ORCID Profile
0000-0001-8493-2208
Current Organisation
University of South Australia
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Publisher: SPIE
Date: 24-11-2016
DOI: 10.1117/12.2242707
Publisher: MDPI AG
Date: 06-04-2023
Abstract: Eighty percent of ovarian cancer patients initially respond to chemotherapy, but the majority eventually experience a relapse and die from the disease with acquired chemoresistance. In addition, 20% of patients do not respond to treatment at all, as their disease is intrinsically chemotherapy resistant. Data-independent acquisition nano-flow liquid chromatography–mass spectrometry (DIA LC-MS) identified the three protein markers: gelsolin (GSN), calmodulin (CALM1), and thioredoxin (TXN), to be elevated in high-grade serous ovarian cancer (HGSOC) tissues from patients that responded to chemotherapy compared to those who did not the differential expression of the three protein markers was confirmed by immunohistochemistry. Analysis of the online GENT2 database showed that mRNA levels of GSN, CALM1, and TXN were decreased in HGSOC compared to fallopian tube epithelium. Elevated levels of GSN and TXN mRNA expression correlated with increased overall and progression-free survival, respectively, in a Kaplan–Meier analysis of a large online repository of HGSOC patient data. Importantly, differential expression of the three protein markers was further confirmed when comparing parental OVCAR-5 cells to carboplatin-resistant OVCAR-5 cells using DIA LC-MS analysis. Our findings suggest that GSN, CALM1, and TXN may be useful biomarkers for predicting chemotherapy response and understanding the mechanisms of chemotherapy resistance. Proteomic data are available via ProteomeXchange with identifier PXD033785.
Publisher: Springer US
Date: 2021
Publisher: MDPI AG
Date: 04-01-2021
DOI: 10.3390/DIAGNOSTICS11010069
Abstract: Ovarian cancer (OC) is commonly diagnosed at advanced stage when prognosis is poor. Consequently, there is an urgent clinical need to identify novel biomarkers for early detection to improve survival. We examined the diagnostic value of the calcium phospholipid binding protein annexin A2 (ANXA2), which plays an important role in OC metastasis. Annexin A2 plasma levels in patients with high grade serous OC (n = 105), benign ovarian lesions (n = 55) and healthy controls (n = 143) were measured by ELISA. Annexin A2 levels were found to be significantly increased in patients with stage I (p 0.0001) and stage IA (p = 0.0027) OC when compared to healthy controls. In the logistic regression models followed by receiver operating characteristics (ROC) curve analyses, plasma annexin A2 showed 46.7% sensitivity at 99.6% specificity in distinguishing stage IA OC patients from healthy controls and 75% sensitivity at 65.5% specificity in the diagnosis of stage IA versus benign ovarian tumors. In the diagnosis of stage IA OC versus normal controls, the combination of plasma annexin A2 and CA125 showed 80% sensitivity at 99.6% specificity (AUC = 0.970) which was significantly higher than for CA125 (53.3% sensitivity at 99.6% specificity AUC = 0.891) alone. The diagnostic accuracy in distinguishing stage IA OC from benign ovarian disease when combining annexin A2 and CA125 (71.4% accuracy at 100% sensitivity) was almost twice as high compared to CA125 (37.1% accuracy at 100% sensitivity) alone. In conclusion, annexin A2 in combination with CA125 has potential as a biomarker for the early detection of OC and to predict malignancy in patients with ovarian lesions, warranting further investigations.
Publisher: Elsevier BV
Date: 2008
Publisher: Wiley
Date: 15-10-2019
Abstract: Malignant ascites is a fluid, which builds up in the abdomen and contains cancer cells in the form of single cells or multicellular clusters called spheroids. Malignant ascites has been observed in patients suffering from ovarian, cervical, gastric, colorectal, pancreatic, endometrial, or primary liver cancer. The spheroids are believed to play a major role in chemo resistance and metastasis of the cancer. To ease the discomfort of patients, malignant ascites (MA) is often drained from the abdomen using a procedure called paracentesis. MA retrieved via this minimal invasive procedure is a great source for cancer spheroids, which can be used for testing chemotherapeutic drugs and drug combinations. Herein, the existing workflow is adapted to make concurrent monitoring of drug accumulation, drug response, and drug metabolites feasible using primary spheroids or spheroids grown without a scaffolding matrix. To achieve this, those spheroids are embedded in matrigel, before fixing them with formalin. This makes it possible to process, store, and ship s les at room temperature. This new approach might be used to choose the best targeted therapy for each patient and thereby facilitate personalized medicine.
Publisher: Springer Science and Business Media LLC
Date: 09-02-2016
DOI: 10.1007/S00253-016-7344-8
Abstract: Beer spoilage microorganisms present a major risk for the brewing industry and can lead to cost-intensive recall of contaminated products and damage to brand reputation. The applicability of molecular profiling using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in combination with Biotyper software was investigated for the identification of beer spoilage microorganisms from routine brewery quality control s les. Reference mass spectrum profiles for three of the most common bacterial beer spoilage microorganisms (Lactobacillus lindneri, Lactobacillus brevis and Pediococcus damnosus), four commercially available brewing yeast strains (top- and bottom-fermenting) and Dekkera/Brettanomyces bruxellensis wild yeast were established, incorporated into the Biotyper reference library and validated by successful identification after inoculation into beer. Each bacterial species could be accurately identified and distinguished from one another and from over 5600 other microorganisms present in the Biotyper database. In addition, wild yeast contaminations were rapidly detected and distinguished from top- and bottom-fermenting brewing strains. The applicability and integration of mass spectrometry profiling using the Biotyper platform into existing brewery quality assurance practices within industry were assessed by analysing routine microbiology control s les from a local brewery, where contaminating microorganisms could be reliably identified. Brewery-isolated microorganisms not present in the Biotyper database were further analysed for identification using LC-MS/MS methods. This renders the Biotyper platform a promising candidate for biological quality control testing within the brewing industry as a more rapid, high-throughput and cost-effective technology that can be tailored for the detection of brewery-specific spoilage organisms from the local environment.
Publisher: Elsevier BV
Date: 2009
Publisher: Elsevier BV
Date: 04-2008
Publisher: Wiley
Date: 07-2003
DOI: 10.1359/JBMR.2003.18.7.1326
Abstract: A PTH gene has been isolated from the fish Fugu rubripes. The encoded protein of 80 amino acid has the lowest homology with any of the PTH family members. Fugu PTH(1-34) had 5-fold lower potency than human PTH(1-34) in a mammalian cell system. Parathyroid hormone (PTH) is the major hypercalcemic hormone in higher vertebrates. Fish lack parathyroid glands, but there have numerous attempts to identify and isolate PTH from fish. Polymerase chain reaction (PCR) was performed with primers based on preliminary data from the Joint Genome Institute database. PCR lification was performed on genomic DNA isolated from Fugu rubripes. PCR products were purified and DNA was sequenced. All sequence was confirmed from more than one independently lified PCR product. Multiple sequence alignments were carried out, and the percentage of identities and similarities were calculated. An unrooted phylogenetic tree, using all the known PTH and PTH-related protein (PTHrP) amino acid sequences, was determined. Synthetic peptides were tested in a biological assay that measured cyclic adenosine 3',5'-monophosphate formation in UMR106.1 cells. Rabbit polyclonal antisera specific for N-terminal human PTHrP and one rabbit polyclonal antiserum specific for N terminus hPTH were used to test the cross-reactivity with fPTH(1-34) in immunoblots.
Publisher: Optica Publishing Group
Date: 24-10-2008
DOI: 10.1364/OE.16.018514
Abstract: Glass microstructured optical fibers have been rendered biologically active for the first time via the immobilization of antibodies within the holes in the fiber cross-section. This has been done by introducing coating layers to the internal surfaces of soft glass fibers. The detection of proteins that bind to these antibodies has been demonstrated experimentally within this system via the use of fluorescence labeling. The approach combines the sensitivity resulting from the long interaction lengths of filled fibers with the selectivity provided by the use of antibodies.
Publisher: Springer Science and Business Media LLC
Date: 24-03-2022
DOI: 10.1038/S41419-022-04693-0
Abstract: Mutations in N -glycanase 1 (NGLY1), which deglycosylates misfolded glycoproteins for degradation, can cause NGLY1 deficiency in patients and their abnormal fetal development in multiple organs, including microcephaly and other neurological disorders. Using cerebral organoids (COs) developed from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), we investigate how NGLY1 dysfunction disturbs early brain development. While NGLY1 loss had limited impact on the undifferentiated cells, COs developed from NGLY1-deficient hESCs showed defective formation of SATB2-positive upper-layer neurons, and attenuation of STAT3 and HES1 signaling critical for sustaining radial glia. Bulk and single-cell transcriptomic analysis revealed premature neuronal differentiation accompanied by downregulation of secreted and transcription factors, including TTR, IGFBP2, and ID4 in NGLY1-deficient COs. NGLY1 malfunction also dysregulated ID4 and enhanced neuronal differentiation in CO transplants developed in vivo. NGLY1-deficient CO cells were more vulnerable to multiple stressors treating the deficient cells with recombinant TTR reduced their susceptibility to stress from proteasome inactivation, likely through LRP2-mediated activation of MAPK signaling. Expressing NGLY1 led to IGFBP2 and ID4 upregulation in CO cells developed from NGLY1-deficiency patient’s hiPSCs. In addition, treatment with recombinant IGFBP2 enhanced ID4 expression, STAT3 signaling, and proliferation of NGLY1-deficient CO cells. Overall, our discoveries suggest that dysregulation of stress responses and neural precursor differentiation underlies the brain abnormalities observed in NGLY1-deficient in iduals.
Publisher: Elsevier BV
Date: 03-2024
Publisher: MDPI AG
Date: 29-09-2015
DOI: 10.3390/S151025090
Publisher: Wiley
Date: 17-05-2016
Abstract: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a mass spectrometry technique used for the analysis of macromolecules on an intact tissue of interest, thereby allowing the assessment of molecular signatures in health and disease in the anatomical context. MALDI-MSI is increasingly used to investigate neurodegenerative and psychiatric disorders at the molecular level, including Alzheimer's disease (AD), Parkinson's disease (PD), and schizophrenia (SCZ). These illnesses are characterized by complex neuropathological processes, and conventional proteomic techniques investigating brain tissue homogenates have inherent limitations in determining the precise anatomical or cellular location of proteomic findings. In this article, we review MALDI-MSI studies on neurodegenerative and psychiatric disorders, and explore whether the technique could accelerate the translation of proteomic information into improved understanding and ultimately better therapeutic applications.
Publisher: American Chemical Society (ACS)
Date: 27-07-2010
DOI: 10.1021/PR9011766
Abstract: Imaging mass spectrometry (IMS) is a powerful technology for mapping distributions of biological molecules like proteins and peptides within tissue sections. It is therefore potentially extremely useful for the analysis of pathological conditions such as neoplastic diseases. The use of IMS is typically limited to fresh frozen tissue specimens. However, there is a high interest in the possibility of being able to analyze the tissue proteome of formalin-fixed paraffin-embedded (FFPE) specimens that have been stored together with the clinicopathological information of patients in huge archives over many decades. We have therefore developed an antigen-retrieval protocol using a high temperature citric acid buffer to allow partial reversal of FFPE protein cross-linking. Coupled with automated deposition of trypsin and matrix, our method allows the generation of meaningful peptide ion distribution images. In situ peptide fragmentation provided identification of high abundance proteins such as Actin and Collagen. Furthermore, downstream application of three different HPLC-MS strategies allowed identification of a maximum of 106 proteins, 67 of which were mass correlated to ions from IMS analysis of archived FFPE ovarian tissue. The CAAR method presented here complements previously described antigen-retrieval protocols and is an important step in being able to fully analyze the proteome of archived FFPE tissue.
Publisher: Wiley
Date: 06-2016
Abstract: Magnetic resonance imaging (MRI) is a non-invasive technique routinely used to investigate pathological changes in knee osteoarthritis (OA) patients. MRI uniquely reveals zones of the most severe change in the subchondral bone (SCB) in OA, called bone marrow lesions (BMLs). BMLs have diagnostic and prognostic significance in OA, but MRI does not provide a molecular understanding of BMLs. Multiple N-glycan structures have been observed to play a pivotal role in the OA disease process. We applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of N-glycans to formalin-fixed paraffin-embedded (FFPE) SCB tissue sections from patients with knee OA, and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was conducted on consecutive sections to structurally characterize and correlate with the N-glycans seen by MALDI-MSI. The application of this novel MALDI-MSI protocol has enabled the first steps to spatially investigate the N-glycome in the SCB of knee OA patients.
Publisher: Elsevier BV
Date: 03-2021
Publisher: American Chemical Society (ACS)
Date: 08-03-2016
DOI: 10.1021/ACS.ANALCHEM.6B00365
Abstract: Biosensing within complex biological s les requires a sensor that can compensate for fluctuations in the signal due to changing environmental conditions and nonspecific binding events. To achieve this, we developed a novel self-referenced biosensor consisting of two almost identically sized dye-doped polystyrene microspheres placed on adjacent holes at the tip of a microstructured optical fiber (MOF). Here self-referenced biosensing is demonstrated with the detection of Neutravidin in undiluted, immunoglobulin-deprived human serum s les. The MOF allows remote excitation and collection of the whispering gallery modes (WGMs) of the microspheres while also providing a robust and easy to manipulate dip-sensing platform. By taking advantage of surface functionalization techniques, one microsphere acts as a dynamic reference, compensating for nonspecific binding events and changes in the environment (such as refractive index and temperature), while the other microsphere is functionalized to detect a specific interaction. The almost identical size allows the two spheres to have virtually identical refractive index sensitivity and surface area, while still having discernible WGM spectra. This ensures their responses to nonspecific binding and environmental changes are almost identical, whereby any specific changes, such as binding events, can be monitored via the relative movement between the two sets of WGM peaks.
