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1
Srivastava, Himangi, Michael J. Lippincott, Jordan Currie, Robert Canfield, Maggie P. Y. Lam, and Edward Lau. "Protein prediction models support widespread post-transcriptional regulation of protein abundance by interacting partners." PLOS Computational Biology 18, no. 11 (November 10, 2022): e1010702. http://dx.doi.org/10.1371/journal.pcbi.1010702.
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Protein and mRNA levels correlate only moderately. The availability of proteogenomics data sets with protein and transcript measurements from matching samples is providing new opportunities to assess the degree to which protein levels in a system can be predicted from mRNA information. Here we examined the contributions of input features in protein abundance prediction models. Using large proteogenomics data from 8 cancer types within the Clinical Proteomic Tumor Analysis Consortium (CPTAC) data set, we trained models to predict the abundance of over 13,000 proteins using matching transcriptome data from up to 958 tumor or normal adjacent tissue samples each, and compared predictive performances across algorithms, data set sizes, and input features. Over one-third of proteins (4,648) showed relatively poor predictability (elastic net r ≤ 0.3) from their cognate transcripts. Moreover, we found widespread occurrences where the abundance of a protein is considerably less well explained by its own cognate transcript level than that of one or more trans locus transcripts. The incorporation of additional trans-locus transcript abundance data as input features increasingly improved the ability to predict sample protein abundance. Transcripts that contribute to non-cognate protein abundance primarily involve those encoding known or predicted interaction partners of the protein of interest, including not only large multi-protein complexes as previously shown, but also small stable complexes in the proteome with only one or few stable interacting partners. Network analysis further shows a complex proteome-wide interdependency of protein abundance on the transcript levels of multiple interacting partners. The predictive model analysis here therefore supports that protein-protein interaction including in small protein complexes exert post-transcriptional influence on proteome compositions more broadly than previously recognized. Moreover, the results suggest mRNA and protein co-expression analysis may have utility for finding gene interactions and predicting expression changes in biological systems.
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Hortin, Glen L. "The MALDI-TOF Mass Spectrometric View of the Plasma Proteome and Peptidome." Clinical Chemistry 52, no. 7 (July 1, 2006): 1223–37. http://dx.doi.org/10.1373/clinchem.2006.069252.
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Abstract Background: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the related technique, surface-enhanced laser desorption/ionization (SELDI)-TOF MS, are being applied widely to analyze serum or plasma specimens for potential disease markers. Methods: Reports on the basic principles and applications of MALDI-TOF MS were reviewed and related to information on abundance and masses of major plasma proteins. Outcomes: MALDI-TOF MS is a particle-counting method that responds to molar abundance, and ranking of plasma proteins by molar abundance increases the rank of small proteins relative to traditional ranking by mass abundance. Detectors for MALDI-TOF MS augment the bias for detecting smaller components by yielding stronger signals for an equivalent number of small vs large ions. Consequently, MALDI-TOF MS is a powerful tool for surveying small proteins and peptides comprising the peptidome or fragmentome, opening this new realm for analysis. It is complementary to techniques such as electrophoresis and HPLC, which have a bias for detecting larger molecules. Virtually all of the potential markers identified by MALDI-TOF MS to date represent forms of the most abundant plasma proteins. Conclusions: Analyses of serum or plasma by MALDI-TOF MS provide new information mainly about small proteins and peptides with high molar abundance. The spectrum of observed proteins and peptides suggests value for applications such as assessment of cardiovascular risk, nutritional status, liver injury, kidney failure, and systemic immune responses rather than early detection of cancer. Extending analysis by MALDI-TOF MS to lower abundance components, such as markers for early-stage cancers, probably will require more extensive specimen fractionation before analysis.
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Askeland, Anders, Anne Borup, Ole Østergaard, Jesper V. Olsen, Sigrid M. Lund, Gunna Christiansen, Søren R. Kristensen, Niels H. H. Heegaard, and Shona Pedersen. "Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation." Biomedicines 8, no. 8 (July 25, 2020): 246. http://dx.doi.org/10.3390/biomedicines8080246.
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Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both.
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Pachane, Bianca Cruz, Ana Carolina Caetano Nunes, Thais Regiani Cataldi, Kelli Cristina Micocci, Bianca Caruso Moreira, Carlos Alberto Labate, Heloisa Sobreiro Selistre-de-Araujo, and Wanessa Fernanda Altei. "Small Extracellular Vesicles from Hypoxic Triple-Negative Breast Cancer Cells Induce Oxygen-Dependent Cell Invasion." International Journal of Molecular Sciences 23, no. 20 (October 21, 2022): 12646. http://dx.doi.org/10.3390/ijms232012646.
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Hypoxia, a condition of low oxygenation frequently found in triple-negative breast tumors (TNBC), promotes extracellular vesicle (EV) secretion and favors cell invasion, a complex process in which cell morphology is altered, dynamic focal adhesion spots are created, and ECM is remodeled. Here, we investigated the invasive properties triggered by TNBC-derived hypoxic small EV (SEVh) in vitro in cells cultured under hypoxic (1% O2) and normoxic (20% O2) conditions, using phenotypical and proteomic approaches. SEVh characterization demonstrated increased protein abundance and diversity over normoxic SEV (SEVn), with enrichment in pro-invasive pathways. In normoxic cells, SEVh promotes invasive behavior through pro-migratory morphology, invadopodia development, ECM degradation, and matrix metalloprotease (MMP) secretion. The proteome profiling of 20% O2-cultured cells exposed to SEVh determined enrichment in metabolic processes and cell cycles, modulating cell health to escape apoptotic pathways. In hypoxia, SEVh was responsible for proteolytic and catabolic pathway inducement, interfering with integrin availability and gelatinase expression. Overall, our results demonstrate the importance of hypoxic signaling via SEV in tumors for the early establishment of metastasis.
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Hortin, Glen L., Denis Sviridov, and N. Leigh Anderson. "High-Abundance Polypeptides of the Human Plasma Proteome Comprising the Top 4 Logs of Polypeptide Abundance." Clinical Chemistry 54, no. 10 (October 1, 2008): 1608–16. http://dx.doi.org/10.1373/clinchem.2008.108175.
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Abstract Background: Plasma contains thousands of proteins, but a small number of these proteins comprise the majority of protein molecules and mass. Content: We surveyed proteomic studies to identify candidates for high-abundance polypeptide chains. We searched the literature for information on the plasma concentrations of the most abundant components in healthy adults and for the molecular mass of the mature polypeptide chains in plasma. Because proteomic studies usually dissociate proteins into polypeptide chains or detect short peptide segments of proteins, we summarized data on individual peptide chains for proteins containing multiple subunits or polypeptides. We collected data on about 150 of the most abundant polypeptides in plasma. The abundant polypeptides span approximately the top 4 logs of concentration in plasma, from 650 to 0.06 μmol/L on a molar basis or from about 50 000 to 1 mg/L mass abundance. Conclusions: Data on the concentrations of the high-abundance peptide chains in plasma assist in understanding the composition of plasma and potential approaches for clinical laboratory or proteomic analysis of plasma proteins. Development of more extensive databases regarding the plasma concentrations of proteins in health and diseases would promote diagnostic and proteomic advances.
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Stöckl, Jan B., Nina Schmid, Florian Flenkenthaler, Charis Drummer, Rüdiger Behr, Artur Mayerhofer, Georg J. Arnold, and Thomas Fröhlich. "Age-Related Alterations in the Testicular Proteome of a Non-Human Primate." Cells 10, no. 6 (May 24, 2021): 1306. http://dx.doi.org/10.3390/cells10061306.
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Aging of human testis and associated cellular changes is difficult to assess. Therefore, we used a translational, non-human primate model to get insights into underlying cellular and biochemical processes. Using proteomics and immunohistochemistry, we analyzed testicular tissue of young (age 2 to 3) and old (age 10 to 12) common marmosets (Callithrix jacchus). Using a mass spectrometry-based proteomics approach, we identified 63,124 peptides, which could be assigned to 5924 proteins. Among them, we found proteins specific for germ cells and somatic cells, such as Leydig and Sertoli cells. Quantitative analysis showed 31 differentially abundant proteins, of which 29 proteins were more abundant in older animals. An increased abundance of anti-proliferative proteins, among them CDKN2A, indicate reduced cell proliferation in old testes. Additionally, an increased abundance of several small leucine rich repeat proteoglycans and other extracellular matrix proteins was observed, which may be related to impaired cell migration and fibrotic events. Furthermore, an increased abundance of proteins with inhibitory roles in smooth muscle cell contraction like CNN1 indicates functional alterations in testicular peritubular cells and may mirror a reduced capacity of these cells to contract in old testes.
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van Ginkel, Jetty, Mike Filius, Malwina Szczepaniak, Pawel Tulinski, Anne S. Meyer, and Chirlmin Joo. "Single-molecule peptide fingerprinting." Proceedings of the National Academy of Sciences 115, no. 13 (March 12, 2018): 3338–43. http://dx.doi.org/10.1073/pnas.1707207115.
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Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.
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Chan, Eric Y., Jennifer N. Sutton, Jon M. Jacobs, Andrey Bondarenko, Richard D. Smith, and Michael G. Katze. "Dynamic Host Energetics and Cytoskeletal Proteomes in Human Immunodeficiency Virus Type 1-Infected Human Primary CD4 Cells: Analysis by Multiplexed Label-Free Mass Spectrometry." Journal of Virology 83, no. 18 (July 8, 2009): 9283–95. http://dx.doi.org/10.1128/jvi.00814-09.
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ABSTRACT We report on a proteomic analysis of ex vivo human immunodeficiency virus (HIV) type 1 infection in human primary CD4 cells by shotgun liquid chromatography-tandem mass spectrometry analysis, revealing two distinct proteomic profiles at two phases of virus replication. Relative to mock-infected cells, 168 signature proteins exhibited abundance changes at the first sign of Gag p24 production (8 h postinfection [p.i.]) or the peak of virus replication (24 h p.i.); interestingly, most of the changes were exclusive to only one phase of virus replication. Based on characterization by functional ontology and known human-HIV protein interactions, we observed the enrichment for protein abundance increases pertaining to protein synthesis and nucleasomal reorganization amid an otherwise placid cellular proteome at the first sign of HIV replication. In contrast, we observed indications of decreased protein turnover, concomitant with heightened DNA repair activities and preludes to apoptosis, in the presence of robust virus replication. We also observed hints of disruptions in protein and small molecule trafficking. Our label-free proteomic strategy allowed us to perform multiplexed comparisons—we buttressed our detection specificity with the use of a reverse transcriptase inhibitor as a counterscreen, enabling highlighting of cellular protein abundance changes unique to robust virus replication as opposed to viral entry. In conjunction with complementary high-throughput screens for cellular partners of HIV, we put forth a model pinpointing specific rerouting of cellular biosynthetic, energetic, and trafficking pathways as HIV replication accelerates in human primary CD4 cells.
