Proteome-wide profiling and mapping of post translational modifications in human hearts.
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ABSTRACT:
SUBMITTER: Bagwan N
PROVIDER: S-EPMC7838296 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundHistones and their post-translational modifications impact cellular function by acting as key regulators in the maintenance and remodeling of chromatin, thus affecting transcription regulation either positively (activation) or negatively (repression). In this study we describe a comprehensive, bottom-up proteomics approach to profiling post-translational modifications (acetylation, mono-, di- and tri-methylation, phosphorylation, biotinylation, ubiquitination, citrullination and ADP-ribosylation) in human macrophages, which are primary cells of the innate immune system. As our knowledge expands, it becomes more evident that macrophages are a heterogeneous population with potentially subtle differences in their responses to various stimuli driven by highly complex epigenetic regulatory mechanisms.MethodsTo profile post-translational modifications (PTMs) of histones in macrophages we used two platforms of liquid chromatography and mass spectrometry. One platform was based on Sciex5600 TripleTof and the second one was based on VelosPro Orbitrap Elite ETD mass spectrometers.ResultsWe provide side-by-side comparison of profiling using two mass spectrometric platforms, ion trap and qTOF, coupled with the application of collisional induced and electron transfer dissociation. We show for the first time methylation of a His residue in macrophages and demonstrate differences in histone PTMs between those currently reported for macrophage cell lines and what we identified in primary cells. We have found a relatively low level of histone PTMs in differentiated but resting human primary monocyte derived macrophages.ConclusionsThis study is the first comprehensive profiling of histone PTMs in primary human MDM. Our study implies that epigenetic regulatory mechanisms operative in transformed cell lines and primary cells are overlapping to a limited extent. Our mass spectrometric approach provides groundwork for the investigation of how histone PTMs contribute to epigenetic regulation in primary human macrophages.
| S-EPMC4582717 | biostudies-literature
Project description:Peroxisomes, which are ubiquitous organelles in all eukaryotes, are highly dynamic organelles that are essential for development and stress responses. Plant peroxisomes are involved in major metabolic pathways, such as fatty acid β-oxidation, photorespiration, ureide and polyamine metabolism, in the biosynthesis of jasmonic, indolacetic, and salicylic acid hormones, as well as in signaling molecules such as reactive oxygen and nitrogen species (ROS/RNS). Peroxisomes are involved in the perception of environmental changes, which is a complex process involving the regulation of gene expression and protein functionality by protein post-translational modifications (PTMs). Although there has been a growing interest in individual PTMs in peroxisomes over the last ten years, their role and cross-talk in the whole peroxisomal proteome remain unclear. This review provides up-to-date information on the function and crosstalk of the main peroxisomal PTMs. Analysis of whole peroxisomal proteomes shows that a very large number of peroxisomal proteins are targeted by multiple PTMs, which affect redox balance, photorespiration, the glyoxylate cycle, and lipid metabolism. This multilevel PTM regulation could boost the plasticity of peroxisomes and their capacity to regulate metabolism in response to environmental changes.
| S-EPMC6801620 | biostudies-literature
Project description:Performing a comprehensive nonbiased proteome analysis is an extraordinary challenge due to sample complexity and wide dynamic range, especially in eukaryotic tissues. Thus, prefractionation steps conducted prior to mass spectrometric analysis are critically important to reduce complex biological matrices and allow in-depth analysis. Here we demonstrated the use of OFFGel prefractionation to identify more low abundant and hydrophobic proteins than in a nonfractionated sample. Moreover, OFFGel prefractionation of a kidney protein sample was able to unveil protein functional relevance by detecting PTMs, especially when prefractionation was augmented with a targeted enrichment strategy such as TiO₂ phospho-enrichment. The OFFGel-TiO₂ combination used in this study was comparable to other global phosphoproteomics approaches (SCX-TiO₂, ERLIC-TiO₂, or HILIC-TiO₂). The detailed mouse kidney proteome with the phosphopeptide enrichment presented here serves as a useful platform for a better understanding of how the renal protein modification machinery works and, ultimately, will contribute to our understanding of pathological processes as well as normal physiological renal functions.