Publisher: American Society for Microbiology
Date: 02-2015
DOI: 10.1128/IAI.02702-14
Abstract: An undetermined feature of Staphylococcus aureus pathogenesis is its persistence and then relapse of disease. This has been explained by its switch to alternative lifestyles, mainly as biofilm or small-colony variants (SCVs). Studying the native characteristics of SCVs has been problematic due to their reversion to the parental lifestyle. We have observed that for a number of S. aureus strains as they switch to an SCV lifestyle, there is the formation of an extracellular matrix. We focused our analysis on one strain, WCH-SK2. For bacterial survival in the host, the combination of low nutrients and the prolonged time frame forms a stress that selects for a specific cell type from the population. In this context, we used steady-state growth conditions with low nutrients and a controlled low growth rate for a prolonged time and with methylglyoxal. These conditions induced S. aureus WCH-SK2 into a stable SCV cell type the cells did not revert after subculturing. Analysis revealed these cells possessed a metabolic and surface profile that was different from those of previously described SCVs or biofilm cells. The extracellular matrix was protein and extracellular DNA but not polysaccharide. The SCV cells induced expression of certain surface proteins (such as Ebh) and synthesis of lantibiotics while downregulating factors that stimulate the immune response (leucocidin, capsule, and carotenoid). Our data reveal cell heterogeneity within an S. aureus population and under conditions that resemble long-term survival in the host have identified a previously unnoticed S. aureus cell type with a distinctive metabolic and molecular profile.
Publisher: Springer Science and Business Media LLC
Date: 15-06-2004
Publisher: Springer Science and Business Media LLC
Date: 15-10-2021
Publisher: Wiley
Date: 12-05-2009
DOI: 10.1002/RCM.4081
Abstract: [M-H](-) anions from small diphosphopeptides (phosphate groups on Ser, Thr or Tyr) show characteristic peaks corresponding to m/z 177 (H(3)P(2)O(7) (-)), 159 (HP(2)O(6) (-)) and sometimes [(M-H)(-)-H(4)P(2)O(7)](-). M/z 177 and m/z 159 are major peaks in the spectra of small peptides with 1,2, 1,3, 1,4, 1,5 and 1,6 diphosphate substitution, which means that the decomposing [M-H](-) anions must have flexible structures in order for the two phosphate groups to interact with each other. Peptides where the two phosphate groups are more than six amino acid residues apart have not been studied. Theoretical calculations indicate that m/z 177 is formed in a strongly exothermic reaction involving facile nucleophilic interaction between the two phosphate groups: m/z 159 is formed by loss of water from energised m/z 177.
Publisher: Springer Science and Business Media LLC
Date: 07-08-2012
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.BBI.2014.07.001
Abstract: Alterations in the neuro-immune axis contribute toward viscerosensory nerve sensitivity and symptoms in Irritable Bowel Syndrome (IBS). Inhibitory factors secreted from immune cells inhibit colo-rectal afferents in health, and loss of this inhibition may lead to hypersensitivity and symptoms. We aimed to determine the immune cell type(s) responsible for opioid secretion in humans and whether this is altered in patients with IBS. The β-endorphin content of specific immune cell lineages in peripheral blood and colonic mucosal biopsies were compared between healthy subjects (HS) and IBS patients. Peripheral blood mononuclear cell (PBMC) supernatants from HS and IBS patients were applied to colo-rectal sensory afferent endings in mice with post-inflammatory chronic visceral hypersensitivity (CVH). β-Endorphin was identified predominantly in monocyte/macrophages relative to T or B cells in human PBMC and colonic lamina propria. Monocyte derived β-endorphin levels and colonic macrophage numbers were lower in IBS patients than healthy subjects. PBMC supernatants from healthy subjects had greater inhibitory effects on colo-rectal afferent mechanosensitivity than those from IBS patients. The inhibitory effects of PBMC supernatants were more prominent in CVH mice compared to healthy mice due to an increase in μ-opioid receptor expression in dorsal root ganglia neurons in CVH mice. Monocyte/macrophages are the predominant immune cell type responsible for β-endorphin secretion in humans. IBS patients have lower monocyte derived β-endorphin levels than healthy subjects, causing less inhibition of colonic afferent endings. Consequently, altered immune function contributes toward visceral hypersensitivity in IBS.
Publisher: MDPI AG
Date: 21-01-2011
DOI: 10.3390/IJMS12010773
Publisher: MDPI AG
Date: 14-01-2011
DOI: 10.3390/IJMS12010410
Publisher: Wiley
Date: 18-04-2023
DOI: 10.1002/YEA.3851
Abstract: Beer refermentation in bottles is an industrial process utilized by breweries where yeast and fermentable extract are added to green beer. The beer is refermented for a minimum of 2 weeks before distribution, with the physiological state of the yeast a critical factor for successful refermentation. Ideally, fresh yeast that is propagated from a dedicated propagation plant should be used for refermentation in bottles. Here, we explored the applicability of the fluorescent and redox‐sensitive dye, resazurin, to assess cellular metabolism in yeast and its ability to differentiate between growth stages. We applied this assay, with other markers of yeast physiology, to evaluate yeast quality during a full‐scale industrial propagation. Resazurin allowed the discrimination between the different growth phases in yeast and afforded a more in‐depth understanding of yeast metabolism during propagation. This assay can be used to optimize the yeast propagation process and cropping time to improve beer quality.
Publisher: American Chemical Society (ACS)
Date: 16-08-2023
Publisher: Elsevier BV
Date: 10-2012
Publisher: Elsevier BV
Date: 05-2008
Publisher: Elsevier BV
Date: 05-2009
Publisher: Elsevier BV
Date: 12-2018
Publisher: Elsevier BV
Date: 02-2024
Publisher: Elsevier BV
Date: 09-2016
Publisher: Institute of Mathematical Statistics
Date: 2014
DOI: 10.1214/14-EJS900REJ
Publisher: Wiley
Date: 09-02-2018
Abstract: Multicellular tumor spheroids (MCTS) are a powerful biological in vitro model, which closely mimics the 3D structure of primary avascularized tumors. Mass spectrometry (MS) has established itself as a powerful analytical tool, not only to better understand and describe the complex structure of MCTS, but also to monitor their response to cancer therapeutics. The first part of this review focuses on traditional mass spectrometry approaches with an emphasis on elucidating the molecular characteristics of these structures. Then the mass spectrometry imaging (MSI) approaches used to obtain spatially defined information from MCTS is described. Finally the analysis of primary spheroids, such as those present in ovarian cancer, and the great potential that mass spectrometry analysis of these structures has for improved understanding of cancer progression and for personalized in vitro therapeutic testing is discussed.
Publisher: Wiley
Date: 03-2005
Abstract: A 35 kDa protein present in mammary tumors from Neu/ErbB2 transgenic mice was detected on the basis of its cross-reactivity with a phosphoserine-specific antibody against the transcription factor FKHR. To isolate this protein from cytosolic extracts derived from human breast carcinoma cells, we used free-flow electrophoresis in the first dimension to separate proteins according to their charge, followed by reversed-phase high-performance liquid chromatography (RP-HPLC) in the second and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the third dimension. Tryptic digests of Coomassie-stained bands were analyzed by nano-spray ionization-quadrupole quadrupole-time of flight-mass spectrometry identifying StarD10, a START domain containing protein, which cross-reacted with the anti-phospho-FKHR antibody. The site of phosphorylation was identified in immunoaffinity purified Flag-tagged StarD10 from 293T cells transiently expressing this protein. Tryptic phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC) and StarD10 Ser-259-phosphate was identified by tandem mass spectrometry. Thus, free-flow electrophoresis is a powerful high-capacity complementary technique to RP-HPLC and SDS-PAGE for the purification of proteins from complex cell lysates.
Publisher: Springer New York
Date: 14-11-2019
DOI: 10.1007/978-1-4939-8793-1_34
Abstract: Free-flow electrophoresis has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient s le recovery, high resolving power, high reproducibility and wide applicability to protein classes. As a stand-alone platform, however, its utility in comparative proteomic analysis is limited as protein s les must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with free-flow electrophoresis to simultaneously separate and identify differentially expressed proteins in a model cell system.
Publisher: Wiley
Date: 16-04-2014
Abstract: Blue native PAGE (BN-PAGE) is a powerful method to separate protein complexes while preserving their native state. However, the resolution of the method is limited as complexes with similar molecular masses cannot be resolved. Here we describe native 2DE using immobilized pH-gradients in combination with BN-PAGE to resolve protein complexes by their pI and molecular mass. This method enables electrophoretic separation of proteins between pI 3 and 10 and can resolve molecular masses up to 1.2 MDa. Visualized gel spots at large molecular weight were identified using MS to confirm potential protein complexes. Several protein complexes could be identified, most prominent GroEL in complex with GroES, parts of the ribosomal machinery and membrane transport system. In summary, this method enables easy high-resolution electrophoretic separation of protein complexes.
Publisher: Wiley
Date: 11-2010
DOI: 10.1002/0471140864.PS1311S62
Abstract: This unit describes methods to detect, identify, and characterize tyrosine phosphorylation in proteins by mass spectrometry, including s le preparation methods, enrichment strategies using phosphotyrosine‐specific antibodies, and chromatographic separation methods. Curr. Protoc. Protein Sci . 62:13.11.1‐13.11.26. © 2010 by John Wiley & Sons, Inc.
Publisher: American Chemical Society (ACS)
Date: 07-07-2023
Publisher: Wiley
Date: 16-08-2019
Abstract: Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex)
Publisher: Wiley
Date: 03-0001
Abstract: Cytoskeletal proteins are essential building blocks of cells. More than 100 cytoskeletal and cytoskeleton-associated proteins are known and for some, their function and regulation are understood in great detail. Apart from cell shape and support, they facilitate many processes such as intracellular signaling and transport, and cancer related processes such as proliferation, migration, and invasion. During the last decade, comparative proteomic studies have identified cytoskeletal proteins as in vitro markers for tumor progression and metastasis. Here, these results are summarized and a number of unrelated studies are highlighted, identifying the same cytoskeletal proteins as potential biomarkers. These findings might indicate that the abundance of these potential markers of tumor progression is associated with the biological outcome and are independent of the cancer origin. This correlates well with recently published results from the Cancer Genome Atlas, indicating that cancers show remarkable similarities in their analyzed molecular information, independent of their organ of origin. It is postulated that the quantification of cytoskeletal proteins in healthy tissues, tumors, in adjacent tissues, and in stroma, is a great source of molecular information, which might not only be used to classify tumors, but more importantly to predict patients' outcome or even best treatment choices.
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.RVSC.2011.08.015
Abstract: The neurological livestock disease annual ryegrass toxicity (ARGT) is caused by the ingestion of the naturally occurring glycolipid toxins - the corynetoxins. Corynetoxins also threaten human health as potential contaminants of the food supply. Presently, there are no routine diagnostic tests for corynetoxins-exposure in humans or livestock. Chronic ingestion of corynetoxins has been modeled in rats exposed to dietary tunicamycins for 12 months and carbohydrate deficient transferrin (CDT) has been previously identified as a candidate disease biomarker. Here, the technique of immuno-capture mass spectrometry (icMS) was used to evaluate serum levels of CDT, discriminating between control and tunicamycins-exposed rats with 85% accuracy. The icMS approach is based on the combination of specific transferrin enrichment with functionalized magnetic beads and automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). With no other clinically-relevant diagnostic tests available icMS could be readily adapted for high-throughput clinical assessment of corynetoxins-exposure in humans or livestock.
Publisher: Springer Science and Business Media LLC
Date: 26-05-2017
DOI: 10.1038/S41598-017-02465-X
Abstract: Streptococcus pneumoniae (the pneumococcus) is a human pathogen, accounting for massive global morbidity and mortality. Although asymptomatic colonization of the nasopharynx almost invariably precedes disease, the critical determinants enabling pneumococcal progression from this niche to cause invasive disease are poorly understood. One mechanism proposed to be central to this transition involves opacity phase variation, whereby pneumococci harvested from the nasopharynx are typically transparent, while those simultaneously harvested from the blood are opaque. Here, we used two dimensional-differential gel electrophoresis (2D-DIGE) to compare protein expression profiles of transparent and opaque variants of 3 pneumococcal strains, D39 (serotype 2), WCH43 (serotype 4) and WCH16 (serotype 6A) in vitro . One spot comprising a mixture of capsular polysaccharide biosynthesis protein and other proteins was significantly up-regulated in the opaque phenotype in all 3 strains other proteins were differentially regulated in a strain-specific manner. We conclude that pneumococcal phase variation is a complex and multifactorial process leading to strain-specific pathogenicity.
Publisher: Elsevier BV
Date: 06-2015
DOI: 10.1016/J.BBAPAP.2014.10.003
Abstract: Retrospective proteomic studies, including those which aim to elucidate the molecular mechanisms driving cancer, require the assembly and characterization of substantial patient tissue cohorts. The difficulty of maintaining and accessing native tissue archives has prompted the development of methods to access archives of formalin-fixed tissue. Formalin-fixed tissue archives, complete with patient meta data, have accumulated for decades, presenting an invaluable resource for these retrospective studies. This review presents the current knowledge concerning formalin-fixed tissue, with descriptions of the mechanisms of formalin fixation, protein extraction, top-down proteomics, bottom-up proteomics, quantitative proteomics, phospho- and glycoproteomics as well as imaging mass spectrometry. Particular attention has been given to the inclusion of proteomic investigations of archived tumour tissue. This article is part of a Special Issue entitled: Medical Proteomics.