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Cui, Yanjun, and Xianhong Gu. "Proteomic changes of the porcine small intestine in response to chronic heat stress." Journal of Molecular Endocrinology 55, no. 3 (September 28, 2015): 277–93. http://dx.doi.org/10.1530/jme-15-0161.
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Acute heat stress (HS) negatively affects intestinal integrity and barrier function. In contrast, chronic mild HS poses a distinct challenge to animals. Therefore, this study integrates biochemical, histological and proteomic approaches to investigate the effects of chronic HS on the intestine in finishing pigs. Castrated male crossbreeds (79.00±1.50 kg BW) were subjected to either thermal neutral (TN, 21 °C; 55%±5% humidity; n=8) or HS conditions (30 °C; 55%±5% humidity; n=8) for 3 weeks. The pigs were sacrificed after 3 weeks of high environmental exposure and the plasma hormones, the intestinal morphology, integrity, and protein profiles of the jejunum mucosa were determined. Chronic HS reduced the free triiodothyronine (FT3) and GH levels. HS damaged intestinal morphology, increased plasma d-lactate concentrations and decreased alkaline phosphatase activity of intestinal mucosa. Proteome analysis of the jejunum mucosa was conducted by 2D gel electrophoresis and mass spectrometry. Fifty-three intestinal proteins were found to be differentially abundant, 18 of which were related to cell structure and motility, and their changes in abundance could comprise intestinal integrity and function. The down-regulation of proteins involved in tricarboxylic acid cycle (TCA cycle), electron transport chain (ETC), and oxidative phosphorylation suggested that chronic HS impaired energy metabolism and thus induced oxidative stress. Moreover, the changes of ten proteins in abundance related to stress response and defense indicated pigs mediated long-term heat exposure and counteracted its negative effects of heat exposure. These findings have important implications for understanding the effect of chronic HS on intestines.
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Donovan, Margaret, Henry Huang, John Blume, Marwin Ko, Ryan Benz, Theodore Platt, Juan Cuevas, Serafim Batzoglou, Asim Siddiqui, and Omid Farokhzad. "Abstract 6340: Deep, unbiased and peptide-centric plasma proteomics with differential analysis of proteoforms enabling proteogenomic studies of NSCLC at scale." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6340. http://dx.doi.org/10.1158/1538-7445.am2022-6340.
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Abstract Introduction: Comprehensive assessment of the proteome remains elusive because of proteoforms arising from alternative splicing, allelic variation, and protein modifications. Characterization of the variable protein forms, or proteoforms will expand our understanding of the molecular mechanisms underlying diseases, however requires unbiased protein coverage at sufficient scale. Scalable, deep and unbiased proteomics studies have been impractical due to cumbersome and lengthy workflows required for complex samples, like blood plasma. Here, we demonstrate the power of Proteograph in a proof-of-concept proteogenomic analysis of 80 healthy controls and 61 early-stage non-small-cell lung cancer (NSCLC) samples to dissect differences between protein isoforms arising from alternative gene splicing, as well as the identification of novel peptides arising from allelic variation. Materials, Methods and Results: Processing the 141 plasma samples with Proteograph yielded 21,959 peptides corresponding to 2,499 protein groups. Using peptides with significant abundance differences (p < 0.05; Benjamini-Hochberg corrected), we extracted proteins comprised of peptides where at least one peptide had significantly higher plasma abundance, and another significantly lower plasma abundance in controls vs. cancer, resulting in a set of putative proteoforms. For three of these proteins, the abundance variation is possibly explained by underlying protein isoforms. To identify protein variants, we performed exome sequencing on 29 individuals from the NSCLC study, created personalized mass spectrometry search libraries for each individual, and identified 464 protein variants. Conclusions: Proteograph can generate unbiased and deep plasma proteome profiles that enable identification of protein variants and peptides present in plasma, at a scale sufficient to enable population-scale proteomic studies. Citation Format: Margaret Donovan, Henry Huang, John Blume, Marwin Ko, Ryan Benz, Theodore Platt, Juan Cuevas, Serafim Batzoglou, Asim Siddiqui, Omid Farokhzad. Deep, unbiased and peptide-centric plasma proteomics with differential analysis of proteoforms enabling proteogenomic studies of NSCLC at scale [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6340.
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Senavirathna, Lakmini, Cheng Ma, Ru Chen, and Sheng Pan. "Spectral Library-Based Single-Cell Proteomics Resolves Cellular Heterogeneity." Cells 11, no. 15 (August 7, 2022): 2450. http://dx.doi.org/10.3390/cells11152450.
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Dissecting the proteome of cell types and states at single-cell resolution, while being highly challenging, has significant implications in basic science and biomedicine. Mass spectrometry (MS)-based single-cell proteomics represents an emerging technology for system-wide, unbiased profiling of proteins in single cells. However, significant challenges remain in analyzing an extremely small amount of proteins collected from a single cell, as a proteome-wide amplification of proteins is not currently feasible. Here, we report an integrated spectral library-based single-cell proteomics (SLB-SCP) platform that is ultrasensitive and well suited for a large-scale analysis. To overcome the low MS/MS signal intensity intrinsically associated with a single-cell analysis, this approach takes an alternative approach by extracting a breadth of information that specifically defines the physicochemical characteristics of a peptide from MS1 spectra, including monoisotopic mass, isotopic distribution, and retention time (hydrophobicity), and uses a spectral library for proteomic identification. This conceptually unique MS platform, coupled with the DIRECT sample preparation method, enabled identification of more than 2000 proteins in a single cell to distinguish different proteome landscapes associated with cellular types and heterogeneity. We characterized individual normal and cancerous pancreatic ductal cells (HPDE and PANC-1, respectively) and demonstrated the substantial difference in the proteomes between HPDE and PANC-1 at the single-cell level. A significant upregulation of multiple protein networks in cancer hallmarks was identified in the PANC-1 cells, functionally discriminating the PANC-1 cells from the HPDE cells. This integrated platform can be built on high-resolution MS and widely accepted proteomic software, making it possible for community-wide applications.
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Völlmy, Franziska, Henk van den Toorn, Riccardo Zenezini Chiozzi, Ottavio Zucchetti, Alberto Papi, Carlo Alberto Volta, Luisa Marracino, et al. "A serum proteome signature to predict mortality in severe COVID-19 patients." Life Science Alliance 4, no. 9 (July 5, 2021): e202101099. http://dx.doi.org/10.26508/lsa.202101099.
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Here, we recorded serum proteome profiles of 33 severe COVID-19 patients admitted to respiratory and intensive care units because of respiratory failure. We received, for most patients, blood samples just after admission and at two more later time points. With the aim to predict treatment outcome, we focused on serum proteins different in abundance between the group of survivors and non-survivors. We observed that a small panel of about a dozen proteins were significantly different in abundance between these two groups. The four structurally and functionally related type-3 cystatins AHSG, FETUB, histidine-rich glycoprotein, and KNG1 were all more abundant in the survivors. The family of inter-α-trypsin inhibitors, ITIH1, ITIH2, ITIH3, and ITIH4, were all found to be differentially abundant in between survivors and non-survivors, whereby ITIH1 and ITIH2 were more abundant in the survivor group and ITIH3 and ITIH4 more abundant in the non-survivors. ITIH1/ITIH2 and ITIH3/ITIH4 also showed opposite trends in protein abundance during disease progression. We defined an optimal panel of nine proteins for mortality risk assessment. The prediction power of this mortality risk panel was evaluated against two recent COVID-19 serum proteomics studies on independent cohorts measured in other laboratories in different countries and observed to perform very well in predicting mortality also in these cohorts. This panel may not be unique for COVID-19 as some of the proteins in the panel have previously been annotated as mortality markers in aging and in other diseases caused by different pathogens, including bacteria.
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Rodríguez-Piñeiro, Ana M., Joakim H. Bergström, Anna Ermund, Jenny K. Gustafsson, André Schütte, Malin E. V. Johansson, and Gunnar C. Hansson. "Studies of mucus in mouse stomach, small intestine, and colon. II. Gastrointestinal mucus proteome reveals Muc2 and Muc5ac accompanied by a set of core proteins." American Journal of Physiology-Gastrointestinal and Liver Physiology 305, no. 5 (September 1, 2013): G348—G356. http://dx.doi.org/10.1152/ajpgi.00047.2013.
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The mucus that protects the surface of the gastrointestinal tract is rich in specialized O-glycoproteins called mucins, but little is known about other mucus proteins or their variability along the gastrointestinal tract. To ensure that only mucus was analyzed, we combined collection from explant tissues mounted in perfusion chambers, liquid sample preparation, single-shot mass spectrometry, and specific bioinformatics tools, to characterize the proteome of the murine mucus from stomach to distal colon. With our approach, we identified ∼1,300 proteins in the mucus. We found no differences in the protein composition or abundance between sexes, but there were clear differences in mucus along the tract. Noticeably, mucus from duodenum showed similarities to the stomach, probably reflecting the normal distal transport. Qualitatively, there were, however, fewer differences than might had been anticipated, suggesting a relatively stable core proteome (∼80% of the total proteins identified). Quantitatively, we found significant differences (∼40% of the proteins) that could reflect mucus specialization throughout the gastrointestinal tract. Hierarchical clustering pinpointed a number of such proteins that correlated with Muc2 (e.g., Clca1, Zg16, Klk1). This study provides a deeper knowledge of the gastrointestinal mucus proteome that will be important in further understanding this poorly studied mucosal protection system.
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Hindle, Allyson G., Katharine R. Grabek, L. Elaine Epperson, Anis Karimpour-Fard, and Sandra L. Martin. "Metabolic changes associated with the long winter fast dominate the liver proteome in 13-lined ground squirrels." Physiological Genomics 46, no. 10 (May 15, 2014): 348–61. http://dx.doi.org/10.1152/physiolgenomics.00190.2013.
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Small-bodied hibernators partition the year between active homeothermy and hibernating heterothermy accompanied by fasting. To define molecular events underlying hibernation that are both dependent and independent of fasting, we analyzed the liver proteome among two active and four hibernation states in 13-lined ground squirrels. We also examined fall animals transitioning between fed homeothermy and fasting heterothermy. Significantly enriched pathways differing between activity and hibernation were biased toward metabolic enzymes, concordant with the fuel shifts accompanying fasting physiology. Although metabolic reprogramming to support fasting dominated these data, arousing (rewarming) animals had the most distinct proteome among the hibernation states. Instead of a dominant metabolic enzyme signature, torpor-arousal cycles featured differences in plasma proteins and intracellular membrane traffic and its regulation. Phosphorylated NSFL1C, a membrane regulator, exhibited this torpor-arousal cycle pattern; its role in autophagosome formation may promote utilization of local substrates upon metabolic reactivation in arousal. Fall animals transitioning to hibernation lagged in their proteomic adjustment, indicating that the liver is more responsive than preparatory to the metabolic reprogramming of hibernation. Specifically, torpor use had little impact on the fall liver proteome, consistent with a dominant role of nutritional status. In contrast to our prediction of reprogramming the transition between activity and hibernation by gene expression and then within-hibernation transitions by posttranslational modification (PTM), we found extremely limited evidence of reversible PTMs within torpor-arousal cycles. Rather, acetylation contributed to seasonal differences, being highest in winter (specifically in torpor), consistent with fasting physiology and decreased abundance of the mitochondrial deacetylase, SIRT3.
