-art MS-based analysis of protein phosphorylation as well as the strategies employed in large-scale phosphoproteomic experiments with focus on the various challenges and limitations this field currently faces.
Selective enrichment of sialic acid–containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry
Publisher: Springer Science and Business Media LLC
Date: 18-11-2010
Abstract: The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in s le preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and the characterization of glycan structures. In this paper, we describe a protocol for the selective enrichment of SA-containing glycopeptides using a combination of titanium dioxide (TiO(2)) and hydrophilic interaction liquid chromatography (HILIC). The selectivity of TiO(2) toward SA-containing glycopeptides is achieved by using a low-pH buffer that contains a substituted acid such as glycolic acid to improve the binding efficiency and selectivity of SA-containing glycopeptides to the TiO(2) resin. By combining TiO(2) enrichment of sialylated glycopeptides with HILIC separation of deglycosylated peptides, a more comprehensive analysis of formerly sialylated glycopeptides by MS can be achieved. Here we illustrate the efficiency of the method by the identification of 1,632 unique formerly sialylated glycopeptides from 817 sialylated glycoproteins. The TiO(2)/HILIC protocol requires 2 d and the entire procedure from protein isolation can be performed in <5 d, depending on the time taken to analyze data.
UHT treatment and storage of liquid infant formula affects protein digestion and release of bioactive peptides
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D1FO02619D
Abstract: UHT treatment and storage affect IF protein digestibility and the release of bioactive peptides due to structural changes and crosslinkings.
SNAP-25 phosphorylation at Ser187 is not involved in Ca2+ or phorbolester-dependent potentiation of synaptic release
Publisher: Elsevier BV
Date: 2020
DOI: 10.1016/J.MCN.2019.103452
Abstract: SNAP-25, one of the three SNARE-proteins responsible for synaptic release, can be phosphorylated by Protein Kinase C on Ser-187, close to the fusion pore. In neuroendocrine cells, this phosphorylation event potentiates vesicle recruitment into releasable pools, whereas the consequences of phosphorylation for synaptic release remain unclear. We mutated Ser-187 and expressed two mutants (S187C and S187E) in the context of the SNAP-25B-isoform in SNAP-25 knockout glutamatergic autaptic neurons. Whole-cell patch cl recordings were performed to assess the effect of Ser-187 phosphorylation on synaptic transmission. Blocking phosphorylation by expressing the S187C mutant did not affect synapse density, basic evoked or spontaneous neurotransmission, the readily-releasable pool size or its Ca
Generation of Aggregates of α-Lactalbumin by UV-B Light Exposure
Publisher: American Chemical Society (ACS)
Date: 12-05-2020
Structural Basis for Phosphorylation and Lysine Acetylation Cross-talk in a Kinase Motif Associated with Myocardial Ischemia and Cardioprotection
Publisher: Elsevier BV
Date: 09-2014
Titanium dioxide as chemo-affinity chromatographic sorbent of biomolecular compounds — Applications in acidic modification-specific proteomics
Publisher: Elsevier BV
Date: 12-2011
DOI: 10.1016/J.JPROT.2011.07.024
Abstract: Characterization of the biomolecules involved in molecular processes occurring in biological systems such as the human cell remains central to biology, biotechnology, and medicine. One of the preferred methods of selectively purifying specific classes of biomolecules from complex biological matrices for further characterization is affinity chromatography, which relies on the specific interaction between an analyte in solution and a solid adsorbent. Titanium dioxide-based affinity chromatography has proven to be a versatile tool in enrichment of various compounds such as phosphorylated biomolecules due to its unique ion and ligand exchange properties and high stability towards pH and temperature. Recently, titanium dioxide chromatography was introduced in proteomics as a highly specific method for enriching phosphorylated peptides - a method, which has been widely adapted by the field of phosphoproteomics. Additional studies have shown the potential of this sorbent in purification of other acidic post-translational modified peptides, such as sialylated glycopeptides, thereby targeting the sialiome, defined as the content of sialic acid containing glycoproteins of a given cell, body fluid or tissue. The development of TiO(2)-based chromatographic strategies for separation of various biomolecules from its introduction for small molecules more than 20 years ago until recent proteomics applications today will be reviewed here.
The temporal profile of activity-dependent presynaptic phospho-signalling reveals long-lasting patterns of poststimulus regulation
Publisher: Public Library of Science (PLoS)
Date: 03-2019
A proteome‐scale study on in vivo protein Nα‐acetylation using an optimized method
Publisher: Wiley
Date: 06-12-2010
Abstract: Protein N-terminal acetylation (N(α) -acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α) -modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of s le to resin had to be determined prior to the analysis since Lys-containing N(α) -modified peptides may be lost. To address this problem, we hereby present an optimized method by the introduction of double incubation at pH 6.0. We demonstrate with the optimized method that the N(α) -modified peptides can be enriched regardless of whether ε-NH(2) is present or not, and the s le to resin ratio optimization is no longer necessary. Another improvement was accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to alleviate the shortcoming of the reduced charge state of N(α) -modified peptides. We employed a duplicate experiment using 80 μg s les each and identified 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini data set acquired from a tryptic digest. Furthermore, the reproducibility of the N(α) -acetyl proteome approach was evaluated and its complementarity to the regular proteome approach was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing peptides via the imidazole group was discovered.
Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications
Publisher: Elsevier BV
Date: 03-2015
A Novel Method for the Simultaneous Enrichment, Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development
Publisher: Elsevier BV
Date: 11-2012
Cysteine residues are responsible for the sulfurous off-flavor formed in heated whey protein solutions
Publisher: Elsevier BV
Date: 12-2022
Author Correction: An analysis-ready and quality controlled resource for pediatric brain white-matter research
Publisher: Springer Science and Business Media LLC
Date: 28-04-2023
The interaction of assembly protein AP180 and clathrin is inhibited by multi-site phospho-mimetics
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.NEUINT.2019.104474
Abstract: Clathrin-mediated endocytosis at the nerve terminal is dependent on assembly protein 180 (AP180) and adapter protein complex 2 (AP2). Both membrane adapter proteins bind to each other and to clathrin, to drive assembly of the clathrin coat over nascent synaptic vesicles. Using knowledge of in vivo phosphorylation sites, AP180 was mutated to determine the effect on binding. N-terminally truncated AP180 exhibited phospho-mimetic (Ser/Thr to Glu)-dependent interaction with AP2, but not clathrin. C-terminally truncated and full length phospho-mutant AP180 bound less AP2 than wild type. However, there was no difference in AP2 binding for the phospho-mimetic or phospho-deficient (Ser/Thr to Ala) AP180 mutants. Thus, the phospho-mutant approach did not provide clarity for the role of phosphorylation in AP180-AP2 binding. Clathrin exhibited a phospho-mimetic-dependent interaction with full-length AP180. Furthermore, phospho-mimetic AP180 was deficient at assembling clathrin cages. These latter discoveries support a model where AP180 phosphorylation inhibits clathrin binding and assembly.
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