Publisher: Wiley
Date: 06-2009
DOI: 10.1002/RCM.4107
Abstract: The following peptides have been examined in this study: GLDFG(OH), caeridin 1.1 [GLLDGLLGLGGL(NH(2))], 11 Ala citropin 1.1 [GLFDVIKKVAAVIGGL(NH(2))], Crinia angiotensin [APGDRIYVHPF(OH)] and their isoAsp isomers. It is not possible to differentiate between Asp- and isoAsp-containing peptides (used in this study) using negative ion electrospray mass spectrometry. This is because the isoAsp residue cleaves to give the same fragment anions as those formed by delta and gamma backbone cleavage of Asp. The isoAsp fragmentations are as follows: RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR')] --> RNH(-)+HO(2)CCH=CHCONHR' and RNHCH(CO(2)H)(-)CHCONHR' --> [RNH(-)(HO(2)CCH=CHCONHR'] --> (-)O(2)CCH=CHCONHR'+RNH(2). Calculations at the HF/6-31+G(d)//AM1 level of theory indicate that the first of these isoAsp cleavage processes is endothermic (by +115 kJ mol(-1)), while the second is exothermic (-85 kJ mol(-1)). The barrier to the highest transition state is 42 kJ mol(-1). No diagnostic cleavage cations were observed in the electrospray mass spectra of the MH(+) ion of the Asp- and isoAsp-containing peptides (used in this study) to allow differentiation between these two amino acid residues.
Publisher: Elsevier BV
Date: 12-2022
Publisher: MDPI AG
Date: 15-08-2023
Abstract: Nanoparticle-based therapeutics are being clinically translated for treating cancer. Even when thought to be biocompatible, nanoparticles are being increasingly identified as altering cell regulation and homeostasis. Antioxidant pathways are important for maintaining cell redox homeostasis and play important roles by maintaining ROS levels within tolerable ranges. Here, we sought to understand how a model of a relatively inert nanoparticle without any therapeutic agent itself could antagonize a cancer cell lines’ antioxidant mechanism. A label-free protein expression approach was used to assess the glutathione-thioredoxin antioxidative pathway in a prostate cancer cell line (PC-3) after exposure to gold nanoparticles conjugated with a targeting moiety (transferrin). The impact of the nanoparticles was also corroborated through morphological analysis with TEM and classification of pro-apoptotic cells by way of the sub-G0/G1 population via the cell cycle and annexin V apoptosis assay. After a two-hour exposure to nanoparticles, major proteins associated with the glutathione-thioredoxin antioxidant pathway were downregulated. However, this response was acute, and in terms of protein expression, cells quickly recovered within 24 h once nanoparticle exposure ceased. The impact on PRDX-family proteins appears as the most influential factor in how these nanoparticles induced an oxidative stress response in the PC-3 cells. An apparent adaptive response was observed if exposure to nanoparticles continued. Acute exposure was observed to have a detrimental effect on cell viability compared to continuously exposed cells. Nanoparticle effects on cell regulation likely provide a compounding therapeutic advantage under some circumstances, in addition to the action of any cytotoxic agents however, any therapeutic advantage offered by nanoparticles themselves with regard to vulnerabilities specific to the glutathione-thioredoxin antioxidative pathway is highly temporal.
Publisher: SPIE
Date: 09-12-2016
DOI: 10.1117/12.2242259
Publisher: Wiley
Date: 18-02-2013
DOI: 10.1002/RCM.6488
Abstract: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry provides the means to map the in situ distribution of tryptic peptides in formalin-fixed clinical tissue s les. The ability to analyze clinical s les is of great importance to further developments in the imaging field. However, there is a requirement in this field of research for additional methods describing the characterization of tryptic peptides by MALDI imaging. This protocol gives highly detailed instructions, with ex les, for (1) successfully performing tryptic peptide MALDI imaging on formalin-fixed cancer tissue using a MALDI-TOF/TOF MS instrument, (2) tentatively generating identifications through nLC/MS/MS, and (3) validating these identifications by in situ MS/MS of peptides of interest. This protocol provides a detailed and straightforward description of the methods required for groups new to MALDI imaging to begin analysis of formalin-fixed clinical s les.
Publisher: Wiley
Date: 19-02-2015
Abstract: Difference gel electrophoresis enables the accurate quantification of changes in the proteome including combinations of PTMs and protein isoform expression. Here, we review recent advances in study design, image acquisition, and statistical analysis. We also compare DIGE to established and emerging mass spectrometric analysis technologies. Despite these recent advances in MS and the still unsolved limitations of 2DE to map hydrophobic, high molecular weight proteins with extreme pIs, DIGE remains the most comprehensive top-down method to study changes in abundance of intact proteins.
Publisher: Wiley
Date: 15-12-2016
Abstract: This review discusses the current status of proteomics technology in endometrial cancer diagnosis, treatment and prognosis. The first part of this review focuses on recently identified biomarkers for endometrial cancer, their importance in clinical use as well as the proteomic methods used in their discovery. The second part highlights some of the emerging mass spectrometry based proteomic technologies that promise to contribute to a better understanding of endometrial cancer by comparing the abundance of hundreds or thousands of proteins simultaneously.
Publisher: Elsevier BV
Date: 09-2023
Publisher: Elsevier BV
Date: 07-2013
Publisher: Elsevier BV
Date: 15-02-2011
DOI: 10.1016/J.YEXCR.2010.11.003
Abstract: Deleted in liver cancer 1 (DLC1) is a tumor suppressor protein that is frequently downregulated in various tumor types. DLC1 contains a Rho GTPase activating protein (GAP) domain that appears to be required for its tumor suppressive functions. Little is known about the molecular mechanisms that regulate DLC1. By mass spectrometry we have mapped a novel phosphorylation site within the DLC1 GAP domain on serine 807. Using a phospho-S807-specific antibody, our results identify protein kinase D (PKD) to phosphorylate this site in DLC1 in intact cells. Although phosphorylation on serine 807 did not directly impact on in vitro GAP activity, a DLC1 serine-to-alanine exchange mutant inhibited colony formation more potently than the wild type protein. Our results thus show that PKD-mediated phosphorylation of DLC1 on serine 807 negatively regulates DLC1 cellular function.
Publisher: American Chemical Society (ACS)
Date: 19-07-2012
DOI: 10.1021/JP301464S
Abstract: Coordination polymers and discrete metallo-supramolecular assemblies of hexaaryl[3]radialene compounds exhibit intriguing structures with short anion to π-centroid distances in the solid-state. Furthermore, these [3]radialene compounds display useful photophysical and electrochemical properties that make them ideal as potential platforms for anion receptors. In this study, hexafluoro[3]radialene was optimized to the MP2/aug-cc-pVTZ level of theory, and its complexes with halide anions were optimized to HF/6-31G++(d,p), MP2/6-31G++(d,p), M06-2X/6-31G++(d,p), and M06-2X/6-311G++(d,p) levels of theory. Hexafluoro[3]radialene was shown to have properties (large positive Qzz and areas of positive electrostatic surface potential) comparable to other compounds that show anion-π interactions. The interaction energies of complexes of hexafluoro[3]radialene with halide anions were calculated and found to be favorable and equivalent to those of fluorinated aromatic compounds. A series of synthetically accessible hexaaryl[3]radialenes were optimized to HF/6-31G++(d,p) theory and their complexes with halides optimized to the M06-2X/6-31G++(d,p) level of theory. The calculated properties of the electron-deficient hexaaryl[3]radialenes also show large positive Qzz quadrupole moments and two areas of positive potential at the [3]radialene core and the acidic aryl hydrogen atoms. The interaction energies of the complexes of hexaaryl[3]radialenes and halide anions were found to follow the trend F(-) > Cl(-) ≈ Br(-) and correlate with the electron-deficient nature of the [3]radialene. Close contacts were observed between the anion and the radialene core and the aryl hydrogen atoms, suggesting a combination of anion-π and hydrogen bonding is important. Mass spectrometry was used to experimentally observe the complexes of a number of hexaaryl[3]radialenes with F(-), Cl(-), and Br(-) predicted computationally. Anion-[3]radialene complexes were successfully detected, and the stability of the complexes in tandem MS/MS experiments was found to support the computational results.
Publisher: Elsevier BV
Date: 07-2023
Publisher: Elsevier BV
Date: 04-2012
DOI: 10.1016/J.IJCARD.2011.09.014
Abstract: The coronary slow flow phenomenon [CSFP] is a coronary microvascular disorder, characterized by delayed distal vessel opacification despite the absence of obstructive coronary artery disease. Patients frequently present with an acute coronary syndrome [ACS] although the pathophysiological mechanisms responsible are unknown. The aim of this study was to identify potential mechanisms for the ACS presentation associated with the CSFP using a plasma proteomic profiling approach. Plasma s les from nine CSFP subjects [56 ± 11years] were assayed for high sensitivity C-reactive protein [hsCRP], troponin T [TnT], creatine kinase [CK], and proteomic analyses (n=6), during an ACS presentation and one month later [chronic phase]. Proteomic analysis involved chromatographic depletion of abundant plasma proteins followed by two-dimensional differential gel electrophoresis [2-D DIGE]. Protein spots demonstrating ±1.5-fold change relative to the control were identified by mass spectrometry and two differentially expressed proteins were selected for validation via Western blotting. During the ACS presentation, hsCRP was elevated [ACS=14.9 ± 3.9 mg/L vs chronic=4.23 ± 1.37 mg/L, p=0.05] but TnT and CK levels were unchanged. Proteomic analysis identified six proteins that were significantly different in abundance between the acute and chronic s les. During the ACS presentation there was a 1.6 ± 0.13 fold increase in the anti-oxidant enzyme paraoxonase-1 and an increase in inflammatory proteins alpha-1-antichymotrypsin [1.65 ± 0.13 fold] and alpha-1-antitrypsin [2.5 ± 0.34 fold]. The latter was confirmed by Western blotting [1.33 ± 0.17 OD acute/chronic ratio, p=0.05]. The findings from this novel detailed approach, implicate an inflammatory/oxidative stress process in the pathogenesis of the ACS presentation associated with the CSFP. Future studies should further elucidate these mechanisms.
Publisher: American Chemical Society (ACS)
Date: 29-10-2019
DOI: 10.1021/ACS.ANALCHEM.9B03091
Abstract: The strength of MALDI-MSI is to analyze and visualize spatial intensities of molecular features from an intact tissue. The distribution of the intensities can then be visualized within a single tissue section or compared in between sections, acquired consecutively. This method can be reliably used to reveal physiological structures and has the potential to identify molecular details, which correlate with biological outcomes. MALDI-MSI implementation in clinical laboratories requires the ability to ensure method quality and validation to meet diagnostic expectations. To be able to get consistent qualitative and quantitative results, standardized s le preparation and data acquisition are of highest priority. We have previously shown that the deposition of internal standards onto the tissue section during s le preparation can be used to improve the mass accuracy of monitored
Publisher: American Chemical Society (ACS)
Date: 13-12-2013
DOI: 10.1021/PR300996X
Abstract: MALDI imaging mass spectrometry is a powerful tool for morphology-based proteomic tissue analysis. However, peptide identification is still a major challenge due to low S/N ratios, low mass accuracy and difficulties in correlating observed m/z species with peptide identities. To address this, we have analyzed tryptic digests of formalin-fixed paraffin-embedded tissue microarray cores, from 31 ovarian cancer patients, by LC-MS/MS. The s le preparation closely resembled the MALDI imaging workflow in order to create representative reference data sets containing peptides also observable in MALDI imaging experiments. This resulted in 3844 distinct peptide sequences, at a false discovery rate of 1%, for the entire cohort and an average of 982 distinct peptide sequences per s le. From this, a total of 840 proteins and, on average, 297 proteins per s le could be inferred. To support the efforts of the Chromosome-centric Human Proteome Project Consortium, we have annotated these proteins with their respective chromosome location. In the presented work, the benefit of using a large cohort of data sets was exemplified by correct identification of several m/z species observed in a MALDI imaging experiment. The tryptic peptide data sets generated will facilitate peptide identification in future MALDI imaging studies on ovarian cancer.
Publisher: Public Library of Science (PLoS)
Date: 17-03-2015
Publisher: Public Library of Science (PLoS)
Date: 12-07-2023
DOI: 10.1371/JOURNAL.PONE.0288084
Abstract: Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the s ling and preparation of animal tissues from the field. Although RNA later is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require s les to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught s les. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNA later can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNA later immediately, 3 h, and 6 h after euthanasia. Processed tissue s les were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of s le treatment. However, nearly 10% additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that s ling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity.
Publisher: OMICS Publishing Group
Date: 13-12-2008
DOI: 10.4172/JPB.1000053
Publisher: American Chemical Society (ACS)
Date: 11-04-2012
DOI: 10.1021/JF205168T
Abstract: The effects of protein oxidation, for ex le of methionine residues, are linked to many diseases, including those of protein misfolding, such as Alzheimer's disease. Protein misfolding diseases are characterized by the accumulation of insoluble proteinaceous aggregates comprised mainly of amyloid fibrils. Amyloid-containing bodies known as corpora amylacea (CA) are also found in mammary secretory tissue, where their presence slows milk flow. The major milk protein κ-casein readily forms amyloid fibrils under physiological conditions. Milk exists in an extracellular oxidizing environment. Accordingly, the two methionine residues in κ-casein (Met(95) and Met(106)) were selectively oxidized and the effects on the fibril-forming propensity, cellular toxicity, chaperone ability, and structure of κ-casein were determined. Oxidation resulted in an increase in the rate of fibril formation and a greater level of cellular toxicity. β-Casein, which inhibits κ-casein fibril formation in vitro, was less effective at suppressing fibril formation of oxidized κ-casein. The ability of κ-casein to prevent the amorphous aggregation of target proteins was slightly enhanced upon methionine oxidation, which may arise from the protein's greater exposed surface hydrophobicity. No significant changes to κ-casein's intrinsically disordered structure occurred upon oxidation. The enhanced rate of fibril formation of oxidized κ-casein, coupled with the reduced chaperone ability of β-casein to prevent this aggregation, may affect casein-casein interaction within the casein micelle and thereby promote κ-casein aggregation and contribute to the formation of CA.