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Wang, Liang, Jianye Yang, Yaping Xu, Xue Piao, and Jichang Lv. "Domain-based Comparative Analysis of Bacterial Proteomes: Uniqueness, Interactions, and the Dark Matter." Current Genomics 20, no. 2 (May 22, 2019): 115–23. http://dx.doi.org/10.2174/1389202920666190320134438.
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Background: Proteins may have none, single, double, or multiple domains, while a single domain may appear in multiple proteins. Their distribution patterns may have impacts on bacterial physiology and lifestyle. Objective: This study aims to understand how domains are distributed and duplicated in bacterial proteomes, in order to better understand bacterial physiology and lifestyles. Methods: In this study, we used 16712 Hidden Markov Models to screen 944 bacterial reference proteomes versus a threshold E-value<0.001. The number of non-redundant domains and duplication rates of redundant domains for each species were calculated. The unique domains, if any, were also identified for each species. In addition, the properties of no-domain proteins were investigated in terms of physicochemical properties. Results: The increasing number of non-redundant domains for a bacterial proteome follows the trend of an asymptotic function. The domain duplication rate is positively correlated with proteome size and increases more rapidly. The high percentage of single-domain proteins is more associated with small proteome size. For each proteome, unique domains were also obtained. Moreover, no-domain proteins show differences with the other three groups for several physicochemical properties analysed in this study. Conclusion: The study confirmed that a low domain duplication rate and a high percentage of singledomain proteins are more likely to be associated with bacterial host-dependent or restricted nicheadapted lifestyle. In addition, the unique lifestyle and physiology were revealed based on the analysis of species-specific domains and core domain interactions or co-occurrences.
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Vann, Christopher G., Paul A. Roberson, Shelby C. Osburn, Petey W. Mumford, Matthew A. Romero, Carlton D. Fox, Johnathon H. Moore, et al. "Skeletal Muscle Myofibrillar Protein Abundance Is Higher in Resistance-Trained Men, and Aging in the Absence of Training May Have an Opposite Effect." Sports 8, no. 1 (January 10, 2020): 7. http://dx.doi.org/10.3390/sports8010007.
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Resistance training generally increases skeletal muscle hypertrophy, whereas aging is associated with a loss in muscle mass. Interestingly, select studies suggest that aging, as well as resistance training, may lead to a reduction in the abundance of skeletal muscle myofibrillar (or contractile) protein (per mg tissue). Proteomic interrogations have also demonstrated that aging, as well as weeks to months of resistance training, lead to appreciable alterations in the muscle proteome. Given this evidence, the purpose of this small pilot study was to examine total myofibrillar as well as total sarcoplasmic protein concentrations (per mg wet muscle) from the vastus lateralis muscle of males who were younger and resistance-trained (denoted as YT, n = 6, 25 ± 4 years old, 10 ± 3 self-reported years of training), younger and untrained (denoted as YU, n = 6, 21 ± 1 years old), and older and untrained (denoted as OU, n = 6, 62 ± 8 years old). The relative abundances of actin and myosin heavy chain (per mg tissue) were also examined using SDS-PAGE and Coomassie staining, and shotgun proteomics was used to interrogate the abundances of individual sarcoplasmic and myofibrillar proteins between cohorts. Whole-body fat-free mass (YT > YU = OU), VL thickness (YT > YU = OU), and leg extensor peak torque (YT > YU = OU) differed between groups (p < 0.05). Total myofibrillar protein concentrations were greater in YT versus OU (p = 0.005), but were not different between YT versus YU (p = 0.325). The abundances of actin and myosin heavy chain were greater in YT versus YU (p < 0.05) and OU (p < 0.001). Total sarcoplasmic protein concentrations were not different between groups. While proteomics indicated that marginal differences existed for individual myofibrillar and sarcoplasmic proteins between YT versus other groups, age-related differences were more prominent for myofibrillar proteins (YT = YU > OU, p < 0.05: 7 proteins; OU > YT = YU, p < 0.05: 11 proteins) and sarcoplasmic proteins (YT = YU > OU, p < 0.05: 8 proteins; OU > YT&YU, p < 0.05: 29 proteins). In summary, our data suggest that modest (~9%) myofibrillar protein packing (on a per mg muscle basis) was evident in the YT group. This study also provides further evidence to suggest that notable skeletal muscle proteome differences exist between younger and older humans. However, given that our n-sizes are low, these results only provide a preliminary phenotyping of the reported protein and proteomic variables.
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Piragasam, Ramanaguru S., S. Faraz Hussain, Steven G. Chaulk, Zaeem A. Siddiqi, and Richard P. Fahlman. "Label-free proteomic analysis reveals large dynamic changes to the cellular proteome upon expression of the miRNA-23a-27a-24-2 microRNA cluster." Biochemistry and Cell Biology 98, no. 1 (February 2020): 61–69. http://dx.doi.org/10.1139/bcb-2019-0014.
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In deciphering the regulatory networks of gene expression controlled by the small non-coding RNAs known as microRNAs (miRNAs), a major challenge has been with the identification of the true mRNA targets by these RNAs within the context of the enormous numbers of predicted targets for each of these small RNAs. To facilitate the system-wide identification of miRNA targets, a variety of system wide methods, such as proteomics, have been implemented. Here we describe the utilization of quantitative label-free proteomics and bioinformatics to identify the most significant changes to the proteome upon expression of the miR-23a-27a-24-2 miRNA cluster. In light of recent work leading to the hypothesis that only the most pronounced regulatory events by miRNAs may be physiologically relevant, our data reveal that label-free analysis circumvents the limitations of proteomic labeling techniques that limit the maximum differences that can be quantified. The result of our analysis identifies a series of novel candidate targets that are reduced in abundance by more than an order of magnitude upon the expression of the miR-23a-27a-24-2 cluster.
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Mukhopadhyay, Aindrila, Alyssa M. Redding, Marcin P. Joachimiak, Adam P. Arkin, Sharon E. Borglin, Paramvir S. Dehal, Romy Chakraborty, et al. "Cell-Wide Responses to Low-Oxygen Exposure in Desulfovibrio vulgaris Hildenborough." Journal of Bacteriology 189, no. 16 (June 1, 2007): 5996–6010. http://dx.doi.org/10.1128/jb.00368-07.
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ABSTRACT The responses of the anaerobic, sulfate-reducing organism Desulfovibrio vulgaris Hildenborough to low-oxygen exposure (0.1% O2) were monitored via transcriptomics and proteomics. Exposure to 0.1% O2 caused a decrease in the growth rate without affecting viability. Concerted upregulation of the predicted peroxide stress response regulon (PerR) genes was observed in response to the 0.1% O2 exposure. Several of the candidates also showed increases in protein abundance. Among the remaining small number of transcript changes was the upregulation of the predicted transmembrane tetraheme cytochrome c 3 complex. Other known oxidative stress response candidates remained unchanged during the low-O2 exposure. To fully understand the results of the 0.1% O2 exposure, transcriptomics and proteomics data were collected for exposure to air using a similar experimental protocol. In contrast to the 0.1% O2 exposure, air exposure was detrimental to both the growth rate and viability and caused dramatic changes at both the transcriptome and proteome levels. Interestingly, the transcripts of the predicted PerR regulon genes were downregulated during air exposure. Our results highlight the differences in the cell-wide responses to low and high O2 levels in D. vulgaris and suggest that while exposure to air is highly detrimental to D. vulgaris, this bacterium can successfully cope with periodic exposure to low O2 levels in its environment.
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Filipecki, Marcin, Marek Żurczak, Mateusz Matuszkiewicz, Magdalena Święcicka, Wojciech Kurek, Jarosław Olszewski, Marek Daniel Koter, Douglas Lamont, and Mirosław Sobczak. "Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots." International Journal of Molecular Sciences 22, no. 22 (November 10, 2021): 12147. http://dx.doi.org/10.3390/ijms222212147.
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Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100–200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.
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Wang, Xiangyu, Xiaofei Guo, Xiaoyun He, Ran Di, Xiaosheng Zhang, Jinlong Zhang, and Mingxing Chu. "Integrated Proteotranscriptomics of the Hypothalamus Reveals Altered Regulation Associated with the FecB Mutation in the BMPR1B Gene that Affects Prolificacy in Small Tail Han Sheep." Biology 12, no. 1 (December 30, 2022): 72. http://dx.doi.org/10.3390/biology12010072.
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The litter size and ovulation rate are different among ewes of different FecB genotypes in Small Tail Han sheep. These variants in reproductive phenotypes may be regulated by hormones released by the hypothalamic–pituitary–ovarian axis. However, there have been few reports on the hypothalamus regarding regulating an increase in ovulation in sheep with FecB mutation at different estrous stages. Thus, we examined the abundance of hypothalamus tissue protein profiles of six FecB mutant homozygous (BB) and six wild-type (WW) ewes at the luteal and follicular phases. We determined this abundance by tandem mass tag-based quantitative analysis and parallel reaction monitoring methods. Furthermore, an integrated proteotranscriptomic analysis was performed by the Data Integration Analysis for Biomarker discovery using the latent variable approaches for Omics studies (DIABLO) framework to examine biological processes and pathway alterations by the FecB mutant. The abundance of 154 proteins was different between the two estrous stages. Growth hormone and prolactin were particularly enriched in the neuroactive ligand–receptor interactions, the prolactin signaling pathway, and the PI3K-Akt signaling pathway which are related to hypothalamic function and reproduction. We combined proteome and transcriptome data from different estrous stages and genotypes. There is a high correlation (Pearson correlation coefficient = 0.99) between the two datasets in the first two components. We applied the traditional single-omic multivariate approach to obtain differentially abundant proteins and differentially expressed genes. The major fertility related biomarkers were selected using the two approaches mentioned above. Several key pathways (GABAergic synapse, neuroactive ligand–receptor interaction, estrogen and MAPK signaling pathways) were enriched, which are central to gonadotrophin-releasing hormone (GnRH) secretion and reproduction. A higher level of gamma-aminobutyric acid type A receptor subunit alpha1 (GABRA1) and gamma-aminobutyric acid type A receptor subunit beta2 (GABRB2) expression was observed in BB ewes as compared to WW ewes. This finding suggested that a greater production of GnRH during follicular development in BB ewes may explain the higher mature follicle number in mutant ewes. FKBP prolyl isomerase 1A (FKBP1A), which was a major feature factor in the proteome selected by DIABLO, was an important switch for activating the transforming growth factor beta (TGFβ) pathway, and its expression was higher in the WW ewes than in the BB ewes. We suggest that BB sheep maintain TGFβ pathway activity by reducing FKBP1A protein abundance. This innovative data integration in the hypothalamus may provide fresh insight into the mechanisms by which the FecB mutation affects sheep fertility, while providing novel biomarkers related to reproductive endocrinology in sheep breeding.