| S-EPMC3993965 | biostudies-literature
Project description:The HUPO Human Proteome Project (HPP) has two overall goals: (1) stepwise completion of the protein parts list-the draft human proteome including confidently identifying and characterizing at least one protein product from each protein-coding gene, with increasing emphasis on sequence variants, post-translational modifications (PTMs), and splice isoforms of those proteins; and (2) making proteomics an integrated counterpart to genomics throughout the biomedical and life sciences community. PeptideAtlas and GPMDB reanalyze all major human mass spectrometry data sets available through ProteomeXchange with standardized protocols and stringent quality filters; neXtProt curates and integrates mass spectrometry and other findings to present the most up to date authorative compendium of the human proteome. The HPP Guidelines for Mass Spectrometry Data Interpretation version 2.1 were applied to manuscripts submitted for this 2016 C-HPP-led special issue [ www.thehpp.org/guidelines ]. The Human Proteome presented as neXtProt version 2016-02 has 16,518 confident protein identifications (Protein Existence [PE] Level 1), up from 13,664 at 2012-12, 15,646 at 2013-09, and 16,491 at 2014-10. There are 485 proteins that would have been PE1 under the Guidelines v1.0 from 2012 but now have insufficient evidence due to the agreed-upon more stringent Guidelines v2.0 to reduce false positives. neXtProt and PeptideAtlas now both require two non-nested, uniquely mapping (proteotypic) peptides of at least 9 aa in length. There are 2,949 missing proteins (PE2+3+4) as the baseline for submissions for this fourth annual C-HPP special issue of Journal of Proteome Research. PeptideAtlas has 14,629 canonical (plus 1187 uncertain and 1755 redundant) entries. GPMDB has 16,190 EC4 entries, and the Human Protein Atlas has 10,475 entries with supportive evidence. neXtProt, PeptideAtlas, and GPMDB are rich resources of information about post-translational modifications (PTMs), single amino acid variants (SAAVSs), and splice isoforms. Meanwhile, the Biology- and Disease-driven (B/D)-HPP has created comprehensive SRM resources, generated popular protein lists to guide targeted proteomics assays for specific diseases, and launched an Early Career Researchers initiative.
| S-EPMC5129622 | biostudies-literature
Project description:BackgroundResearch on the human urine proteome may lay the foundation for the discovery of relevant disease biomarkers. Post-translational modifications (PTMs) have important effects on the functions of protein biomarkers. Identifying PTMs without enrichment adds no extra steps to conventional identification procedures for urine proteomics. The only difference is that this method requires software that can conduct unrestrictive identifications of PTMs. In this study, routine urine proteomics techniques were used to identify urine proteins. Unspecified PTMs were searched by MODa and PEAKS 6 automated software, followed by a manual search to screen out in vivo PTMs by removing all in vitro PTMs and amino acid substitutions.ResultsThere were 75 peptides with 6 in vivo PTMs that were found by both MODa and PEAKS 6. Of these, 34 peptides in 18 proteins have novel in vivo PTMs compared with the annotation information of these proteins on the Universal Protein Resource website. These new in vivo PTMs had undergone methylation, dehydration, oxidation, hydroxylation, phosphorylation, or dihydroxylation.ConclusionsIn this study, we identified PTMs of urine proteins without the need for enrichment. Our investigation may provide a useful reference for biomarker discovery in the future.
| S-EPMC3585864 | biostudies-literature
Project description:Cardiovascular complications are the leading cause of death in diabetic patients. Decades of research has focused on altered gene expression, altered cellular signaling, and altered metabolism. This work has led to better understanding of disease progression and treatments aimed at reversing or stopping this deadly process. However, one of the pieces needed to complete the puzzle and bridge the gap between altered gene expression and changes in signaling/metabolism is the proteome and its host of modifications. Defining the mechanisms of regulation includes examining protein levels, localization, and activity of the functional component of cellular machinery. Excess or misutilization of nutrients in obesity and diabetes may lead to PTMs contributing to cardiovascular disease progression. PTMs link regulation of metabolic changes in the healthy and diseased heart with regulation of gene expression itself (e.g. epigenetics), protein enzymatic activity (e.g. mitochondrial oxidative capacity), and function (e.g. contractile machinery). Although a number of PTMs are involved in each of these pathways, we will highlight the role of the serine and threonine O-linked addition of β-N-acetyl-glucosamine or O-GlcNAcylation. This nexus of nutrient supply, utilization, and storage allows for the modification and translation of mitochondrial function to many other aspects of the cell.