Publisher: Wiley
Date: 07-2010
Abstract: The quality of MALDI-TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix-deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix-deposition strategy for LC-MALDI-TOF/TOF MS using an automated instrument that produces a nebulised matrix "mist" under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5-DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix-deposition strategy with LC-MALDI-TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC-ESI-IT-MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor-stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC-MALDI-TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.
Publisher: SPIE
Date: 05-03-2013
DOI: 10.1117/12.2003522
Publisher: Oxford University Press (OUP)
Date: 2022
Abstract: Enterococcus faecalis is able to adapt to alkaline conditions and is commonly recovered from teeth in which endodontic treatment has failed. The role that E. faecalis membrane proteins play in survival strategies to extreme alkaline conditions is unclear. We grew E. faecalis V583 in a chemostat at pH 8 and 11 at one-tenth the organism’s relative maximum growth rate. Following membrane shaving, isotope-coding protein labels were added at the peptide level to s les and then combined. The relative proportion of membrane proteins were identified using LC-ESI mass spectrometry and MaxQuant analysis. Ratios of membrane proteins were log2 transformed, with proteins deviating by more than 1 SD of the mean considered to be up- or down-regulated. A total of six proteins were up-regulated in pH 11 including: EF0669 (polysaccharide biosynthesis family) EF1927 (glycerol uptake facilitator), and EF0114 (glycosyl hydrolase). A total of five proteins were down-regulated including: EF0108 (C4-dicarboxylate transporter) EF1838 (PTS system IIC component) EF0456 (PTS system IID component) and EF0022 (PTS mannose-specific IID component). In extreme alkaline conditions, the membrane proteins of E. faecalis seem to be involved in a shift of carbohydrate metabolism from the PTS system to glycerol, which supports the formation of a protective capsule protecting the cell.
Publisher: Springer Science and Business Media LLC
Date: 21-11-2020
Publisher: Springer Science and Business Media LLC
Date: 21-04-2015
DOI: 10.1007/S10585-015-9718-1
Abstract: Ovarian cancer, the most lethal gynaecological cancer, is characterised by the shedding of epithelial cells from the ovarian surface, followed by metastasis and implantation onto the peritoneal surfaces of abdominal organs. Our proteomic studies investigating the interactions between peritoneal (LP-9) and ovarian cancer (OVCAR-5) cells found transketolase (TKT) to be regulated in the co-culture system. This study characterized TKT expression in advanced stage (III/IV) serous ovarian cancers (n = 125 primary and n = 54 peritoneal metastases), normal ovaries (n = 6) and benign serous cystadenomas (n = 10) by immunohistochemistry. In addition, we also evaluated the function of TKT in ovarian cancer cells in vitro. Nuclear TKT was present in all primary serous ovarian cancer tissues examined (median 82.0 %, range 16.5-100 %) and was significantly increased in peritoneal metastases compared with matching primary cancers (P = 0.01, Wilcoxon Rank test). Kaplan-Meier survival and Cox regression analyses showed that high nuclear TKT positivity in peritoneal metastases (>94 %) was significantly associated with reduced overall survival (P = 0.006) and a 2.8 fold increased risk of ovarian cancer death (95 % CI 1.29-5.90, P = 0.009). Knockdown of TKT by siRNAs significantly reduced SKOV-3 cell proliferation but had no effect on their motility or invasion. Oxythiamine, an inhibitor of TKT activity, significantly inhibited the proliferation of four ovarian cancer cell lines (OV-90, SKOV-3, OVCAR-3 and OVCAR-5) and primary serous ovarian cancer cells isolated from patient ascites. In conclusion, these findings indicate that TKT plays an important role in the proliferation of metastatic ovarian cancer cells and could be used as novel therapeutic target for advanced disease.
Publisher: Impact Journals, LLC
Date: 27-01-2017
Publisher: Wiley
Date: 18-04-2017
DOI: 10.1002/RCM.7845
Abstract: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) of the proteome of a tissue has been an established technique for the past decade. In the last few years, MALDI-MSI of the N-glycome has emerged as a novel MALDI-MSI technique. To assess the accuracy and clinical significance of the N-linked glycan spatial distribution, we have developed a method that utilises MALDI-MSI followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) in order to assign glycan structures to the differentiating MALDI-MSI glycan masses released from the tissue glycoproteins. Our workflow presents a comprehensive list of instructions on how to (i) apply MALDI-MSI to spatially map the N-glycome across formalin-fixed paraffin-embedded (FFPE) clinical s les, (ii) structurally characterise N-glycans extracted from consecutive FFPE tissue sections by LC/MS/MS, and (iii) match relevant N-glycan masses from MALDI-MSI with confirmed N-glycan structures determined by LC/MS/MS. Our protocol provides groups that are new to this technique with instructions how to establish N-glycan MALDI-MSI in their laboratory. Furthermore, the method assigns N-glycan structural detail to the masses obtained in the MALDI-MS image. Copyright © 2017 John Wiley & Sons, Ltd.
Publisher: SPIE
Date: 15-05-2011
DOI: 10.1117/12.885066
Publisher: MDPI AG
Date: 10-07-2022
DOI: 10.3390/MPS5040057
Abstract: The molecular analysis of small or rare patient tissue s les is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) generates spatial intensity maps of molecular features, which can be easily correlated with histology. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) can identify and quantify proteins of interest from a consecutive section of the same tissue. Here, we present data from concurrent precancerous lesions from the endometrium and fallopian tube of a single patient. Using this complementary approach, we monitored the abundance of hundreds of proteins within the precancerous and neighboring healthy regions. The method described here represents a useful tool to maximize the number of molecular data acquired from small s le sizes or even from a single case. Our initial data are indicative of a migratory phenotype in these lesions and warrant further research into their malignant capabilities.
Publisher: American Chemical Society (ACS)
Date: 23-04-2009
DOI: 10.1021/PR801111A
Abstract: A model for chronic corynetoxins poisoning has been established in rats exposed to the toxicologically bioequivalent inhibitor of N-linked glycosylation the tunicamycins. Consumption of corynetoxins in contaminated pasture can result in the often fatal neurological disease of grazing livestock annual ryegrass toxicity (ARGT). Corynetoxins may also threaten human health as potential contaminants of the food supply via grain or products derived from subclinically exposed animals. The serum proteomes of four dose groups plus a control group following 6, 9, and 12 months dietary tunicamycins exposure were compared by one-dimensional electrophoresis. Numerous differences were observed between the control and the highest dose group (40.5 mug tunicamycins/kg of body weight/day), designated as High). Accordingly, these s les were further examined using two-dimensional electrophoresis. Thirty-three protein spots were found to be differentially displayed between the Control and High dose sera based on univariate statistics (p < 0.05 for log(10) transformed normalized volumes) and significant fold-changes in spot volume (+/-2.3-fold as determined by posthoc power analysis). Identities for 28 spots were obtained by MALDI-TOF MS corresponding to 13 different proteins. An increasing population of carbohydrate deficient transferrin was identified in the High dose sera using a combination of antibody and lectin detection and confirmed by ESI-IT MS/MS. The functionalities of other identified proteins were consistent with the oxidative stress and acute phase responses. The biomarkers identified in this study may not only play a useful role in diagnosing toxin exposure but could be helpful in identifying new treatment strategies for ARGT and equivalent human diseases.
Publisher: Wiley
Date: 27-11-2020
DOI: 10.1002/JMS.4689
Publisher: MDPI AG
Date: 20-04-2023
DOI: 10.3390/CIMB45040235
Abstract: Nearly 90% of cervical cancers are linked to human papillomavirus (HPV). Uncovering the protein signatures in each histological phase of cervical oncogenesis provides a path to biomarker discovery. The proteomes extracted from formalin-fixed paraffin-embedded tissues of the normal cervix, HPV16/18-associated squamous intraepithelial lesion (SIL), and squamous cell carcinoma (SCC) were compared using liquid chromatography-mass spectrometry (LC-MS). A total of 3597 proteins were identified, with 589, 550, and 1570 proteins unique to the normal cervix, SIL, and SCC groups, respectively, while 332 proteins overlapped between the three groups. In the transition from normal cervix to SIL, all 39 differentially expressed proteins were downregulated, while all 51 proteins discovered were upregulated in SIL to SCC. The binding process was the top molecular function, while chromatin silencing in the SIL vs. normal group, and nucleosome assembly in SCC vs. SIL groups was the top biological process. The PI3 kinase pathway appears crucial in initiating neoplastic transformation, while viral carcinogenesis and necroptosis are important for cell proliferation, migration, and metastasis in cervical cancer development. Annexin A2 and cornulin were selected for validation based on LC-MS results. The former was downregulated in the SIL vs. normal cervix and upregulated in the progression from SIL to SCC. In contrast, cornulin exhibited the highest expression in the normal cervix and lowest in SCC. Although other proteins, such as histones, collagen, and vimentin, were differentially expressed, their ubiquitous expression in most cells precluded further analysis. Immunohistochemical analysis of tissue microarrays found no significant difference in Annexin A2 expression between the groups. Conversely, cornulin exhibited the strongest expression in the normal cervix and lowest in SCC, supporting its role as a tumor suppressor and potential biomarker for disease progression.
Publisher: American Chemical Society (ACS)
Date: 13-04-2017
DOI: 10.1021/ACS.JPROTEOME.6B01032
Abstract: The evolutionary conserved family of 14-3-3 proteins appears to have a role in integrating numerous intracellular pathways, including signal transduction, intracellular trafficking, and metabolism. However, little is known about how this interactive network might be affected by the direct abrogation of 14-3-3 function. The loss of Drosophila 14-3-3ε resulted in reduced survival of mutants during larval-to-adult transition, which is known to depend on an energy supply coming from the histolysis of fat body tissue. Here we report a differential proteomic analysis of larval fat body tissue at the onset of larval-to-adult transition, with the loss of 14-3-3ε resulting in the altered abundance of 16 proteins. These included proteins linked to protein biosynthesis, glycolysis, tricarboxylic acid cycle, and lipid metabolic pathways. The ecdysone receptor (EcR), which is responsible for initiating the larval-to-adult transition, colocalized with 14-3-3ε in wild-type fat body tissues. The altered protein abundance in 14-3-3ε mutant fat body tissue was associated with transcriptional deregulation of alcohol dehydrogenase, fat body protein 1, and lamin genes, which are known targets of the EcR. This study indicates that 14-3-3ε has a critical role in cellular metabolism involving either molecular crosstalk with the EcR or direct interaction with metabolic proteins.
Publisher: Wiley
Date: 08-11-2011
DOI: 10.1002/RCM.5261
Abstract: A joint experimental and theoretical investigation of the fragmentation behaviour of energised [M-H](-) anions from selected phosphorylated peptides has confirmed some of the most complex rearrangement processes yet to be reported for peptide negative ions. In particular: pSer and pThr (like pTyr) may transfer phosphate groups to C-terminal carboxyl anions and to the carboxyl anion side chains of Asp and Glu, and characteristic nucleophilic/cleavage reactions accompany or follow these rearrangements. pTyr may transfer phosphate to the side chains of Ser and Thr. The reverse reaction, namely transfer of a phosphate group from pSer or pThr to Tyr, is energetically unfavourable in comparison. pSer can transfer phosphate to a non-phosphorylated Ser. The non-rearranged [M-H](-) species yields more abundant product anions than its rearranged counterpart. If a peptide containing any or all of Ser, Thr and Tyr is not completely phosphorylated, negative-ion cleavages can determine the number of phosphated residues, and normally the positions of Ser, Thr and Tyr, but not which specific residues are phosphorylated. This is in accord with comments made earlier by Lehmann and coworkers.
Publisher: Springer Science and Business Media LLC
Date: 20-09-2022
DOI: 10.1007/S00216-022-04289-9
Abstract: N -Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of N -glycans in knee osteoarthritis (KOA) progression at the tissue level, especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific N -glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients ( n =3) and CTL in iduals ( n =3), aged years old, were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22 N -glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15 N -glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type N -glycans, (Hex) 4 (HexNAc) 3 , (Hex) 4 (HexNAc) 4 , and (Hex) 5 (HexNAc) 4 , were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that N -glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these N -glycans could further be evaluated as potential diagnostic and prognostic biomarkers.