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Arcia, David, Denise S. Boulanger, Edward N. James, and Tim Elliott. "Integration of affinity and abundance parameters for the identification of novel CD8+ T cell specificities in the CT26 tumor model." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 104.11. http://dx.doi.org/10.4049/jimmunol.206.supp.104.11.
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Abstract The CT26 model is one of the most widely tested systems for cancer immunotherapy. Over 70% of the CD8+ CTL responses in CT26 tumors are directed against two peptides: SPSYVYHQF (AH1) presented by H-2Ld, or GGPESFYCASW (GSW11) presented by H-2Dd. Both peptides derive from the ecotropic murine leukaemia virus gp70 envelope glycoprotein, which is the highest expressed gene in CT26, and have relative low affinity values for their respective MHC-I. However, there is a lack of knowledge about other CTL specificities in CT26, the relationship between abundance and affinity of such peptides, and their roles in tumor rejection. We identified 96 potential epitopes restricted by MHC-I molecules in BALB/c mice based on previously published immuno-transcriptomic data. We used a mathematical algorithm integrating the peptides relative affinity to MHC-I measured by NetMHC4.0, and their abundance in the proteome, which resulted in a prediction of the likelihood of peptide presentation. Screening was performed in splenocytes from Treg-depleted CT26-challenged mice via IFNγ intracellular cytokine staining and T cell proliferation assays. Our approach led to the identification of a novel CTL specificity restricted by H-2Kd (Kd34), which shows moderate IFNγ responses in splenocytes from mice responding to the therapy. Moreover, using Kd34-specific dextramers, we were able to detect a small population of tumor-infiltrating lymphocytes (TILs) specific for this peptide, with similar phenotypic traits to AH1- and GSW1-specific TILs. The identification of such novel CD8+ T cell specificities will improve the understanding about the relationship between immunodominance and epitope abundance/affinity to better tailor vaccine strategies in cancer.
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Barnholtz-Sloan, Jill S. "BIOM-20. PROTEOMICS PROVIDES POTENTIAL THERAPEUTIC APPROACHES TO GLIOBLASTOMA TREATMENT: RESULTS FROM THE CLINICAL PROTEOMICS TUMOR ANALYSIS CONSORTIUM (CPTAC)." Neuro-Oncology 22, Supplement_2 (November 2020): ii6. http://dx.doi.org/10.1093/neuonc/noaa215.020.
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Abstract Glioblastoma (GBM) is the most common type of primary malignant tumor in adults and contributes disproportionately to cancer morbidity and mortality. The ability to augment current knowledge based on the genome and transcriptome with information regarding protein abundance and post-translational modifications with small molecule and lipid abundance has provided novel biological insights into GBM tumorigenesis. In this presentation we will discuss results from proteogenomic and metabolomic investigations of prospectively recruited, newly diagnosed, treatment-naive glioblastomas (GBMs; N=99). Multiple key findings emerged that were not known from traditional genomic analyses as implemented in most currently available datasets. Analysis of protein phosphorylation identified key signaling intermediates in the RTK/RAS pathway common to multiple RTK genomic alterations, potentially offering common therapeutic targets for different oncogenic drivers in GBM. Phosphoproteomics also identified potential druggable targets based on kinase-substrate pathway analysis, as well as novel phosphoprotein targets associated with the regulation of telomere length by ATRX in IDH mutants. Identification of distinct Immune High and Immune Low phenotypes in GBM from whole genome interrogation of the proteome were driven by tumor-associated macrophage markers, and associated with distinct epigenetic modifications and histone acetylation patterns. Identification of key metabolic changes in IDH mutants facilitating the accumulation of oncometabolite 2-HG were also associated with lipidomic changes. Taken all together this work identifies additional therapeutic channels for GBM and novel information useful for more accurate stratification patients for effective treatment.
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Joost, Sarah, Stefan Mikkat, Michael Wille, Antje Schümann, and Oliver Schmitt. "Membrane Protein Identification in Rodent Brain Tissue Samples and Acute Brain Slices." Cells 8, no. 5 (May 8, 2019): 423. http://dx.doi.org/10.3390/cells8050423.
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Acute brain slices are a sample format for electrophysiology, disease modeling, and organotypic cultures. Proteome analyses based on mass spectrometric measurements are seldom used on acute slices, although they offer high-content protein analyses and explorative approaches. In neuroscience, membrane proteins are of special interest for proteome-based analysis as they are necessary for metabolic, electrical, and signaling functions, including myelin maintenance and regeneration. A previously published protocol for the enrichment of plasma membrane proteins based on aqueous two-phase polymer systems followed by mass spectrometric protein identification was adjusted to the small sample size of single acute murine slices from newborn animals and the reproducibility of the results was analyzed. For this, plasma membrane proteins of 12 acute slice samples from six animals were enriched and analyzed by liquid chromatography-mass spectrometry. A total of 1161 proteins were identified, of which 369 were assigned to membranes. Protein abundances showed high reproducibility between samples. The plasma membrane protein separation protocol can be applied to single acute slices despite the low sample size and offers a high yield of identifiable proteins. This is not only the prerequisite for proteome analysis of organotypic slice cultures but also allows for the analysis of small-sized isolated brain regions at the proteome level.
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Burat, Bastien, Audrey Reynaerts, Dominique Baiwir, Maximilien Fléron, Sophie Gohy, Gauthier Eppe, Teresinha Leal, and Gabriel Mazzucchelli. "Sweat Proteomics in Cystic Fibrosis: Discovering Companion Biomarkers for Precision Medicine and Therapeutic Development." Cells 11, no. 15 (July 31, 2022): 2358. http://dx.doi.org/10.3390/cells11152358.
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In clinical routine, the diagnosis of cystic fibrosis (CF) is still challenging regardless of international consensus on diagnosis guidelines and tests. For decades, the classical Gibson and Cooke test measuring sweat chloride concentration has been a keystone, yet, it may provide normal or equivocal results. As of now, despite the combination of sweat testing, CFTR genotyping, and CFTR functional testing, a small fraction (1–2%) of inconclusive diagnoses are reported and justifies the search for new CF biomarkers. More importantly, in the context of precision medicine, with a view to early diagnosis, better prognosis, appropriate clinical follow-up, and new therapeutic development, discovering companion biomarkers of CF severity and phenotypic rescue are of utmost interest. To date, previous sweat proteomic studies have already documented disease-specific variations of sweat proteins (e.g., in schizophrenia and tuberculosis). In the current study, sweat samples from 28 healthy control subjects and 14 patients with CF were analyzed by nanoUHPLC-Q-Orbitrap-based shotgun proteomics, to look for CF-associated changes in sweat protein composition and abundance. A total of 1057 proteins were identified and quantified at an individual level, by a shotgun label-free approach. Notwithstanding similar proteome composition, enrichment, and functional annotations, control and CF samples featured distinct quantitative proteome profiles significantly correlated with CF, accounting for the respective inter-individual variabilities of control and CF sweat. All in all: (i) 402 sweat proteins were differentially abundant between controls and patients with CF, (ii) 68 proteins varied in abundance between F508del homozygous patients and patients with another genotype, (iii) 71 proteins were differentially abundant according to the pancreatic function, and iv) 54 proteins changed in abundance depending on the lung function. The functional annotation of pathophysiological biomarkers highlighted eccrine gland cell perturbations in: (i) protein biosynthesis and trafficking, (ii) CFTR proteostasis and membrane stability, and (iii) cell-cell adherence, membrane integrity, and cytoskeleton crosstalk. Cytoskeleton-related biomarkers were of utmost interest because of the consistency between variations observed here in CF sweat and variations previously documented in other CF tissues. From a clinical stance, nine candidate biomarkers of CF diagnosis (CUTA, ARG1, EZR, AGA, FLNA, MAN1A1, MIA3, LFNG, SIAE) and seven candidate biomarkers of CF severity (ARG1, GPT, MDH2, EML4 (F508del homozygous), MGAT1 (pancreatic insufficiency), IGJ, TOLLIP (lung function impairment)) were deemed suitable for further verification.
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Mosen, Peter, Anne Sanner, Jasjot Singh, and Dominic Winter. "Targeted Quantification of the Lysosomal Proteome in Complex Samples." Proteomes 9, no. 1 (January 26, 2021): 4. http://dx.doi.org/10.3390/proteomes9010004.
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In eukaryotic cells, lysosomes play a crucial role in the breakdown of a variety of components ranging from small molecules to complex structures, ascertaining the continuous turnover of cellular building blocks. Furthermore, they act as a regulatory hub for metabolism, being crucially involved in the regulation of major signaling pathways. Currently, ~450 lysosomal proteins can be reproducibly identified in a single cell line by mass spectrometry, most of which are low-abundant, restricting their unbiased proteomic analysis to lysosome-enriched fractions. In the current study, we applied two strategies for the targeted investigation of the lysosomal proteome in complex samples: data-independent acquisition (DIA) and parallel reaction monitoring (PRM). Using a lysosome-enriched fraction, mouse embryonic fibroblast whole cell lysate, and mouse liver whole tissue lysate, we investigated the capabilities of DIA and PRM to investigate the lysosomal proteome. While both approaches identified and quantified lysosomal proteins in all sample types, and their data largely correlated, DIA identified on average more proteins, especially for lower complex samples and longer chromatographic gradients. For the highly complex tissue sample and shorter gradients, however, PRM delivered a better performance regarding both identification and quantification of lysosomal proteins. All data are available via ProteomeXchange with identifier PXDD023278.
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Leon, Ramon G., Diane C. Bassham, and Micheal D. K. Owen. "Germination and proteome analyses reveal intraspecific variation in seed dormancy regulation in common waterhemp (Amaranthus tuberculatus)." Weed Science 54, no. 02 (April 2006): 305–15. http://dx.doi.org/10.1614/ws-05-115r1.1.