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Project description:We present the data for the global proteome and post-translational modification mapping of Labeo rohita (Rohu) which consists of mass-spectrometric (MS) data for 8498 proteins at 1% false discovery rate, which constitutes 26% of the total protein-coding sequences in Rohu. This data consists of deep proteomics of 17 normal tissues including eye, spinal cord, brain, male gonad, female gonad, gill, air bladder, gall bladder, gut, liver, heart, kidney, skin, scales, muscle, fin, spleen, as well as blood plasma and embryo of Rohu. The data from SRM-based targeted analysis to validate the presence of few key proteins is also included. Global post translational modification-based analysis (global PTM) was also performed in the studied tissues and its background data is also publicly accessible. This data and the web-based proteome map may aid applied and basic research endeavors in aquaculture to meet the food demands and nutritional security challenges of an increasing world population. The data here is related to the research article "Organ-based proteome and post-translational modification profiling of a widely cultivated tropical water fish, Labeo rohita" in the Journal of Proteome Research [1].
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Project description:The cytoskeletal protein tubulin plays an integral role in the functional specialization of many cell types. In the central nervous system, post-translational modifications and the expression of specific tubulin isotypes in neurons have been analyzed in greater detail than in their astrocytic counterparts. In this study, we characterized post-translational specifications of tubulin in human astrocytes using the normal human astrocyte (NHA; Lonza) commercial cell line of fetal origin. Immunocytochemical techniques were implemented in conjunction with confocal microscopy to image class III ?-tubulin (?III-tubulin), acetylated tubulin, and polyglutamylated tubulin using fluorescent antibody probes. Fluorescent probe intensity differences and colocalization were quantitatively assessed with the 'EBImage' package for the statistical programming language R. Colocalization analysis revealed that, although both acetylated tubulin and polyglutamylated tubulin showed a high degree of correlation with ?III-tubulin, the correlation with acetylated tubulin was stronger. Quantification and statistical analysis of fluorescence intensity demonstrated that the fluorescence probe intensity ratio for acetylated tubulin/?III-tubulin was greater than the ratio for polyglutamylated tubulin/?III-tubulin. The open source GEODATA set GSE819950, comprising RNA sequencing data for the NHA cell line, was mined for the expression of enzymes responsible for tubulin modifications. Our analysis uncovered greater expression at the mRNA level for enzymes reported to function in acetylation and deacetylation as compared to enzymes implicated in glutamylation and deglutamylation. Taken together, the results represent a step toward unraveling the tubulin isotypic expression profile and post-translational modification patterns in astrocytes during human brain development.
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Project description:The data reported here are associated with the article entitled "Analysis of Post-Translational Modification Dynamics Unveiled Novel Insights into Rice Responses to MSP1" [1]. pathogen-associated molecular pattern (PAMP) -triggered immunity (PTI) serves as the fundamental defense mechanism in plants, providing innate protection against pathogen invasion. The fungus Magnaporthe oryzae (M. oryzae) secretes MSP1, a protein recognized as a PAMP that induces PTI responses in rice. However, the comprehensive characterization of MSP1-induced post-translational modifications (PTMs) and their contribution to PTI responses remains elusive thus far. In this manuscript, we report the analysis of the phosphoproteome, ubiquitinome, and acetylproteome to investigate the alterations in MSP1-induced changes in these PTMs in MSP1 overexpressed and wild-type rice, utilizing the QExactiveTM Orbitrap High-Resolution Mass Spectrometer [1]. Our data primarily focuses on unraveling the PTMs of MSP1-overexpressing transgenic rice, with the goal of elucidating MSP1-induced signaling cascades and deciphering their regulatory mechanisms.
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Project description:Protein post-translational modifications are an important biological regulatory mechanism, and the rate of their discovery using high throughput techniques is rapidly increasingly. To make use of this wealth of sequence data, we introduce a new general strategy designed to predict a variety of post-translational modifications in several organisms. We used the motif-x program to determine phosphorylation motifs in yeast, fly, mouse, and man and lysine acetylation motifs in man. These motifs were then scanned against proteomic sequence data using a newly developed tool called scan-x to globally predict other potential modification sites within these organisms. 10-fold cross-validation was used to determine the sensitivity and minimum specificity for each set of predictions, all of which showed improvement over other available tools for phosphoprediction. New motif discovery is a byproduct of this approach, and the phosphorylation motif analyses provide strong evidence of evolutionary conservation of both known and novel kinase motifs.
| S-EPMC2634583 | biostudies-literature