Publisher: Informa UK Limited
Date: 16-05-2021
Publisher: Mary Ann Liebert Inc
Date: 10-2010
Abstract: Postnatal mesenchymal stem/stromal-like cells (MSCs) including periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and bone marrow stromal cells (BMSCs) are capable of self-renewal and differentiation into multiple mesenchymal cell lineages. Despite their similar expression of MSC-associated and osteoblastic markers, MSCs retain the capacity to generate structures resembling the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical setting. With this in mind, systematic approaches are required to identify the differential protein expression patterns responsible for lineage commitment and mediating the formation of these complex structures. This is the first study to compare the differential proteomic expression profiles of ex vivo-expanded ovine PDLSCs, DPSCs, and BMSCs derived from an in idual donor. The two-dimensional electrophoresis was performed and regulated proteins were identified by liquid chromatography--electrospray-ionization tandem mass spectrometry (MS and MS/MS), database searching, and de novo sequencing. In total, 58 proteins were differentially expressed between at least 2 MSC populations in both sheep, 12 of which were up-regulated in one MSC population relative to the other two. In addition, the regulation of selected proteins was also conserved between equivalent human MSC populations. We anticipate that differential protein expression profiling will provide a basis for elucidating the protein expression patterns and molecular cues that are crucial in specifying the characteristic growth and developmental capacity of dental and non-dental tissue-derived MSC populations. These expression patterns can serve as important tools for the regeneration of particular tissues in future stem cell-based tissue engineering studies using animal models.
Publisher: Impact Journals, LLC
Date: 14-07-2013
Publisher: Bentham Science Publishers Ltd.
Date: 05-2007
DOI: 10.2174/092986607780782849
Abstract: The detection and identification of O-phosphorylation sites in proteins with mass spectrometry remains a challenge. A common approach to analyse these modifications is to enrich phosphopeptides by immobilized metal affinity chromatography (IMAC) prior to mass spectrometric analysis. In this study two commercially available IMAC kits based on Fe(III)-ions immobilized on magnetic beads and Ga(III)-ions immobilized on a chelate-resin, have been investigated and the binding efficiency of peptide mixtures containing non-phosphorylated, singly, doubly and triply phosphorylated peptides have been tested.
Publisher: Hindawi Limited
Date: 31-01-2012
DOI: 10.5402/2012/706545
Abstract: Hyperphosphorylated keratin (K) 8 acts as a phosphate “sponge” for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2D DIGE showed 3 isoforms of K8 significantly increased in tumor ≥2-fold in 6/8 pairs. Metal oxide affinity chromatography mass spectrometry and bioinformatics were used to identify phosphorylated serine residues. Levels of PS24, PS432, and PS74 by western blotting were found to be significantly increased in tumor versus matched normal. Blocking of EGFR signaling in Caco2 cells showed a significant decrease ( P 0.0001 ) in K8 PS74 and PS432 levels by 59% and 66%, respectively, resulting in increased apoptosis.
Publisher: IEEE
Date: 06-2007
Publisher: Humana Press
Date: 2012
DOI: 10.1007/978-1-61779-573-2_15
Abstract: Serum is unarguably the most used diagnostic fluid. As it circulates throughout the body, leakage peptides roteins from damaged and dying cells, host-response proteins including inflammatory mediators, and aberrant secretions from tumors and diseased tissues are released into serum, potentially providing a rich source of disease biomarkers. Here, a method for extending access to the serum proteome by removing highly abundant proteins prior to comparative two-dimensional difference gel electrophoresis (2D DIGE) and subsequent protein digestion for identification by mass spectrometry is described.
Publisher: Springer Science and Business Media LLC
Date: 12-12-2018
DOI: 10.1007/S10495-017-1431-X
Abstract: The original version of this article unfortunately contained a mistake. The affiliation of first author Dr. Ibrahim Alanazi was incorrect.
Publisher: American Society for Microbiology
Date: 09-2010
DOI: 10.1128/JB.00064-10
Abstract: The importance of Mn 2+ for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn 2+ is required are yet to be fully elucidated. Here, we analyzed the effect of Mn 2+ limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn 2+ transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn 2+ -dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn 2+ -SodA and virulence-associated genes pcpA and prtA . We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-response gene cluster, comprising adhC , nmlR , and czcD , in response to Mn 2+ stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn 2+ -replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn 2+ having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn 2+ transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn 2+ stress.
Publisher: Wiley
Date: 28-07-2009
DOI: 10.1002/RCM.4167
Abstract: Mass spectrometry (MS) profiling of the proteome and peptidome for disease-associated patterns is a new concept in clinical diagnostics. The technique, however, is highly sensitive to external sources of variation leading to potentially unacceptable numbers of false positive and false negative results. Before MS profiling can be confidently implemented in a medical setting, standard experimental methods must be developed that minimize technical variance. Past studies of variance have focused largely on pre-analytical variation (i.e., s le collection, handling, etc.). Here, we examined how factors at the analytical stage including the matrix and solid-phase extraction influence MS profiling. Firstly, a standard peptide rotein s le was measured automatically by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS across five consecutive days using two different preparation methods, dried droplet and s le/matrix, of four types of matrix: alpha-cyano-4-hydroxycinnamic acid (HCCA), sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB) and 2,5-dihydroxyacetophenone (DHAP). The results indicated that the matrix preparation greatly influenced a number of key parameters of the spectra including repeatability (within-day variability), reproducibility (inter-day variability), resolution, signal strength, background intensity and detectability. Secondly, an investigation into the variance associated with C8 magnetic bead extraction of the standard s le prior to automated MS profiling demonstrated that the process did not adversely affect these same parameters. In fact, the spectra were generally more robust following extraction. Thirdly, the best performing matrix preparations were evaluated using C8 magnetic bead extracted human plasma. We conclude that the DHAP prepared according to the dried-droplet method is the most appropriate matrix to use when performing automated MS profiling.
Publisher: American Chemical Society (ACS)
Date: 19-09-2016
DOI: 10.1021/ACS.JPROTEOME.6B00053
Abstract: Although acetylation is regarded as a common protein modification, a detailed proteome-wide profile of this post-translational modification may reveal important biological insight regarding differential acetylation of in idual proteins. Here we optimized a novel peptide IEF fractionation method for use prior to LC-MS/MS analysis to obtain a more in depth coverage of N-terminally acetylated proteins from complex s les. Application of the method to the analysis of the serous ovarian cancer cell line OVCAR-5 identified 344 N-terminally acetylated proteins, 12 of which are previously unreported. The protein peptidyl-prolyl cis-trans isomerase A (PPIA) was detected in both the N-terminally acetylated and unmodified forms and was further analyzed by data-independent acquisition in carboplatin-responsive parental OVCAR-5 cells and carboplatin-resistant OVCAR-5 cells. This revealed a higher ratio of unacetylated to acetylated N-terminal PPIA in the parental compared with the carboplatin-resistant OVCAR-5 cells and a 4.1-fold increase in PPIA abundance overall in the parental cells relative to carboplatin-resistant OVCAR-5 cells (P = 0.015). In summary, the novel IEF peptide fractionation method presented here is robust, reproducible, and can be applied to the profiling of N-terminally acetylated proteins. All mass spectrometry data is available as a ProteomeXchange repository (PXD003547).
Publisher: Wiley
Date: 28-04-2015
DOI: 10.1038/ICB.2015.35
Abstract: Phosphoinositide 3-kinase γ (PI3Kγ) consists of the catalytic subunit p110γ that forms a mutually exclusive heterodimer with one of the two adaptor subunits, p101 or p84. Although activation of PI3Kγ is necessary for cell migration downstream of G-protein-coupled receptor engagement, particularly within the immune system, aberrant PI3Kγ signalling has been associated with transformation, increased migration and the progression of multiple cancer types. Regulation of PI3Kγ signal activation and duration is critical to controlling and maintaining coordinated cellular migration however, the mechanistic basis for this is not well understood. We have recently demonstrated that, in contrast to the tumour-promoting potential of p110γ and p101, p84 possesses tumour-suppressor activity, suggesting a negative regulatory role within PI3Kγ signalling. The present study investigated the role of p84 phosphorylation in the context of PI3Kγ signalling, cell migration and p84-mediated tumour suppression. Two putative phosphorylation sites were characterised within p84, Ser358 and Thr607. Expression of wild-type p84 reduced the oncogenic potential of MDA.MB.231 cells and inhibited metastatic lung colonisation in vivo, effects that were dependent on Thr607. Furthermore, loss of Thr607 enhanced migration of MDA.MB.231 cells in vitro and prevented the induction of p84 110γ dimers. The dimerisation of wild-type p84 with p110γ was not detected at the plasma membrane, indicating an inhibitory interaction preventing PI3Kγ lipid-kinase activity. In contrast, Ser358 phosphorylation was not determined to be critical for p84 activity in the context of migration. Our findings suggest that p84 binding to p110γ may represent a novel negative feedback signal that terminates PI3Kγ activity.
Publisher: Institute of Mathematical Statistics
Date: 2014
DOI: 10.1214/14-EJS900
Publisher: Wiley
Date: 20-10-2009
DOI: 10.1002/RCM.4284
Abstract: The demand for analysis of oral fluid for illicit drugs has arisen with the increased adoption of roadside testing, particularly in countries where changes in legislation allow random roadside testing of drivers for the presence of a palette of illicit drugs such as meth hetamine (MA), 3,4-methylenedioxymeth hetamine (MDMA) and Delta9-tetrahydrocannabinol (THC). Oral s les are currently tested for such drugs at the roadside using an immunoassay-based commercial test kit. Positive roadside tests are sent for confirmatory laboratory analysis, traditionally by means of gas chromatography/mass spectrometry (GC/MS). We present here an alternative rapid analysis technique, porous silicon assisted laser desorption/ionization time-of-flight mass spectrometry (pSi LDI-MS), for the high-throughput analysis of oral fluids. This technique alleviates the need for s le derivatization, requires only sub-microliter s le volumes and allows fast analysis (of the order of seconds). In this study, the application of the technique is demonstrated with real s les from actual roadside testing. The analysis of oral s les resulted in detection of MA and MDMA with no extraction and analysis of THC after ethyl acetate extraction. We propose that, subject to miniaturization of a suitable mass spectrometer, this technique is well suited to underpin the deployment of oral fluid testing in the clinic, workplace and on the roadside.
Publisher: MDPI AG
Date: 08-07-2016
DOI: 10.3390/IJMS17071088
Publisher: Wiley
Date: 19-05-2016
Publisher: Wiley
Date: 16-03-2021
Abstract: The applicability of mass spectrometry imaging (MSI) has exponentially increased with the improvement of s le preparation, instrumentation (spatial resolution) and data analysis. The number of MSI publications listed in PubMed continues to grow with 378 published articles in 2020‐2021. Initially, MSI was just sensitive enough to identify molecular features correlating with distinct tissue regions, similar to the resolution achieved by visual inspection after standard immunohistochemical staining. Although the spatial resolution was limited compared with other imaging modalities, the molecular intensity mapping added a new exciting capability. Over the past decade, significant improvements in every step of the workflow and most importantly in instrumentation were made, which now enables the molecular analysis at a cellular and even subcellular level. Here, we summarize the latest developments in MSI, with a focus on the latest approaches for tissue‐based imaging described in 2020.
Publisher: MDPI AG
Date: 08-04-2022
DOI: 10.3390/IJMS23084139
Abstract: The accurate segregation of sister chromatids is complex, and errors that arise throughout this process can drive chromosomal instability and tumorigenesis. We recently showed that methylglyoxal (MGO), a glycolytic by-product, can cause chromosome missegregation events in lymphocytes. However, the underlying mechanisms of this were not explored. Therefore, in this study, we utilised shotgun proteomics to identify MGO-modified proteins, and label-free quantitation to measure changes in protein abundance following exposure to MGO. We identified numerous mitotic proteins that were modified by MGO, including those involved in the separation and cohesion of sister chromatids. Furthermore, the protein abundance of Securin, an inhibitor of sister chromatid separation, was increased following treatment with MGO. Cytological examination of chromosome spreads showed MGO prevented sister chromatid separation, which was associated with the formation of complex nuclear anomalies. Therefore, results from this study suggest MGO may drive chromosomal instability by preventing sister chromatid separation.
Publisher: Frontiers Media SA
Date: 15-12-2020
Abstract: Serous endometrial cancer (SEC) and high grade serous ovarian cancer (HGSOC) are aggressive gynecological malignancies with high rates of metastasis and poor prognosis. Endometrial intraepithelial carcinoma (EIC), the precursor for SEC, and serous tubal intraepithelial carcinoma (STIC), believed to be the precursor lesion for HGSOC, can also be associated with intraabdominal spread. To provide insight into the etiology of these precancerous lesions and to explore the potential molecular mechanisms underlying their metastatic behavior, we performed a proteomic mass spectrometry analysis in a patient with synchronous EIC and STIC. Through histological and molecular identification of precancerous lesions followed by laser capture microdissection, we were able to identify over 450 proteins within the precancerous lesions and adjacent healthy tissue. The proteomic analysis of STIC and EIC showed remarkable overlap in the proteomic patterns, reflecting early neoplastic changes in proliferation, loss of polarity and attachment. Our proteomic analysis showed that both EIC and STIC, despite being regarded as premalignant lesions, have metastatic potential, which correlates with the common presentation of invasive serous gynecological malignancies at advanced stage.
Publisher: Cold Spring Harbor Laboratory
Date: 05-2022
DOI: 10.1101/2022.05.01.490183
Abstract: Immuno-specific enrichment of extracellular vesicles (EVs) originating from specific cells/tissues is a promising source of information towards improving insights into cellular pathways underpinning various pathologies and developing novel non-invasive diagnostic methods. Enrichment is an important aspect in mass spectrometry-based analyses of EVs. Herein, we report a protocol for immuno-magnetic enrichment of subtype specific EVs and their subsequent processing for mass spectrometry. Specifically, we conjugated placental alkaline phosphatase (PLAP) antibodies to magnetic iron oxide nanowires (NWs) derived from bacterial biofilms and demonstrated the utility of this approach by enriching placental specific EVs (containing PLAP) from cell culture media. We demonstrate efficient PLAP+ve EV enrichment for both NW-PLAP and Dynabeads™-PLAP, with PLAP protein recovery (83.7±8.9% and 83.2±5.9%, respectively), high particle-to-protein ratio (7.5±0.7×10 9 and 7.1 ± 1.2×10 9, respectively), and low non-specific binding of non-target EVs (7±3.2% and 5.4±2.2%, respectively). Furthermore, our optimized EV enrichment and processing approach identified 2518 and 2545 protein groups with mass spectrometry for NW-PLAP and Dynabead™-PLAP, respectively, with excellent reproducibility (Pearson correlation 0.986 and 0.988). The proposed immuno-specific EVs enrichment and liquid chromatography-tandem mass spectrometry method using naturally occurring iron oxide magnetic NWs or gold-standard Dynabeads™ enables high-quality EV proteomic studies.