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Common waterhemp is an obligate outcrosser that has high genetic variability. However, under selection pressure, this weed shows population differentiation for adaptive traits. Intraspecific variation for herbicide resistance has been studied, but no studies have been conducted to determine the existence of variation for other adaptive traits that could influence weed management. The objective of this study was to examine the existence of different seed dormancy regulatory mechanisms in common waterhemp. Seed dormancy regulation, in response to different temperature and moisture regimes, was studied through germination experiments and proteome analysis using two common waterhemp biotypes (Ames and Everly) collected from agricultural fields in Iowa, and one biotype (Ohio) collected from a pristine area in Ohio. Without stratification, germination percentage among the different biotypes was 9, 29 and 88% for Ames, Everly, and Ohio respectively. The germination rate of seeds from Ames was dramatically increased after incubation at either 4 or 25 C under wet conditions, whereas germination of seeds from Everly was only increased at 25 C under wet conditions. The Ohio biotype showed no change in germination response to any of the incubation treatments. Germination studies indicated that the rate of seed dormancy alleviation differed between biotypes. Seed protein profiles obtained from the three biotypes differed in protein abundance, number, and type. A putative small heat-shock protein (sHSP) of 17.6 kDa and isoelectric point (pI) 6.1 increased whereas a putative glyceraldehyde-3-phosphate dehydrogenase (G3PDH) of 30.9 kDa and pI 6.4 decreased in abundance in the Ames biotype as seed dormancy was reduced in response to incubation at 4 C and wet conditions. These two proteins did not change in the Everly and Ohio biotypes, suggesting that these proteins changed their abundance in response to seed dormancy alleviation. The results of this study suggest that differences in seed dormancy levels between the biotypes were due to different physiological regulatory mechanisms.
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Pieper, Rembert, Shih-Ting Huang, Jeffrey M. Robinson, David J. Clark, Hamid Alami, Prashanth P. Parmar, Robert D. Perry, Robert D. Fleischmann, and Scott N. Peterson. "Temperature and growth phase influence the outer-membrane proteome and the expression of a type VI secretion system in Yersinia pestis." Microbiology 155, no. 2 (February 1, 2009): 498–512. http://dx.doi.org/10.1099/mic.0.022160-0.
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Yersinia pestis cells were grown in vitro at 26 and 37 °C, the ambient temperatures of its flea vector and its mammalian hosts, respectively, and subjected to subcellular fractionation. Abundance changes at 26 vs 37 °C were observed for many outer-membrane (OM) proteins. The cell adhesion protein Ail (y1324) and three putative small β-barrel OM proteins (y1795, y2167 and y4083) were strongly increased at 37 °C. The Ail/Lom family protein y1682 (OmpX) was strongly increased at 26 °C. Several porins and TonB-dependent receptors, which control small molecule transport through the OM, were also altered in abundance in a temperature-dependent manner. These marked differences in the composition of the OM proteome are probably important for the adaptation of Y. pestis to its in vivo life stages. Thirteen proteins that appear to be part of an intact type VI secretion system (T6SS) were identified in membrane fractions of stationary-phase cells grown at 26 °C, but not at 37 °C. The corresponding genes are clustered in the Y. pestis KIM gene locus y3658–y3677. The proteins y3674 and y3675 were particularly abundant and co-fractionated in a M r range indicative of participation in a multi-subunit complex. The soluble haemolysin-coregulated protein y3673 was even more abundant. Its release into the extracellular medium was triggered by treatment of Y. pestis cells with trypsin. Proteases and other stress-response-inducing factors may constitute environmental cues resulting in the activation of the T6SS in Y. pestis.
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Watkins, Ryan, Roberto Alva-Ruiz, Caitlin Conboy, Dong-Gi Mun, Erik Jessen, Diep Vu, Jos de Man, et al. "Abstract 3094: Proteomic profiling of cholangiocarcinoma predicts response to a novel small molecule inhibitor in preclinical models." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3094. http://dx.doi.org/10.1158/1538-7445.am2022-3094.
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Abstract Background: Cholangiocarcinoma is an aggressive malignancy with limited systemic therapeutics. We have recently identified LCK, a non-receptor tyrosine kinase, as a potential therapeutic target in select subtypes of cholangiocarcinoma. Proteomic profiling in other cancer types has proven beneficial in predicting response to systemic therapies. Herein, we present proteomic profiling of cholangiocarcinoma can predict sensitivity to a novel LCK inhibitor in patient derived xenograft models. Methods: Proteins from 28 unique cholangiocarcinoma patient derived xenograft models (PDX) were isolated, labeled with tandem mass tags (TMT) and then analyzed by liquid chromatography and mass spectroscopy (LC/MS). Phosphopeptides were separately evaluated both by Immobilized Metal Affinity Chromatography and pY-enrichment techniques. Following normalization, the global proteome, serine/threonine phosphoproteome, and tyrosine phosphoproteome were analyzed and a proteomic signature was created for each tumor. These signatures were compared to PDX models with known response profiles to NTRC0652-0, an LCK inhibitor (1 resistant and 1 sensitive). The top 200 differentially abundant proteins (log2FC 2.6) were then utilized to develop a sensitive and a resistant signature. Correlation indices were then calculated across the 28 PDX model library. An additional predicted resistant and predicted sensitive model was expanded. Tumor bearing mice were randomized based on tumor volume to either treatment with NTRC (30mg/kg PO daily), n=5, or vehicle, n=4, (10% Kolliphor, 10% DMSO, 80% Water, 2 mol equivalents HCl). Tumors were measured weekly with calipers and then weighed at the end of the study. Results: In the top 200 differentially abundant proteins that represented the proteomic signature there was over representation of proteins with decreased abundance. There were only 12 proteins with higher abundance in the sensitive model as compared to the resistant. Utilizing the signature developed from the global proteomics data, nine tumors were predicted to be sensitive while 17 were predicted to be resistant to NTRC. PDX115 is a an intrahepatic CCA PDX known to contain an IDH1 mutation and was predicted to be resistant by modeling. The model was resistant to single agent NTRC with no demonstrable effect on tumor growth in vivo. PDX175 is a distal CCA PDX and was predicted to be sensitive by modeling. In vivo growth was inhibited by NTRC as compared to vehicle treated mice following two weeks of treatment (983% vs 435%, p=0.03). Additionally, final tumor weight was significantly lower in NTRC treated mice (0.651 g vs 0.317 g, p=0.04). Conclusion: Cholangiocarcinoma proteomic signatures can be used to predict therapeutic response to a novel LCK inhibitor in preclinical models. Whether or not this could be applied to other treatment modalities in cholangiocarcinoma remains to be determined. Citation Format: Ryan Watkins, Roberto Alva-Ruiz, Caitlin Conboy, Dong-Gi Mun, Erik Jessen, Diep Vu, Jos de Man, Rogier Buijsman, Akhilesh Pandey, Mitesh Borad, Gregory Gores, Rory Smoot. Proteomic profiling of cholangiocarcinoma predicts response to a novel small molecule inhibitor in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3094.
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Georgieva, Veronika S., Julia Etich, Björn Bluhm, Mengjie Zhu, Christian Frie, Richard Wilson, Frank Zaucke, John Bateman, and Bent Brachvogel. "Ablation of the miRNA Cluster 24 Has Profound Effects on Extracellular Matrix Protein Abundance in Cartilage." International Journal of Molecular Sciences 21, no. 11 (June 9, 2020): 4112. http://dx.doi.org/10.3390/ijms21114112.
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MicroRNAs (miRNAs) regulate cartilage differentiation and contribute to the onset and progression of joint degeneration. These small RNA molecules may affect extracellular matrix organization (ECM) in cartilage, but for only a few miRNAs has this role been defined in vivo. Previously, we showed that cartilage-specific genetic ablation of the Mirc24 cluster in mice leads to impaired cartilage development due to increased RAF/MEK/ERK pathway activation. Here, we studied the expression of the cluster in cartilage by LacZ reporter gene assays and determined its role for extracellular matrix homeostasis by proteome and immunoblot analysis. The cluster is expressed in prehypertrophic/hypertrophic chondrocytes of the growth plate and we now show that the cluster is also highly expressed in articular cartilage. Cartilage-specific loss of the cluster leads to increased proteoglycan 4 and matrix metallopeptidase 13 levels and decreased aggrecan and collagen X levels in epiphyseal cartilage. Interestingly, these changes are linked to a decrease in SRY-related HMG box-containing (SOX) transcription factors 6 and 9, which regulate ECM production in chondrocytes. Our data suggests that the Mirc24 cluster is important for ECM homoeostasis and the expression of transcriptional regulators of matrix production in cartilage.
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Blankenburg, Sascha, Christian Hentschker, Anna Nagel, Petra Hildebrandt, Stephan Michalik, Denise Dittmar, Kristin Surmann, and Uwe Völker. "Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers." PROTEOMICS 19, no. 23 (October 3, 2019): 1900192. http://dx.doi.org/10.1002/pmic.201900192.
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Liou, Gunn-Guang, Anna Chao Kaberdina, Wei-Syuan Wang, Vladimir R. Kaberdin, and Sue Lin-Chao. "Combined Transcriptomic and Proteomic Profiling of E. coli under Microaerobic versus Aerobic Conditions: The Multifaceted Roles of Noncoding Small RNAs and Oxygen-Dependent Sensing in Global Gene Expression Control." International Journal of Molecular Sciences 23, no. 5 (February 25, 2022): 2570. http://dx.doi.org/10.3390/ijms23052570.
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Adaptive mechanisms that facilitate intestinal colonization by the human microbiota, including Escherichia coli, may be better understood by analyzing the physiology and gene expression of bacteria in low-oxygen environments. We used high-throughput transcriptomics and proteomics to compare the expression profiles of E. coli grown under aerobic versus microaerobic conditions. Clustering of high-abundance transcripts under microaerobiosis highlighted genes controlling acid-stress adaptation (gadAXW, gadAB, hdeAB-yhiD and hdeD operons), cell adhesion/biofilm formation (pgaABCD and csgDEFG operons), electron transport (cydAB), oligopeptide transport (oppABCDF), and anaerobic respiration/fermentation (hyaABCDEF and hycABCDEFGHI operons). In contrast, downregulated genes were involved in iron transport (fhuABCD, feoABC and fepA-entD operons), iron-sulfur cluster assembly (iscRSUA and sufABCDSE operons), aerobic respiration (sdhDAB and sucABCDSE operons), and de novo nucleotide synthesis (nrdHIEF). Additionally, quantitative proteomics showed that the products (proteins) of these high- or low-abundance transcripts were expressed consistently. Our findings highlight interrelationships among energy production, carbon metabolism, and iron homeostasis. Moreover, we have identified and validated a subset of differentially expressed noncoding small RNAs (i.e., CsrC, RyhB, RprA and GcvB), and we discuss their regulatory functions during microaerobic growth. Collectively, we reveal key changes in gene expression at the transcriptional and post-transcriptional levels that sustain E. coli growth when oxygen levels are low.
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Chen, Rui, Yuduo Song, Mi Yang, Chao Wen, Qiang Liu, Su Zhuang, and Yanmin Zhou. "Effect of Dietary Betaine on Muscle Protein Deposition, Nucleic Acid and Amino Acid Contents, and Proteomes of Broilers." Animals 12, no. 6 (March 15, 2022): 736. http://dx.doi.org/10.3390/ani12060736.