Publisher: Humana Press
Date: 2012
DOI: 10.1007/978-1-61779-821-4_12
Abstract: Free flow electrophoresis (FFE) has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient s le recovery, high resolving power, high reproducibility, and wide applicability to protein classes. As a stand-alone platform however, its utility in comparative proteomic analysis is limited as protein s les must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with FFE to simultaneously separate and identify differentially expressed proteins in a model cell system.
Publisher: Wiley
Date: 11-10-2005
Abstract: Epithelial ovarian cancer is the fourth leading cause of cancer death among women. Due to the asymptomatic nature and poor survival characteristic of the disease, screening for specific biomarkers for ovarian cancer is a major health priority. Differentially expressed proteins in the serum of ovarian cancer patients have the potential to be used as cancer-specific biomarkers. In this study, proteomic methods were used to screen 24 serum s les from women with high-grade ovarian cancer and compared to a control group of 11 healthy women. Affigel-Blue treated serum s les were processed either by linear (pH 4-7) or narrow range (pH 5.5-6.7) IEF strips for the first dimension. Proteins separated in first dimension were resolved by 8-16% gradient SDS-PAGE. Protein spots were visualized by SYPRO Ruby staining, imaged by FX-imager and compared and analyzed by PDQuest software. Twenty-two protein spots were consistently differentially expressed between normal and ovarian cancer patients by resolving proteins in a linear pH strip of 4-7 for the first dimension. Six of the protein spots, significantly up-regulated in grade 3 ovarian cancer patients (p < 0.05), were identified by MALDI-TOF MS and Western blotting as the isoforms of haptoglobin precursor. When serum proteins were resolved on narrow pH range strips (5.5-6.7), 23 spots were consistently differentially expressed between normal and grade 3 ovarian cancer patients. Of these, 4 protein spots significantly down regulated in grade 3 ovarian cancer patients (p < 0.05) were identified by MALDI-TOF MS and Western blotting, as isoforms of transferrin precursor. Increased expression of serum haptoglobin and transferrin was also identified in peritoneal tumor fluid obtained from women diagnosed with grade 2/3 ovarian cancer (n = 7). Changes in the expression of haptoglobin and transferrin in the serum of women with different pathological grades of ovarian cancer was examined by one-dimensional Western blotting method. Serum s les collected from women suffering from benign, borderline, grade 1, grade 2 and grade 3 cancer (n = 4 for haptoglobin and n = 5 for transferrin in each group) were analyzed and compared to the serum of normal healthy women. The mean serum haptoglobin expression in grade 3 ovarian cancer patients was fourfold higher than in the control subjects (p < 0.05). On the other hand, transferrin expression in grade 3 ovarian cancer patients was decreased by twofold than in normal healthy women (p < 0.05). Haptoglobin expression in the serum of cancer patients (n = 7) decreased following chemotherapy (six cycles of taxol/carboplatin). Concomitant with the decrease of haptoglobin, transferrin expression remained constant in four patients, but increased in three out of seven patients included in the study. Changes in serum expression of haptoglobin correlated with the change of CA 125 levels before and after chemotherapy. In conclusion, proteomic profiling of differentially expressed proteins in the sera of normal women compared to women with ovarian cancer can greatly facilitate the discovery of a panel of biomarkers that may aid in the detection of ovarian cancer with greater specificity.
Publisher: Elsevier BV
Date: 06-2019
DOI: 10.1016/J.CELLSIG.2019.03.002
Abstract: Endothelial cell injury and death precede atherosclerosis development. Thus, it is important to understand the mechanisms that lead to these early changes in endothelial cells. Although members of the MAP kinase/ERK kinase (MEK) kinase 3 (MEKK3)-MEK5-ERK5 module play an essential role in underpinning endothelial cell survival, how they execute these actions remain poorly understood. Furthermore, there is poor understanding of death-inducing pathways in endothelial cells and it is also unclear whether there are direct interactions between the kinase module and death-inducing pathways. Using immunoprecipitation and liquid chromatography-electrospray ionisation tandem mass spectrometry approaches, we show in human umbilical vein endothelial cells that the MEKK3-MEK5-ERK5 ternary complex contains glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme that can trigger the death of certain cell-types. GAPDH binds directly to MEKK3. Interestingly, serum depletion, a trigger of endothelial cell death, results in a rapid loss of cytosolic MEKK3 and MEKK3-GAPDH interaction. MEKK3 rapidly reappears in the cytosol upon serum replenishment, accompanied by the restoration of MEKK3-GAPDH interaction. During serum starvation or exposure to cytotoxic concentrations of H
Publisher: CSIRO Publishing
Date: 2009
DOI: 10.1071/SRB09ABS510
Abstract: Ovarian cancer is characterized by metastases to the peritoneal surface lining the abdominal cavity. It remains unclear which factors promote the implantation of ovarian cancer cells onto the peritoneal lining. We have recently investigated interactions between ovarian cancer cells (OVCAR-5, OVCAR-3, and SKOV-3) and mesothelial cells isolated from omental tissues (LP-9). We conducted a proteomic screen of the conditioned medium of co-cultures of ovarian cancer and mesothelial cells. One of the molecules identified to be modulated is the extracellular matrix adhesion protein, transforming growth factor-beta-induced protein (TGFβI, also known as b ig-H3 or keratoepithelin) which is induced by transforming growth factor-beta in many cell types which has been shown to promote adhesion and migration of hepatoma and astrocytoma cells and enhance colon cancer cell extravasation. In this study we investigated the expression of TGFβI in ovarian cancer tissues and the effects of recombinant TGFβI on ovarian cancer motility and adhesion to peritoneal mesothelial cells. In functional assays, treatment with recombinant TGFβI significantly increased adhesion of all three ovarian cancer cell lines to LP-9 mesothelial cells by up to 25% (P .01) and increased motility in OVCAR-5 cells by 62% (P .001). Furthermore, addition of a neutralising TGFβI antibody reduced OVCAR-5 adhesion to LP-9 to 79% of control level (P .001). TGFβI produced by LP-9 cells was processed to smaller forms when co-cultured with ovarian cancer cell lines by western blotting. MALDI-TOF/TOF mass spectrometry identified TGFβI processing at both the N and C terminal domains. The addition of broad spectrum protease inhibitors blocked the TGFβI processing and reduced OVCAR-5 adhesion to LP-9 cells to 60% of control level (P .001). We conclude that although some ovarian cancer cells produce low levels of TGFβI, TGFβI abundantly expressed by peritoneal mesothelial cells can promote ovarian cancer cell adhesion and motility.
Publisher: Wiley
Date: 09-05-2016
Abstract: Applying MALDI-MS imaging to tissue microarrays (TMAs) provides access to proteomics data from large cohorts of patients in a cost- and time-efficient way, and opens the potential for applying this technology in clinical diagnosis. The complexity of these TMA data-high-dimensional low s le size-provides challenges for the statistical analysis, as classical methods typically require a nonsingular covariance matrix that cannot be satisfied if the dimension is greater than the s le size. We use TMAs to collect data from endometrial primary carcinomas from 43 patients. Each patient has a lymph node metastasis (LNM) status of positive or negative, which we predict on the basis of the MALDI-MS imaging TMA data. We propose a variable selection approach based on canonical correlation analysis that explicitly uses the LNM information. We apply LDA to the selected variables only. Our method misclassifies 2.3-20.9% of patients by leave-one-out cross-validation and strongly outperforms LDA after reduction of the original data with principle component analysis.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D2NR05619D
Abstract: Magnetic extracellular vesicle (EV) enrichment using antibody conjugated bacteria-derived iron oxide nanowires coupled with mass spectrometry-based proteome profiling enables efficient EV subtype enrichment and reproducible proteomics.
Publisher: Elsevier BV
Date: 05-2014
DOI: 10.1016/J.BBAPAP.2014.01.018
Abstract: The timely detection of gastric cancer will contribute significantly towards effective treatment and is aided by the availability and reliability of appropriate biomarkers. A combination of several biomarkers can improve the sensitivity and specificity of cancer detection and this work reports results from a panel of 4 proteins. By combining a validated preclinical mouse model with a proteomic approach we have recently discovered novel biomarkers for the detection of gastric cancer. Here, we investigate the specificity of four of those biomarkers (afamin, clusterin, VDBP and haptoglobin) for the detection of gastric cancer using two independent methods of validation: ELISA, and a non antibody based method: Multiple Reaction Monitoring with High Resolution Mass Spectrometry (MRM-HR). All four biomarkers reliably differentiated GC from benign patient serum, and also in a small cohort of 11 early stage cases. We also present a novel isoform specific biomarker alpha-1-antitrypsin (A1AT) that was identified using a mouse model for gastric cancer. This isoform is distinct in charge and mobility in a pH gradient and was validated using human s les by isoelectric focussing and Western-blot (IEF-WB). This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.
Publisher: Wiley
Date: 10-05-2016
Abstract: Metastasis is a crucial step of malignant progression and is the primary cause of death from endometrial cancer. However, clinicians presently face the challenge that conventional surgical-pathological variables, such as tumour size, depth of myometrial invasion, histological grade, lymphovascular space invasion or radiological imaging are unable to predict with accuracy if the primary tumour has metastasized. In the current retrospective study, we have used primary tumour s les of endometrial cancer patients diagnosed with (n = 16) and without (n = 27) lymph node metastasis to identify potential discriminators. Using peptide matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI), we have identified m/z values which can classify 88% of all tumours correctly. The top discriminative m/z values were identified using a combination of in situ sequencing and LC-MS/MS from digested tumour s les. Two of the proteins identified, plectin and α-Actin-2, were used for validation studies using LC-MS/MS data independent analysis (DIA) and immunohistochemistry. In summary, MALDI-MSI has the potential to identify discriminators of metastasis using primary tumour s les.
Publisher: Humana Press
Date: 2012
DOI: 10.1007/978-1-61779-573-2_22
Abstract: Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel.
Publisher: MDPI AG
Date: 03-09-2020
DOI: 10.3390/IJMS21176414
Abstract: Osteoarthritis (OA) is the most common degenerative joint disease, predicted to increase in incidence year by year due to an ageing population. Due to the biological complexity of the disease, OA remains highly heterogeneous. Although much work has been undertaken in the past few years, underlying molecular mechanisms leading to joint tissue structural deterioration are not fully understood, with only few validated markers for disease diagnosis and progression being available. Discovery and quantitation of various OA-specific biomarkers is still largely focused on the bodily fluids which does not appear to be reliable and sensitive enough. However, with the advancement of spatial proteomic techniques, several novel peptides and proteins, as well as N-glycans, can be identified and localised in a reliable and sensitive manner. To summarise the important findings from OA biomarker studies, papers published between 2000 and 2020 were searched via Google Scholar and PubMed. Medical subject heading (MeSH) terms ‘osteoarthritis’, ‘biomarker’, ‘synovial fluid’, ‘serum’, ‘urine’, ’matrix-assisted laser desorption/ionisation’, ‘mass spectrometry imaging’, ‘proteomic’, ‘glycomic’, ‘cartilage’, ‘synovium’ AND ‘subchondral bone’ were selectively used. The literature search was restricted to full-text original research articles and written only in English. Two main areas were reviewed for OA biomarker studies: (1) an overview of disease-specific markers detected from different types of OA bio-s les, and (2) an up-to-date summary of the tissue-specific OA studies that have utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). Overall, these OA biomarkers could provide clinicians with information for better the diagnosis, and prognosis of in idual patients, and ultimately help facilitate the development of disease-modifying treatments.
Publisher: Elsevier BV
Date: 10-2000
DOI: 10.1016/S0021-9673(00)00410-6
Abstract: Separation of the enantiomers of chlorpheniramine and methadone in acidic buffers containing carboxymethyl-betacyclodextrin (CMCD) as chiral selector was investigated by capillary zone electrophoresis. For a range of pH and CMCD concentrations, the mobility difference and resolution of the enantiomers were determined. Then, conditions known to provide well resolved enantiomers and optimized chiral separation were applied to chiral continuous flow electrophoresis. In that approach, a thin film of fluid flowing between two parallel plates is employed as carrier for electrophoresis. The electrolytes and the s le are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. The number of pure enantiomeric fractions obtained by chiral continuous flow electrophoresis was found to be directly dependent on the enantiomeric mobility difference. For racemic chlorpheniramine separated in a betaine-acetic acid buffer at a total throughput of 5 mg/h, complete enantiomeric separation is shown to require a mobility difference of about 3 x 10(-9) m2/V s. Furthermore, compared to the previous investigations with hydroxypropyl-beta-cyclodextrin, CMCD was found to permit improved fractionation of methadone enantiomers. With a total racemic drug throughput of about 15 mg/h, continuous flow zone electrophoresis processing with CMCD as chiral selector is shown to have the potential of providing pure enantiomers on a mg/h scale. The results indicate that chiral capillary zone electrophoresis data can be employed as predictor for preparative scale chiral separations based upon continuous flow zone electrophoresis.
Publisher: Wiley
Date: 24-10-2006
DOI: 10.1002/JMS.1117
Abstract: Nonenzymatic glycosylation (or glycation) is a common nonenzymatic side-chain specific sequence-independent posttranslational modification formed by the reaction of reducing carbohydrates with free amino groups. Thus, proteins can react with aldoses or ketoses to yield Amadori or Heynes compounds, respectively. Here, the fragmentation behavior of D-glucose and D-ribose-derived Amadori peptides as well as D-fructose-derived Heynes peptides were studied by collision-induced fragmentation (CID) after electrospray (ESI) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). All three sugar moieties displayed characteristic fragmentation patterns accompanying the parent and the fragment ions, which could be explained by consecutive losses of water and formaldehyde. Glucose-derived Amadori parent and fragment ions displayed losses of 18, 36, 54, 72, and 84 u at a characteristic intensity distribution compared with losses of 18, 36, 54, 72, 84, and 96 u for D-fructose-derived ions and losses of 18, 36, and 54 u for ribose-derived ions. Furthermore, each sugar moiety produced indicative lysine-derived immonium ions that were successfully used in a precursor ion scan analysis to identify Amadori peptides in a tryptic digest of bovine serum albumin (BSA) glycated with D-glucose. BSA was modified on lysine residues at positions 36, 160, 235, 256, 401, and 548.
Publisher: SPIE
Date: 07-2015
DOI: 10.1117/12.2186140
Publisher: MDPI AG
Date: 28-03-2022
DOI: 10.3390/IJMS23073689
Abstract: Methylglyoxal (MGO) is a highly reactive cellular metabolite that glycates lysine and arginine residues to form post-translational modifications known as advanced glycation end products. Because of their low abundance and low stoichiometry, few studies have reported their occurrence and site-specific locations in proteins. Proteomic analysis of WIL2-NS B lymphoblastoid cells in the absence and presence of exogenous MGO was conducted to investigate the extent of MGO modifications. We found over 500 MGO modified proteins, revealing an over-representation of these modifications on many glycolytic enzymes, as well as ribosomal and spliceosome proteins. Moreover, MGO modifications were observed on the active site residues of glycolytic enzymes that could alter their activity. We similarly observed modification of glycolytic enzymes across several epithelial cell lines and peripheral blood lymphocytes, with modification of fructose bisphosphate aldolase being observed in all s les. These results indicate that glycolytic proteins could be particularly prone to the formation of MGO adducts.
Publisher: Springer Science and Business Media LLC
Date: 02-12-2015
Publisher: Elsevier BV
Date: 08-2007
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9LC00152B
Abstract: Microfluidics and MALDI-TOF MS is a rapid, high-throughput, and accurate method for the identification of beer spoilage bacteria.
Publisher: Frontiers Media SA
Date: 25-09-2020
Publisher: American Chemical Society (ACS)
Date: 15-06-2009
DOI: 10.1021/JF9008372
Abstract: Milk casein proteins can act as molecular chaperones: under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. In this study, alpha(s)- and beta-caseins were dephosphorylated using alkaline phosphatase. A structural and functional investigation was undertaken to determine the effect of dephosphorylation on the chaperone activity of alpha(s)- and beta-caseins against two types of protein misfolding, i.e., amorphous aggregation and amyloid fibril assembly. The dephosphorylation of alpha(s)- and beta-caseins resulted in a decrease in the chaperone efficiency against both heat- and reduction-induced amorphously aggregating target proteins. In contrast, dephosphorylation had no effect on the chaperone activity of alpha(s)- and beta-caseins against the amyloid-forming target protein kappa-casein. Circular dichroism and fluorescence spectroscopic data indicated that the loss of negative charge associated with dephosphorylation led to an increase in ordered structure of alpha(s)- and beta-caseins. It is concluded that the flexible, dynamic, and relatively unstructured and hiphatic nature of alpha(s)- and beta-caseins is important in their chaperone action.
Publisher: MDPI AG
Date: 27-10-2021
Abstract: Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) can determine the spatial distribution of analytes such as protein distributions in a tissue section according to their mass-to-charge ratio. Here, we explored the clinical potential of machine learning (ML) applied to MALDI MSI data for cancer diagnostic classification using tissue microarrays (TMAs) on 302 colorectal (CRC) and 257 endometrial cancer (EC)) patients. ML based on deep neural networks discriminated colorectal tumour from normal tissue with an overall accuracy of 98% in balanced cross-validation (98.2% sensitivity and 98.6% specificity). Moreover, our machine learning approach predicted the presence of lymph node metastasis (LNM) for primary tumours of EC with an accuracy of 80% (90% sensitivity and 69% specificity). Our results demonstrate the capability of MALDI MSI for complementing classic histopathological examination for cancer diagnostic applications.
Publisher: Elsevier
Date: 2017
DOI: 10.1016/BS.ACR.2016.11.002
Abstract: Pathologists play an essential role in the diagnosis and prognosis of benign and cancerous tumors. Clinicians provide tissue s les, for ex le, from a biopsy, which are then processed and thin sections are placed onto glass slides, followed by staining of the tissue with visible dyes. Upon processing and microscopic examination, a pathology report is provided, which relies on the pathologist's interpretation of the phenotypical presentation of the tissue. Targeted analysis of single proteins provide further insight and together with clinical data these results influence clinical decision making. Recent developments in mass spectrometry facilitate the collection of molecular information about such tissue specimens. These relatively new techniques generate label-free mass spectra across tissue sections providing nonbiased, nontargeted molecular information. At each pixel with spatial coordinates (x/y) a mass spectrum is acquired. The acquired mass spectrums can be visualized as intensity maps displaying the distribution of single m/z values of interest. Based on the s le preparation, proteins, peptides, lipids, small molecules, or glycans can be analyzed. The generated intensity maps/images allow new insights into tumor tissues. The technique has the ability to detect and characterize tumor cells and their environment in a spatial context and combined with histological staining, can be used to aid pathologists and clinicians in the diagnosis and management of cancer. Moreover, such data may help classify patients to aid therapy decisions and predict outcomes. The novel complementary mass spectrometry-based methods described in this chapter will contribute to the transformation of pathology services around the world.
Publisher: SPIE
Date: 14-04-2008
DOI: 10.1117/12.801887
Publisher: Oxford University Press (OUP)
Date: 08-2010
Publisher: American Chemical Society (ACS)
Date: 09-2020
Publisher: The Institute of Brewing & Distilling
Date: 23-05-2017
DOI: 10.1002/JIB.428
Publisher: Wiley
Date: 06-2009
Abstract: Phosphorylation is one of the most important PTMs and is estimated to occur on 30% of the mammalian proteome. Its perturbed regulation has been implicated in many pathologies. The rarity of phosphotyrosine compared with phosphoserine or phosphothreonine is prompting the development of more sensitive approaches because proteomic technologies that are currently used to assess tyrosine phosphorylation in proteins are inadequate, identifying only a fraction of the predicted tyrosine phosphoproteome. Here we describe the development of a reproducible, high-sensitivity methodology for the detection and mapping of phosphotyrosine residues by MS. The anti-phosphotyrosine antibody 4G10 was coupled covalently to super para-magnetic beads or by affinity to super para-magnetic beads with protein G covalently attached. Using this approach, we successfully enriched phosphotyrosine peptides mixed with non-phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were subsequently used to enrich tyrosine phosphopeptides from a digest of the in vitro-phosphorylated recombinant beta-intracellular region of the granulocyte-macrophage colony-stimulating factor receptor, which was subsequently analysed by MALDI-TOF/TOF MS. Our results define this methodology as a sensitive approach for tyrosine phosphoproteome analysis.
Publisher: Institute of Mathematical Statistics
Date: 12-2015
DOI: 10.1214/15-AOAS870
Publisher: Wiley
Date: 29-09-2008
DOI: 10.1002/RCM.3736
Abstract: Peptides and proteins may contain post-translationally modified phosphorylated amino acid residues, in particular phosphorylated serine (pSer), threonine (pThr) and tyrosine (pTyr). Following earlier work by Lehmann et al., the [M-H]- anions of peptides containing pSer and pThr functionality show loss of the elements of H3PO4. This process, illustrated for Ser (and using a model system), is CH3CONH-C(CH2OPO3H2)CONHCH(3) --> [CH3CONHC(==CH2)CONHCH3 (-OPO3H2)] (a) --> [CH3CONHC(==CH2)CONHCH3-H]- + H3PO4, a process endothermic by 83 kJ mol(-1) at the MP2/6-31++G(d,p)//HF/6-31++G(d,p) level of theory. In addition, intermediate (a) may decompose to yield CH3CONHC(==CH2)CONHCH3 + H2PO4 - in a process exothermic by 3 kJ mol(-1). The barrier to the transition state for these two processes is 49 kJ mol(-1). Characteristic cleavages of pSer and pThr are more energetically favourable than the negative ion backbone cleavages of peptides described previously. In contrast, loss of HPO3 from [M-H]- is characteristic of pTyr. The cleavage [NH2CH(CH2-C6H4-OPO3H-)CO2H] --> [NH2C(CH2-C6H4-O-)CO2H (HPO3)] (b) --> NH2CH(CH2-C6H4-O-)CO2H + HPO3 is endothermic by 318 kJ mol(-1) at the HF/6-31+G(d)//AM1 level of theory. In addition, intermediate (b) also yields NH2CH(CH2-C6H4-OH)CO2H + PO3 - (reaction endothermic by 137 kJ mol(-1)). The two negative ion cleavages of pTyr have a barrier to the transition state of 198 kJ mol(-1) (at the HF/6-31+G(d)//AM1 level of theory) comparable with those already reported for negative ion backbone cleavages.
Publisher: SPIE
Date: 09-02-2012
DOI: 10.1117/12.909681
Publisher: Springer Science and Business Media LLC
Date: 02-11-2018
Publisher: Wiley
Date: 07-2001
DOI: 10.1002/1615-9861(200107)1:7<807::AID-PROT807>3.0.CO;2-6
Publisher: Oxford University Press (OUP)
Date: 2016
DOI: 10.1039/C6MT00142D
Abstract: The metal-resistant β-proteobacterium Cupriavidus metallidurans drives gold (Au) biomineralisation and the (trans)formation of Au nuggets largely via unknown biochemical processes, ultimately leading to the reductive precipitation of mobile, toxic Au(i/iii)-complexes. In this study proteomic responses of C. metallidurans CH34 to mobile, toxic Au(iii)-chloride are investigated. Cells were grown in the presence of 10 and 50 μM Au(iii)-chloride, 50 μM Cu(ii)-chloride and without additional metals. Differentially expressed proteins were detected by difference gel electrophoresis and identified by liquid chromatography coupled mass spectrometry. Proteins that were more abundant in the presence of Au(iii)-chloride are involved in a range of important cellular functions, e.g., metabolic activities, transcriptional regulation, efflux and metal transport. To identify Au-binding proteins, protein extracts were separated by native 2D gel electrophoresis and Au in protein spots was detected by laser absorption inductively coupled plasma mass spectrometry. A chaperon protein commonly understood to bind copper (Cu), CupC, was identified and shown to bind Au. This indicates that it forms part of a multi-metal detoxification system and suggests that similar/shared detoxification pathways for Au and Cu exist. Overall, this means that C. metallidurans CH34 is able to mollify the toxic effects of cytoplasmic Au(iii) by sequestering this Au-species. This effect may in the future be used to develop CupC-based biosensing capabilities for the in-field detection of Au in exploration s les.
Publisher: Oxford University Press (OUP)
Date: 14-08-2018
Publisher: Wiley
Date: 09-11-2019
Abstract: Protein glycosylation, particularly N-linked glycosylation, is a complex posttranslational modification (PTM), which plays an important role in protein folding and conformation, regulating protein stability and activity, cell-cell interaction, and cell signaling pathways. This review focuses on analytical techniques, primarily MS-based techniques, to qualitatively and quantitatively assess N-glycosylation while successfully characterizing compositional, structural, and linkage features with high specificity and sensitivity. The analytical techniques explored in this review include LC-ESI-MS/MS and MALDI time-of-flight MS (MALDI-TOF-MS), which have been used to analyze clinical s les, such as serum, plasma, ascites, and tissue. Targeting the aberrant N-glycosylation patterns observed in MALDI-MS imaging (MSI) offers a platform to visualize N-glycans in tissue-specific regions. The studies on the intra-patient (i.e., a comparison of tissue-specific regions from the same patient) and inter-patient (i.e., a comparison of tissue-specific regions between different patients) variation of early- and late-stage ovarian cancer (OC) patients identify specific N-glycan differences that improve understanding of the tumor microenvironment and potentially improve therapeutic strategies for the clinic.
Publisher: Optica Publishing Group
Date: 2007
DOI: 10.1364/OE.15.017819
Abstract: The detection of quantum-dot labeled proteins is demonstrated within lead silicate soft glass microstructured optical fibers using near infrared light. The protein concentration is measured using a new fluorescence capture approach. Light guided within the fiber is used both to excite and collect fluorescent photons, and the detection limit achieved without optimization of the fiber geometry is 1 nM, using just 3% of the guided mode of the fiber. Issues that currently restrict the detection of lower protein concentrations are discussed.