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To investigate the effect of betaine supplementation on growth performance, muscle protein deposition, muscle nucleic acid and amino acid contents, and muscle proteome of broilers, 160 one-day-old male partridge shank broiler chickens were randomly divided into 2 groups with 8 replicates of 10 broilers each. Broilers were fed a basal diet alone, or a basal diet supplemented with 1000 mg/kg betaine. Compared with the control group, the betaine group significantly increased (p < 0.05) the broilers average daily gain, the levels of serum insulin-like growth factor-1 (IGF-1), growth hormone (GH), total protein (TP), the contents of muscle absolute protein deposition, RNA, Ser, Glu, Met, and Phe, and the ratio of RNA/DNA, and decreased (p < 0.05) the feed conversion ratio and serum blood urea nitrogen content. Moreover, proteomic analysis revealed 35 differentially abundant proteins (DAPs) in the betaine group compared with the control group, including 27 upregulated proteins and 8 downregulated proteins (p < 0.05). These DAPs were mainly related to cell differentiation, small molecule metabolic process, and tissue development. In conclusion, diets supplemented with 1000 mg/kg betaine improved growth performance and muscle protein deposition of broilers. Increased serum GH, IGF-1, and TP contents, and alterations in muscle nucleic acids, amino acids, and protein abundance levels were involved in this process.
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Krajina, Brad A., Ami Yamamoto, Kevin J. Cheung, and Samuel Madasu. "Abstract P2-23-19: Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform." Cancer Research 83, no. 5_Supplement (March 1, 2023): P2–23–19—P2–23–19. http://dx.doi.org/10.1158/1538-7445.sabcs22-p2-23-19.
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Abstract Tumors are multicellular ecosystems that communicate through the exchange of extracellular signaling molecules. In breast cancer, this multicellular communication can enable tumor cells to cooperate in a range of contexts, including tumor invasion and the outgrowth of distal metastases. Illuminating the mechanisms by which tumor cells communicate may reveal new therapies. To this end, three-dimensional tumor organoid models have emerged as versatile platforms for modeling multicellular behavior ex vivo. However, organoid culture typically requires the use of poorly defined, animal-derived extracellular matrices, such as Matrigel. These exogenous matrices contain thousands of proteins that dominate over and conceal cell-secreted factors in conventional proteomics approaches. Revealing low abundance cell-secreted factors from this complex exogenous background presents a formidable challenge. To surmount this challenge, we develop a new method to isolate the pericellular proteome in 3D organotypic culture models. This technology harnesses biorthogonal click chemistry to bypass exogenous factors, infiltrate intercellular spaces, and retrieve the endogenous intercellular proteome. This approach requires no genetic manipulation, requires only 1 million cells, and can be adapted to diverse organotypic models. These capabilities enable isolation of intercellular signaling factors in models that yield precious amounts of material and that can sustain only limited manipulation ex vivo, which we demonstrate using organoids established from patient-derived xenografts. To establish the generalizability of our approach, we apply this technology to a panel of breast cancer and small-cell lung cancer organoid models. Furthermore, we demonstrate that this method can be readily adapted to different extracellular matrix environments that are widely employed in organoid research, including basement membrane matrices and collagen gels. Taken together, our results establish this technology as a scalable and generalizable platform that may open opportunities for researchers investigating diverse questions in organoid models of cancer. Moving forward, we are leveraging these capabilities to investigate changes in the intercellular proteome that emerge after the acquisition of therapy resistance. We hope to uncover new mechanisms of collective signaling that may be targeted to overcome therapy resistance. Citation Format: Brad A. Krajina, Ami Yamamoto, Kevin J. Cheung, Samuel Madasu. Interrogating multicellular signaling in breast cancer using a bio-orthogonal chemistry-based proteomics platform [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-19.
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Donowitz, M., S. Singh, P. Singh, F. F. Salahuddin, Y. Chen, M. Chakraborty, R. Murtazina, et al. "Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles." Physiological Genomics 42A, no. 3 (November 2010): 200–210. http://dx.doi.org/10.1152/physiolgenomics.00001.2010.
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Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.
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Handtke, Stefan, Leif Steil, Raghavendra Palankar, Jane Conrad, Simran Cauhan, Luise Kraus, Myriam Ferrara, et al. "Role of Platelet Size Revisited—Function and Protein Composition of Large and Small Platelets." Thrombosis and Haemostasis 119, no. 03 (February 6, 2019): 407–20. http://dx.doi.org/10.1055/s-0039-1677875.
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AbstractEpidemiological studies found an association between increased platelet size and the risk for thrombotic complications, but functional differences of large and small platelets remain poorly understood due to a lack of standardized protocols separating platelets with different size. We designed a protocol to separate large and small platelets from 15 mL whole blood. Separated large and small platelet fractions differed in mean platelet volume: 12.1 fl (10.3–13.8 fl) versus 7.7 fl (6.8–9.5 fl, p < 0.01), and forward scatter mean fluorescence intensity: 24.75 (19.9–30.9) versus 16.85 (11.3–20.6; p < 0.01). Similar fold differences were observed in cell diameter and plateletcrit. Large platelets express 30 to 50% more glycoprotein (GP) Ia, GPIb, GPIIIa, GPVI and P2Y12 on their membranes compared with small ones. Single large platelets covered a 50% larger area on a collagen surface. Adhesion to collagen was faster in large platelets compared with small ones indicating enhanced outside-in signal transduction in large platelets via collagen receptors. In contrast, integrin activation was more pronounced in small platelets after ADP stimulation. Proteome analysis revealed that 80 of the 894 proteins quantified differed in abundance: ADP-ribosylation factor 1/3, guanosine triphosphate-binding protein SAR1a, Voltage-dependent anion-selective channel protein 3 and guanylate cyclase soluble sub-unit α-3 were higher abundant in large, whereas immunoglobulins, haptoglobin, hemopexin, α-1-antitrypsin, serotransferrin and vitronectin were more abundant in small platelets. We conclude that some functions and the protein composition of large and small platelets differ, which cannot only be explained by the size difference. Our data suggest different functional roles of large and small platelets.
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Perez-Pujol, Silvia, Lorraine B. Anderson, Michael B. Martinez, LeeAnn Higgins, James G. White, Gary L. Nelsestuen, and Nigel S. Key. "Proteomic Analysis of Gray Platelet Syndrome by iTRAQ Labelling and Mass Spectroscopy: A Potential New Diagnostic Strategy for Platelet Disorders." Blood 106, no. 11 (November 16, 2005): 2161. http://dx.doi.org/10.1182/blood.v106.11.2161.2161.
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Abstract The recent introduction of novel proteomic techniques to evaluate normal human platelet proteins and their modifications following activation may also help to elucidate the causes and improve diagnoses of platelet-related bleeding disorders. Previous studies from this and other laboratories have demonstrated the sensitivity and reliability of this new technique to detect minor changes (+/−20%) in the protein content of platelets. More than 600 proteins shared by normal platelets were identified, and >200 in platelet-derived microparticles (MPs) generated by ionophore or Thrombin Receptor Activating Peptide (TRAP) activation. As a prelude to our ultimate goal of establishing this new technology, we applied the iTRAQ method (multiplexed relative protein quantitation by mass spectrometry) in the study of two unrelated patients with the Gray Platelet Syndrome (GPS). Megakaryocytes of patients with GPS can synthesize alpha-granule proteins and enclose them within membranes. However, the membranes lack structure linked latency, and enclosed proteins leak out of the organelles before platelets are delivered to circulating blood. Thus, GPS platelets contain empty alpha-granule vacuoles instead of alpha-granules. Due to large changes in platelet properties, it was surprising to find that cytoskeletal proteins of GPS were present in similar abundance to normal platelets (i.e. in the range of 0.80–1.20). Receptor proteins showed a similar distribution. As expected, the content of most alpha-granule proteins in the platelets from the 2 GPS patients was markedly reduced, with individual protein abundance ratios of 0.10 to 0.52 compared to normal platelets. However, certain trans-membrane proteins of alpha-granules (such as vesicle-associated proteins and P-selectin) were better preserved in GPS platelets, with ratios in the range of 0.73 to 1.16 compared to normal platelets. These results showed that many structural components (such as cytoskeleton and receptor proteins) are unchanged in GPS platelets. The results support our previous data indicating that detection of a change of as little as 20% may indicate a pathological change in the platelet proteome that may impact normal function. Our results therefore demonstrate that highly sensitive methods, capable of detecting small protein changes, may be needed to fully appreciate pathological disorders. The iTRAQ technique is able to detect low level changes and may be a leading tool that can generate new insights into the molecular pathophysiology and genetic variations of platelet disorders.
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Zhang, Jiao, Jin Wan, Daiwen Chen, Bing Yu, and Jun He. "Low-Molecular-Weight Chitosan Attenuates Lipopolysaccharide-Induced Inflammation in IPEC-J2 Cells by Inhibiting the Nuclear Factor-κB Signalling Pathway." Molecules 26, no. 3 (January 22, 2021): 569. http://dx.doi.org/10.3390/molecules26030569.
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Low-molecular-weight chitosan (LMWC), a product of chitosan deacetylation, possesses anti-inflammatory effects. In the present study, a porcine small intestinal epithelial cell line, IPEC-J2, was used to assess the protective effects of LMWC on lipopolysaccharide (LPS)-induced intestinal epithelial cell injury. IPEC-J2 cells were pretreated with or without LMWC (400 μg/mL) in the presence or absence of LPS (5 μg/mL) for 6 h. LMWC pretreatment increased (p < 0.05) the occludin abundance and decreased (p < 0.05) the tumour necrosis factor-α (TNF-α) production, apoptosis rate and cleaved cysteinyl aspartate-specific protease-3 (caspase-3) and -8 contents in LPS-treated IPEC-J2 cells. Moreover, LMWC pretreatment downregulated (p < 0.05) the expression levels of TNF receptor 1 (TNFR1) and TNFR-associated death domain and decreased (p < 0.05) the nuclear and cytoplasmic abundance of nuclear factor-κB (NF-κB) p65 in LPS-stimulated IPEC-J2 cells. These results suggest that LMWC exerts a mitigation effect on LPS-induced intestinal epithelial cell damage by suppressing TNFR1-mediated apoptosis and decreasing the production of proinflammatory cytokines via the inhibition of NF-κB signalling pathway.
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Wang, Yue, Hsin Jou Yang, and Paul M. Harrison. "The relationship between protein domains and homopeptides in the Plasmodium falciparum proteome." PeerJ 8 (October 2, 2020): e9940. http://dx.doi.org/10.7717/peerj.9940.