Publisher: Elsevier BV
Date: 07-2017
DOI: 10.1016/J.BBAPAP.2016.10.010
Abstract: The prediction of lymph node metastasis using clinic-pathological data and molecular information from endometrial cancers lacks accuracy and is therefore currently not routinely used in patient management. Consequently, although only a small percentage of patients with endometrial cancers suffer from metastasis, the majority undergo radical surgery including removal of pelvic lymph nodes. Upon analysis of publically available data and published research, we compiled a list of 60 proteins having the potential to display differential abundance between primary endometrial cancers with versus those without lymph node metastasis. Using data dependent acquisition LC-ESI-MS/MS we were able to detect 23 of these proteins in endometrial cancers, and using data independent LC-ESI-MS/MS the differential abundance of five of those proteins was observed. The localization of the differentially expressed proteins, was visualized using peptide MALDI MSI in whole tissue sections as well as tissue microarrays of 43 patients. The proteins identified were further validated by immunohistochemistry. Our data indicate that annexin A2 protein level is upregulated, whereas annexin A1 and α actinin 4 expression are downregulated in tumours with lymph node metastasis compared to those without lymphatic spread. Moreover, our analysis confirmed the potential of these markers, to be included in a statistical model for prediction of lymph node metastasis. The predictive model using highly ranked m/z values identified by MALDI MSI showed significantly higher predictive accuracy than the model using immunohistochemistry data. In summary, using publicly available data and complementary proteomics approaches, we were able to improve the prediction model for lymph node metastasis in EC.
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.JPROT.2012.04.054
Abstract: One of the important challenges for MALDI imaging mass spectrometry (MALDI-IMS) is the unambiguous identification of measured analytes. One way to do this is to match tryptic peptide MALDI-IMS m/z values with LC-MS/MS identified m/z values. Matching using current MALDI-TOF/TOF MS instruments is difficult due to the variability of in situ time-of-flight (TOF) m/z measurements. This variability is currently addressed using external calibration, which limits achievable mass accuracy for MALDI-IMS and makes it difficult to match these data to downstream LC-MS/MS results. To overcome this challenge, the work presented here details a method for internally calibrating data sets generated from tryptic peptide MALDI-IMS on formalin-fixed paraffin-embedded sections of ovarian cancer. By calibrating all spectra to internal peak features the m/z error for matches made between MALDI-IMS m/z values and LC-MS/MS identified peptide m/z values was significantly reduced. This improvement was confirmed by follow up matching of LC-MS/MS spectra to in situ MS/MS spectra from the same m/z peak features. The sum of the data presented here indicates that internal calibrants should be a standard component of tryptic peptide MALDI-IMS experiments.
Publisher: Wiley
Date: 16-10-2019
Abstract: While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man
Publisher: Springer Science and Business Media LLC
Date: 28-07-2013
DOI: 10.1007/S10495-013-0887-6
Abstract: A431 cells, derived from epidermoid carcinoma, overexpress the epidermal growth factor receptor (EGFR) and when treated with a high dose of EGF will undergo apoptosis. We exploited microarray and proteomics techniques and network prediction to study the regulatory mechanisms of EGF-induced apoptosis in A431 cells. We observed significant changes in gene expression in 162 genes, approximately evenly split between pro-apoptotic and anti-apoptotic genes and identified 30 proteins from the proteomic data that had either pro or anti-apoptotic annotation. Our correlation analysis of gene expression and proteome modeled a number of distinct sub-networks that are associated with the onset of apoptosis, allowing us to identify specific pathways and components. These include components of the interferon signalling pathway, and down stream components, including cytokines and suppressors of cytokine signalling. A central component of almost all gene expression sub-networks identified was TP53, which is mutated in A431 cells, and was down regulated. This down regulation of TP53 appeared to be correlated with proteomic sub-networks of cytoskeletal or cell adhesion components that might induce apoptosis by triggering cytochrome C release. Of the only three genes also differentially expressed as proteins, only serpinb1 had a known association with apoptosis. We confirmed that up regulation and cleavage of serpinb1 into L-DNAaseII was correlated with the induction of apoptosis. It is unlikely that a single pathway, but more likely a combination of pathways is needed to trigger EGF induced apoptosis in A431cells.
Publisher: Wiley
Date: 28-01-2011
DOI: 10.1002/IJC.25494
Abstract: Ovarian cancer metastasis is characterized by the shedding of malignant cells from the surface of the ovary and their implantation onto the peritoneal surface, which lines the abdominal cavity. As the factors promoting this process are poorly understood, we investigated the ovarian cancer-peritoneal interaction by means of in vitro coculture experiments with ovarian cancer (OVCAR-5 and SKOV-3) and peritoneal (LP-9) cells. One of the proteins differentially expressed in the coculture secretome was identified by MALDI-TOF/TOF mass spectrometry as the extracellular matrix protein transforming growth factor-beta-induced protein (TGFBIp, also known as βig-H3). Immunohistochemistry showed high TGFBIp levels in normal surface ovarian epithelial and peritoneal cells, whereas TGFBIp levels in primary serous ovarian carcinomas and matching metastatic implants was very low. In functional in vitro experiments, treatment with recombinant TGFBIp significantly increased the motility and invasiveness of OVCAR-5 and SKOV-3 cells and significantly increased ovarian cancer cell (OVCAR-5, OVCAR-3 and SKOV-3) adhesion to LP-9 cells. TGFBIp was found to be processed at both the N- and C-terminus in the secretome of the ovarian cancer-peritoneal cell coculture. Plasmin inhibitors blocked TGFBIp processing and significantly reduced OVCAR-5 cell adhesion to peritoneal cells. We conclude that TGFBIp expressed by peritoneal cells increases the metastatic potential of ovarian cancer cells. TGFBIp is therefore a potential novel therapeutic target against ovarian cancer.
Publisher: Proceedings of the National Academy of Sciences
Date: 19-04-2021
Abstract: The immune system relies on coordinated interactions between motile cells guided by molecules known as chemokines. However, processes that control chemokine distribution in complex in vivo microenvironments are poorly understood. Dendritic cells in barrier tissues require the chemokine CCL21 to enter lymphatic vessels during tissue egress. Here, we demonstrate that ACKR4 shapes CCL21 distribution in barrier tissues and prevents leakage of CCL21 from the tissue. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes lymph nodes. The results increase understanding of regulation of dendritic cell egress and chemokine distribution in vivo and raise new questions regarding the function of solubilized CCL21.
Publisher: MDPI AG
Date: 09-08-2012
DOI: 10.3390/IJMS13089942
Publisher: American Chemical Society (ACS)
Date: 07-2014
DOI: 10.1021/PR500158R
Abstract: Experimental autoimmune encephalomyelitis (EAE) is a murine model of multiple sclerosis, a chronic neurodegenerative and inflammatory autoimmune condition of the central nervous system (CNS). Pathology is driven by the infiltration of autoreactive CD4(+) lymphocytes into the CNS, where they attack neuronal sheaths causing ascending paralysis. We used an isotope-coded protein labeling approach to investigate the proteome of CD4(+) cells isolated from the spinal cord and brain of mice at various stages of EAE progression in two EAE disease models: PLP139-151-induced relapsing-remitting EAE and MOG35-55-induced chronic EAE, which emulate the two forms of human multiple sclerosis. A total of 1120 proteins were quantified across disease onset, peak-disease, and remission phases of disease, and of these 13 up-regulated proteins of interest were identified with functions relating to the regulation of inflammation, leukocyte adhesion and migration, tissue repair, and the regulation of transcription/translation. Proteins implicated in processes such as inflammation (S100A4 and S100A9) and tissue repair (annexin A1), which represent key events during EAE progression, were validated by quantitative PCR. This is the first targeted analysis of autoreactive cells purified from the CNS during EAE, highlighting fundamental CD4(+) cell-driven processes that occur during the initiation of relapse and remission stages of disease.
Publisher: Springer Science and Business Media LLC
Date: 05-08-2019
DOI: 10.1007/S00216-019-02047-Y
Abstract: The application of proteomic liquid chromatography mass spectrometry (LC-MS) for identifying proteins and peptides associated with human disease is rapidly growing in clinical diagnostics. However, the ability to accurately and consistently detect disease-associated peptides remains clinically uncertain. Variability in diagnostic testing occurs in part due to the absence of appropriate reference testing materials and standardised clinical guidelines for proteomic testing. In addition, multiple proteomic testing pipelines have not been fully assessed through external quality assurance (EQA). This trial was therefore devised to evaluate the performance of a small number of mass spectrometry (MS) testing facilities to (i) evaluate the EQA material for potential usage in a proteomic quality assurance program, and to (ii) identify key problem areas associated with human peptide testing. Five laboratories were sent six peptide reference testing s les formulated to contain a total of 35 peptides in differing ratios of light (natural) to heavy (labelled) peptides. Proficiency assessment of laboratory data used a modified approach to similarity and dissimilarity testing that was based on Bray-Curtis and Sorensen indices. Proficiency EQA concordant consensus values could not be derived from the assessed data since none of the laboratories correctly identified all reference testing peptides in all s les. However, the produced data may be reflective of specific inter-laboratory differences for detecting multiple peptides since no two testing pipelines used were the same for any laboratory. In addition, laboratory feedback indicated that peptide filtering of the reference material was a common key problem area prior to analysis. These data highlight the importance of an EQA programme for identifying underlying testing issues so that improvements can be made and confidence for clinical diagnostic analysis can be attained.
Publisher: American Chemical Society (ACS)
Date: 02-04-1999
DOI: 10.1021/AC981178V
Abstract: Continuous- or free-flow electrophoresis is based upon a thin film of fluid flowing between two parallel plates. The electrolytes and the s le are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. Using the Octopus apparatus in a horizontal position, continuous preparative separation of methadone enantiomers in the presence of (2-hydroxypropyl)-β-cyclodextrin as a chiral selector was investigated under conditions of continuous-flow zone electrophoresis and continuous-flow isotachophoresis. The enantiomeric composition of methadone in the collected fractions was assessed by chiral capillary electrophoresis and circular-dichroism spectroscopy. In both electrophoretic modes, partial separation of the two enantiomers with an enrichment of about 80% and a throughput of 10-20 mg of racemic methadone per hour was obtained. Operating the Octopus apparatus with interrupted buffer flow during electrophoresis, a process termed interval-flow electrophoresis, resulted in complete separation of milligram quantities of the two methadone enantiomers. Furthermore, commencing with racemic methadone, continuous multistage isotachophoretic processing is shown to be suitable to purify (R)-(-)-methadone, the enantiomer with higher pharmacological activity, on a mg/h scale and at a mM concentration in the collected product stream.
Publisher: The Optical Society
Date: 03-05-2013
DOI: 10.1364/OE.21.011492
Publisher: MDPI AG
Date: 22-08-2012
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.JPROT.2018.02.023
Abstract: In order to accelerate the understanding of pathophysiological mechanisms and clinical biomarker discovery and in psychiatry, approaches that integrate multiple -omics platforms are needed. We introduce a workflow that investigates a narrowly defined psychiatric phenotype, makes use of the potent and cost-effective discovery technology of gene expression microarrays, applies Weighted Gene Co-Expression Network Analysis (WGCNA) to better capture complex and polygenic traits, and finally explores gene expression findings on the proteomic level using targeted mass-spectrometry (MS) technologies. To illustrate the effectiveness of the workflow, we present a proteomic analysis of peripheral blood plasma from patient's remitted major depressive disorder (MDD) who experience ongoing cognitive deficits. We show that co-expression patterns previous detected on the transcript level could be replicated for plasma proteins, as could the module eigengene correlation with cognitive performance. Further, we demonstrate that functional analysis of multi-omics data has the potential to point to cellular mechanisms and candidate biomarkers for cognitive dysfunction in MDD, implicating cell cycle regulation by cyclin D3 (CCND3), regulation of protein processing in the endoplasmatic reticulum by Thioredoxin domain-containing protein 5 (TXND5), and modulation of inflammatory cytokines by Tripartite Motif Containing 26 (TRI26). This paper discusses how data from multiple -omics platforms can be integrated to accelerate biomarker discovery in psychiatry. Using the phenotype of cognitive impairment in remitted major depressive disorder (MDD) as an ex le, we show that the application of a systems biology approach - weighted gene co-expression network analysis (WGCNA) - in the discovery phase, and targeted proteomic follow-up of results, provides a structured avenue towards uncovering novel candidate markers and pathways for personalized clinical psychiatry.
Publisher: American Association for Cancer Research (AACR)
Date: 15-05-2004
DOI: 10.1158/0008-5472.CAN-03-3731
Abstract: We have identified that StarD10, a member of the START protein family, is overexpressed in both mouse and human breast tumors. StarD10 was initially discovered on the basis of its cross-reactivity with a phosphoserine-specific antibody in mammary tumors from Neu/ErbB2 transgenic mice and subsequently isolated from SKBR3 human breast carcinoma cells using a multistep biochemical purification strategy. We have shown that StarD10 is capable of binding lipids. StarD10 was found to be overexpressed in 35% of primary breast carcinomas and 64% of human breast cancer cell lines, correlating with their ErbB2/Her2 status. Coexpression of StarD10 with ErbB1/epidermal growth factor receptor in murine fibroblasts enhanced anchorage-independent growth in soft agar, providing evidence for functional cooperation between StarD10 and ErbB receptor signaling. Taken together, these data suggest that overexpression of this lipid-binding protein contributes to breast oncogenesis.
Location: Germany
Location: Australia
Start Date: 2009
End Date: 2009
Funder: Australian Research Council
View Funded ActivityStart Date: 2016
End Date: 2018
Funder: Australian Research Council
View Funded ActivityStart Date: 2009
End Date: 2012
Funder: Australian Research Council
View Funded ActivityStart Date: 2011
End Date: 2013
Funder: Australian Research Council
View Funded ActivityStart Date: 2013
End Date: 2013
Funder: Australian Research Council
View Funded ActivityStart Date: 2010
End Date: 2013
Funder: Australian Research Council
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