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The proteome of the malaria parasite Plasmodium falciparum is notable for the pervasive occurrence of homopeptides or low-complexity regions (i.e., regions that are made from a small subset of amino-acid residue types). The most prevalent of these are made from residues encoded by adenine/thymidine (AT)-rich codons, in particular asparagine. We examined homopeptide occurrences within protein domains in P. falciparum. Homopeptide enrichments occur for hydrophobic (e.g., valine), or small residues (alanine or glycine) in short spans (<5 residues), but these enrichments disappear for longer lengths. We observe that short asparagine homopeptides (<10 residues long) have a dramatic relative depletion inside protein domains, indicating some selective constraint to keep them from forming. We surmise that this is possibly linked to co-translational protein folding, although there are specific protein domains that are enriched in longer asparagine homopeptides (≥10 residues) indicating a functional linkage for specific poly-asparagine tracts. Top gene ontology functional category enrichments for homopeptides associated with diverse protein domains include “vesicle-mediated transport”, and “DNA-directed 5′-3′ RNA polymerase activity”, with various categories linked to “binding” evidencing significant homopeptide depletions. Also, in general homopeptides are substantially enriched in the parts of protein domains that are near/in IDRs. The implications of these findings are discussed.
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Tucker, Aimee M., Lonnie O. Driskell, Lewis K. Pannell, and David O. Wood. "Differential Proteomic Analysis of Rickettsia prowazekii Propagated in Diverse Host Backgrounds." Applied and Environmental Microbiology 77, no. 14 (June 3, 2011): 4712–18. http://dx.doi.org/10.1128/aem.05140-11.
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ABSTRACTThe obligate intracellular growth ofRickettsia prowazekiiplaces severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile ofR. prowazekiigrown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.
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Okai, Charles A., Manuela Russ, Manja Wölter, Kristin Andresen, Werner Rath, Michael O. Glocker, and Ulrich Pecks. "Precision Diagnostics by Affinity-Mass Spectrometry: A Novel Approach for Fetal Growth Restriction Screening during Pregnancy." Journal of Clinical Medicine 9, no. 5 (May 7, 2020): 1374. http://dx.doi.org/10.3390/jcm9051374.
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Fetal growth restriction (FGR) affects about 3% to 8% of pregnancies, leading to higher perinatal mortality and morbidity. Current strategies for detecting fetal growth impairment are based on ultrasound inspections. However, antenatal detection rates are insufficient and critical in countries with substandard care. To overcome difficulties with detection and to better discriminate between high risk FGR and low risk small for gestational age (SGA) fetuses, we investigated the suitability of risk assessment based on the analysis of a recently developed proteome profile derived from maternal serum in different study groups. Maternal serum, collected at around 31 weeks of gestation, was analyzed in 30 FGR, 15 SGA, and 30 control (CTRL) pregnant women who delivered between 31 and 40 weeks of gestation. From the 75 pregnant women of this study, 2 were excluded because of deficient raw data and 2 patients could not be grouped due to indeterminate results. Consistency between proteome profile and sonography results was obtained for 59 patients (26 true positive and 33 true negative). Of the proteome profiling 12 contrarious grouped individuals, 3 were false negative and 9 were false positive cases with respect to ultrasound data. Both true positive and false positive grouping transfer the respective patients to closer surveillance and thorough pregnancy management. Accuracy of the test is considered high with an area-under-curve value of 0.88 in receiver-operator-characteristics analysis. Proteome profiling by affinity-mass spectrometry during pregnancy provides a reliable method for risk assessment of impaired development in fetuses and consumes just minute volumes of maternal peripheral blood. In addition to clinical testing proteome profiling by affinity-mass spectrometry may improve risk assessment, referring pregnant women to specialists early, thereby improving perinatal outcomes.
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Burnap, Sean A., Katherine Sattler, Raimund Pechlaner, Elisa Duregotti, Ruifang Lu, Konstantinos Theofilatos, Kaloyan Takov, et al. "PCSK9 Activity Is Potentiated Through HDL Binding." Circulation Research 129, no. 11 (November 12, 2021): 1039–53. http://dx.doi.org/10.1161/circresaha.121.319272.
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Rationale: PCSK9 (proprotein convertase subtilisin/kexin type 9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and HDL (high-density lipoprotein) remains unexplored. Objective: This study sought to interrogate the novel relationship between PCSK9 and HDL in humans. Methods and Results: Comparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance spectroscopy and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck study (n=656) revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein C3 (apoC3), an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk; n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 time points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apoC3 from HDL, and lower levels of small HDL as measured by nuclear magnetic resonance spectroscopy. Crosslinking mass spectrometry upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin, and apolipoprotein E within the HDL proteome that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalization and multimerization. Conclusions: This study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipemia as a driver of PCSK9 and apoC3 release from HDL. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03191513; URL: https://www.isrctn.com ; Unique identifier: ISRCTN20774126.
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Datta, Arnab, Christopher Chen, Yong-Gui Gao, and Siu Kwan Sze. "Quantitative Proteomics of Medium-Sized Extracellular Vesicle-Enriched Plasma of Lacunar Infarction for the Discovery of Prognostic Biomarkers." International Journal of Molecular Sciences 23, no. 19 (October 1, 2022): 11670. http://dx.doi.org/10.3390/ijms231911670.
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Lacunar infarction (LACI), a subtype of acute ischemic stroke, has poor mid- to long-term prognosis due to recurrent vascular events or incident dementia which is difficult to predict using existing clinical data. Herein, we aim to discover blood-based biomarkers for LACI as a complementary prognostic tool. Convalescent plasma was collected from forty-five patients following a non-disabling LACI along with seventeen matched control subjects. The patients were followed up prospectively for up to five years to record an occurrence of adverse outcome and grouped accordingly (i.e., LACI-no adverse outcome, LACI-recurrent vascular event, and LACI-cognitive decline without any recurrence of vascular events). Medium-sized extracellular vesicles (MEVs), isolated from the pooled plasma of four groups, were analyzed by stable isotope labeling and 2D-LC-MS/MS. Out of 573 (FDR < 1%) quantified proteins, 146 showed significant changes in at least one LACI group when compared to matched healthy control. A systems analysis revealed that major elements (~85%) of the MEV proteome are different from the proteome of small-sized extracellular vesicles obtained from the same pooled plasma. The altered MEV proteins in LACI patients are mostly reduced in abundance. The majority of the shortlisted MEV proteins are not linked to commonly studied biological processes such as coagulation, fibrinolysis, or inflammation. Instead, they are linked to oxygen-glucose deprivation, endo-lysosomal trafficking, glucose transport, and iron homeostasis. The dataset is provided as a web-based data resource to facilitate meta-analysis, data integration, and targeted large-scale validation.
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O’Connell, Grant C., Kyle B. Walsh, Emily Burrage, Opeolu Adeoye, Paul D. Chantler, and Taura L. Barr. "High-throughput profiling of the circulating proteome suggests sexually dimorphic corticosteroid signaling following ischemic stroke." Physiological Genomics 50, no. 10 (October 1, 2018): 876–83. http://dx.doi.org/10.1152/physiolgenomics.00058.2018.
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Increasing evidence suggests that there are innate differences between sexes with respect to stroke pathophysiology; however, the molecular mechanisms underlying these differences remain unclear. In this investigation, we employed a shotgun approach to broadly profile sex-associated differences in the plasma proteomes of a small group of male ( n = 6) and female ( n = 4) ischemic stroke patients. Peripheral blood was sampled during the acute phase of care, and liquid chromatography electrospray ionization mass spectrometry was used to quantify plasma proteins. We observed widespread differences in plasma composition, as 77 out of 294 detected proteins were significantly differentially expressed between sexes. Corticosteroid-binding globulin (CBG), a negative acute-phase reactant that inversely regulates levels of bioactive free cortisol, was the most dramatically differentially regulated, exhibiting 16-fold higher abundance in plasma from women relative to men. Furthermore, functional annotation analysis revealed that the remaining differentially expressed proteins were significantly enriched for those involved in response to corticosteroid signaling. Plasma CBG levels were further examined in an additional group of male ( n = 19) and female ( n = 28) ischemic stroke patients, as well as a group of male ( n = 13) and female ( n = 18) neurologically normal controls. CBG levels were significantly reduced in male stroke patients relative to male controls; however, no differences were observed between female stroke patients and female controls, suggesting that women may exhibit an attenuated cortisol response to stroke. Collectively, our findings reinforce the idea that there are sex-associated differences in stroke pathophysiology and suggest that cortisol signaling should be investigated further as a potential molecular mediator.
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Helle, Stanislas, Fabrice Bray, Jean-Luc Putaux, Jérémy Verbeke, Stéphanie Flament, Christian Rolando, Christophe D’Hulst, and Nicolas Szydlowski. "Intra-Sample Heterogeneity of Potato Starch Reveals Fluctuation of Starch-Binding Proteins According to Granule Morphology." Plants 8, no. 9 (September 4, 2019): 324. http://dx.doi.org/10.3390/plants8090324.
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Starch granule morphology is highly variable depending on the botanical origin. Moreover, all investigated plant species display intra-tissular variability of granule size. In potato tubers, the size distribution of starch granules follows a unimodal pattern with diameters ranging from 5 to 100 µm. Several evidences indicate that granule morphology in plants is related to the complex starch metabolic pathway. However, the intra-sample variability of starch-binding metabolic proteins remains unknown. Here, we report on the molecular characterization of size-fractionated potato starch granules with average diameters of 14.2 ± 3.7 µm, 24.5 ± 6.5 µm, 47.7 ± 12.8 µm, and 61.8 ± 17.4 µm. In addition to changes in the phosphate contents as well as small differences in the amylopectin structure, we found that the starch-binding protein stoichiometry varies significantly according to granule size. Label-free quantitative proteomics of each granule fraction revealed that individual proteins can be grouped according to four distinct abundance patterns. This study corroborates that the starch proteome may influence starch granule growth and architecture and opens up new perspectives in understanding the dynamics of starch biosynthesis.
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Wei, Chen-Xuan, Michael Francis Burrow, Michael George Botelho, and W. Keung Leung. "Analysing Complex Oral Protein Samples: Complete Workflow and Case Analysis of Salivary Pellicles." Journal of Clinical Medicine 10, no. 13 (June 25, 2021): 2801. http://dx.doi.org/10.3390/jcm10132801.
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Studies on small quantity, highly complex protein samples, such as salivary pellicle, have been enabled by recent major technological and analytical breakthroughs. Advances in mass spectrometry-based computational proteomics such as Multidimensional Protein Identification Technology have allowed precise identification and quantification of complex protein samples on a proteome-wide scale, which has enabled the determination of corresponding genes and cellular functions at the protein level. The latter was achieved via protein-protein interaction mapping with Gene Ontology annotation. In recent years, the application of these technologies has broken various barriers in small-quantity-complex-protein research such as salivary pellicle. This review provides a concise summary of contemporary proteomic techniques contributing to (1) increased complex protein (up to hundreds) identification using minute sample sizes (µg level), (2) precise protein quantification by advanced stable isotope labelling or label-free approaches and (3) the emerging concepts and techniques regarding computational integration, such as the Gene Ontology Consortium and protein-protein interaction mapping. The latter integrates the structural, genomic, and biological context of proteins and genes to predict protein interactions and functional connections in a given biological context. The same technological breakthroughs and computational integration concepts can also be applied to other low-volume oral protein complexes such as gingival crevicular or peri-implant sulcular fluids.
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Kleinwort, Kristina J. H., Stefanie M. Hauck, Roxane L. Degroote, Armin M. Scholz, Christina Hölzel, Erwin P. Maertlbauer, and Cornelia Deeg. "Peripheral blood bovine lymphocytes and MAP show distinctly different proteome changes and immune pathways in host-pathogen interaction." PeerJ 7 (November 25, 2019): e8130. http://dx.doi.org/10.7717/peerj.8130.
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Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogen causing paratuberculosis in cattle and small ruminants. During the long asymptomatic subclinical stage, high numbers of MAP are excreted and can be transmitted to food for human consumption, where they survive many of the standard techniques of food decontamination. Whether MAP is a human pathogen is currently under debate. The aim of this study was a better understanding of the host-pathogen response by analyzing the interaction of peripheral blood lymphocytes (PBL) from cattle with MAP in their exoproteomes/secretomes to gain more information about the pathogenic mechanisms of MAP. Because in other mycobacterial infections, the immune phenotype correlates with susceptibility, we additionally tested the interaction of MAP with recently detected cattle with a different immune capacity referred as immune deviant (ID) cows. In PBL, different biological pathways were enhanced in response to MAP dependent on the immune phenotype of the host. PBL of control cows activated members of cell activation and chemotaxis of leukocytes pathway as well as IL-12 mediated signaling. In contrast, in ID cows CNOT1 was detected as highly abundant protein, pointing to a different immune response, which could be favorable for MAP. Additionally, MAP exoproteomes differed in either GroEL1 or DnaK abundance, depending on the interacting host immune response. These finding point to an interdependent, tightly regulated response of the bovine immune system to MAP and vise versa.
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Coelho, Ana Cristina, Rosa Pires, Gabriela Schütz, Cátia Santa, Bruno Manadas, and Patrícia Pinto. "Disclosing proteins in the leaves of cork oak plants associated with the immune response to Phytophthora cinnamomi inoculation in the roots: A long-term proteomics approach." PLOS ONE 16, no. 1 (January 22, 2021): e0245148. http://dx.doi.org/10.1371/journal.pone.0245148.
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The pathological interaction between oak trees and Phytophthora cinnamomi has implications in the cork oak decline observed over the last decades in the Iberian Peninsula. During host colonization, the phytopathogen secretes effector molecules like elicitins to increase disease effectiveness. The objective of this study was to unravel the proteome changes associated with the cork oak immune response triggered by P. cinnamomi inoculation in a long-term assay, through SWATH-MS quantitative proteomics performed in the oak leaves. Using the Arabidopis proteome database as a reference, 424 proteins were confidently quantified in cork oak leaves, of which 80 proteins showed a p-value below 0.05 or a fold-change greater than 2 or less than 0.5 in their levels between inoculated and control samples being considered as altered. The inoculation of cork oak roots with P. cinnamomi increased the levels of proteins associated with protein-DNA complex assembly, lipid oxidation, response to endoplasmic reticulum stress, and pyridine-containing compound metabolic process in the leaves. In opposition, several proteins associated with cellular metabolic compound salvage and monosaccharide catabolic process had significantly decreased abundances. The most significant abundance variations were observed for the Ribulose 1,5-Bisphosphate Carboxylase small subunit (RBCS1A), Heat Shock protein 90–1 (Hsp90-1), Lipoxygenase 2 (LOX2) and Histone superfamily protein H3.3 (A8MRLO/At4G40030) revealing a pertinent role for these proteins in the host-pathogen interaction mechanism. This work represents the first SWATH-MS analysis performed in cork oak plants inoculated with P. cinnamomi and highlights host proteins that have a relevant action in the homeostatic states that emerge from the interaction between the oomycete and the host in the long term and in a distal organ.
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Brocco, Davide, Paola Lanuti, Damiana Pieragostino, Maria Concetta Cufaro, Pasquale Simeone, Giuseppina Bologna, Pietro Di Marino, et al. "Phenotypic and Proteomic Analysis Identifies Hallmarks of Blood Circulating Extracellular Vesicles in NSCLC Responders to Immune Checkpoint Inhibitors." Cancers 13, no. 4 (February 3, 2021): 585. http://dx.doi.org/10.3390/cancers13040585.
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Immune checkpoint inhibitors (ICIs) induce durable clinical responses only in a subset of advanced non-small cell lung cancer (NSCLC) patients. There is a need to identify mechanisms of ICI resistance and immunotherapy biomarkers to improve clinical benefit. In this study, we evaluated the prognostic and predictive value of circulating endothelial and leukocyte-derived extracellular vesicles (EV) in patients with advanced NSCLC treated with anti-PD-1/PD-L1 agents. In addition, the relationship between total blood circulating EV proteome and response to ICIs was investigated. An optimized flow cytometry method was employed for the identification and subtyping of blood circulating EVs in 59 patients with advanced NSCLC. Blood samples were collected from patients receiving anti-PD-1/PD-L1 inhibitors (n = 31) or chemotherapy (n = 28). An exploratory proteomic analysis of sorted blood EVs was conducted in a subset of patients. Our results show that a low blood concentration of circulating endothelial-derived EVs before treatment was strongly associated to longer overall survival (p = 0.0004) and higher disease control rate (p = 0.045) in patients treated with ICIs. Interestingly, shotgun proteomics revealed that EVs of responders to anti-PD-1 therapy had a specific protein cargo before treatment. In addition, EV protein cargo was specifically modulated during immunotherapy. We identified a previously unknown association between circulating endothelial-derived extracellular vesicle concentration and immunotherapy-related clinical outcomes. We also observed differences in circulating extracellular vesicle proteome according to anti-PD-1-based treatment response in NSCLC patients. Overall, these results may contribute to the identification of novel circulating biomarkers for rational immunotherapy approaches in patients affected by NSCLC.
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Harel, Michal, Coren Lahav, Eyal Jacob, Itamar Sela, Yehonatan Elon, Galit Yahalom, Iris Kamer, et al. "300 Longitudinal plasma proteomic profiling of non-small cell lung cancer patients undergoing immune checkpoint blockade-based therapy." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A323. http://dx.doi.org/10.1136/jitc-2021-sitc2021.300.
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BackgroundImmune checkpoint inhibitors (ICIs) have revolutionized cancer treatment by shifting the focus from the tumor to the immune system of the host. Despite durable response to ICIs, only a small proportion of non-small lung cancer (NSCLC) patients respond to this treatment. Thus, great effort is currently focused on uncovering mechanisms of resistance and identifying predictive biomarkers for outcome.MethodsBlood plasma was obtained from 143 NSCLC patients treated with ICI-based therapy at baseline and early on-treatment (following the first treatment), and the levels of approximately 800 proteins were determined using ELISA-based arrays. Bioinformatic analysis was performed in order to detect novel patterns of resistance to ICI-based therapy. To identify a signature that predicts clinical outcome, a machine learning algorithm was applied.ResultsUnsupervised bioinformatic analysis of the plasma proteomic profiles classified the patients into 3 clusters with distinct clinical and biological features. Patients in cluster #1 exhibited resistance to therapy, bone metastasis and high TNM (tumors, nodes and metastasis) staging; this cluster displayed high levels of proteins related to glycan and pyrimidine metabolism and cell-adhesion. Cluster #2 was enriched with responders, males, and patients with low TNM staging; this cluster displayed a strong representation of desmoglein proteins. Cluster #3 was enriched with female patients while the proteome of these patients displayed high levels of MAPK signaling related proteins. Patient clusters were largely unchanged when comparing baseline and on-treatment data, suggesting pre-existing rather than acquired resistance to therapy. A further comparison between responders and non-responders identified six significantly differentially expressed proteins comprised of both host- and tumor-related proteins, with non-responders displaying a significant enrichment of neutrophil proteins at baseline and early-on-treatment. Notably, there was no significant difference in the neutrophil count between responders and non-responders, suggesting a functional shift in neutrophils upon treatment in non-responders. Lastly, we identified a predictive signature for response comprised of two proteins and two clinical features. The performance of the predictive signature reached an area under the curve (AUC) of the receiver operating characteristics (ROC) plot of 0.8 in an independent validation subset of the cohort, indicating a high predictive power.ConclusionsHere we performed a deep bioinformatic analysis of plasma proteome profiles of 143 NSCLC patients undergoing ICI-based therapy. Our study sheds light on underlying mechanisms of resistance to ICI-based therapy and reveals a predictive signature for response in NSCLC patients.Ethics ApprovalData and study specimens were purchased from Indivumed and Sheba medical center, approval number 0226-13-SMC (institutional review board). Participants gave informed consent before taking part.
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Adur, Malavika K., Yunsheng Li, and Jason W. Ross. "83 MicroRNA574-3p Influences Porcine Oocyte Maturation and Regulates Abundance of Proteins Critical to Early Embryo Development." Journal of Animal Science 99, Supplement_1 (May 1, 2021): 108. http://dx.doi.org/10.1093/jas/skab054.176.
Full textAbstract:
Abstract MicroRNA are small non-coding RNA involved in post-transcriptional gene regulation impacting oocyte maturation and embryo development. MIR574-3p abundance decreases during oocyte in vitro maturation (IVM) and blastocyst development. The study objective was to evaluate the role of MIR574-3p during porcine oocyte maturation and early embryo development. To assess the function of MIR574-3p during these processes, denuded GV stage oocytes injected with MIR574-3p mimic (MIR574-3p-M), MIR574-3p inhibitor (MIR574-3p-I) or negative control oligo (NC) underwent IVM for 42 hours. The number of MII arrested oocytes was decreased (P = 0.06) in the MIR574-3p-M group (67.7 ± 1.4%) as compared to the NC group (76.1 ± 1.3%), whereas maturation was not affected by MIR574-3p-I (75.6 ± 1.5%) as compared to the NC group (73.1 ± 3.6%). MII arrested oocytes were parthenogenetically activated and cultured for 7 days. Neither mimic nor inhibitor affected the cleavage or blastocyst rate. Using LC-MS/MS we evaluated changes in global protein abundance in injected oocytes after 42 hours of IVM. We identified 971 proteins in MIR574-3p-M injected oocytes, of which 57 were differentially abundant as compared to the control group. In MIR574-3p-I injected oocytes, 1007 proteins were identified, of which 107 were differentially abundant as compared to the control group. Overall, MIR574-3p-M upregulated proteins critical to membrane binding mediating sperm receptors on the zona pellucida, while it downregulated intranuclear activity such as nucleotide biosynthesis, mitotic spindle assembly and orientation; whereas MIR574-3p-I induced upregulation of proteins involved in the processes between and including protein biosynthesis and metabolism, while downregulating proteins critical to ATP, RNA, DNA and protein binding. These data suggest artificially increasing MIR574-3p abundance during IVM alters the oocyte proteome and influences meiotic progression to MII. Project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2017-67015-26459 from the USDA National Institute of Food and Agriculture.