Global Lysine Methylome Profiling Using Systematically Characterized Affinity Reagents
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus, Orbitrap Exploris 480
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Evan Cornett
PROVIDER: MSV000090519 | MassIVE | Wed Oct 12 12:34:00 BST 2022
REPOSITORIES: MassIVE
Similar Datasets
Project description:Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria, the results indicate a success rate up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents.
2016-04-13 | PXD003370 | Pride
Project description:Dynamic modification of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) regulates transcription-coupled processes in eukaryotes. The CTD in mammals is composed of 52 heptad-repeats with the consensus sequence Y1-S2-P3-T4-S5-P6-S7. Repeats in the distal part of CTD deviate from the consensus sequence and have frequently replaced serine at position 7 by lysine residues (K7). Mass spectrometry analysis revealed modification of K7 residues in heptad-repeats 39, 42, and 47/49 by acetylation and in heptad-repeats 38, 39, 40, 42, and 47/49 by mono-, di-, or trimethylation. Notably, acetylated as well as di- and tri-methylated K7 residues were found exclusively in phosphorylated CTD peptides, while mono-methylated K7 residues occurred also in non-phosphorylated CTD peptides. Methylation of K7 residues was further studied with the monoclonal antibody (mAb) 1F5, which recognizes mono- and di-methylated K7 in CTD. ChIP experiments revealed high levels of K7 methylation at the transcriptional start site of genes. The low levels of K7 methylation in the body of genes results from impairment of epitope recognition in hyper-phosphorylated CTD by mAb 1F5. In agreement with this notion, phosphatase treatment of hyper-phosphorylated CTD or treatment of cells with kinase inhibitor flavopiridol restored the reactivity of mAb 1F5 towards methylated K7 residues in CTD. We conclude that methylation and acetylation of K7 residues further expand the mammalian CTD code and potentially contribute to regulation of gene expression.
2015-12-01 | GSE72698 | GEO
Project description:Protein methylation is involved in different processes including gene expression regulation, epigenetics, and nucleotide metabolism. Identification of methylated proteins is difficult as methyl groups are small, and do not introduce significant changes in protein hydrophobicity or charge state. The most effective analytical method to date is relative enrichment by pan-specific antibodies and methylation specific binding domains. Here, we present a novel and unbiased chemical strategy to enrich and identify sites of lysine and arginine methylations. This approach makes use of the property that methylation does not alter the charge state of lysine and arginine. This approach revealed over 793 methylation events including 211 arginine and 585 lysine methylation sites in HEK 293 Cells. This strategy proves to be convenient, effective and versatile, with the potential of analyzing other PTMs on both lysine and arginine.
2016-08-16 | PXD001689 | Pride
Project description:Comparison of commercially available histone antibodies
| PRJNA278079 | ENA
Project description:We developed a quantitative assay for carefully selected VP40 peptide variants. We selected two peptides, LGPGIPDHPLR from EBOV VP40 and LRPILLPGK from BDBV VP40 to generate pan-peptide variants antibodies. Potential structural difference in virion particles affect the efficiency of VP40 liberation during sample inactivation, and the sequence variations also affect the affinity of peptide against the pan-variants antibody. Therefore, we quantified the mass contents of VP40 in EBOV and SUDV VLPs and used the VLPs from each species to build the species-specific standard curves. This improves the accuracy of VP40 quantitation. The feasibility of using miniature mass spectrometer to collect the tandem MS spectra for both peptides was validated.
2021-11-02 | PXD026048 | Pride
Project description:Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) is a prevalent posttranslational modification that regulates diverse cellular processes. Aberrant expression of type I PRMTs that catalyze asymmetric arginine demethylation (ADMA) is often found in cancer; however, little is known about the ADMA status of substrate proteins in tumors. Using LC-MS/MS along with pan-specific ADMA antibodies, we performed global mapping of ADMA in five patient-derived xenograft (PDX) tumors representing different subtypes of human breast cancer and identified 415 methylated peptides from 213 proteins. Approximately 70% of the putative substrates were validated using peptide arrays in vitro methylated by PRMT1, PRMT4, and PRMT6, among which 48% of substrates varied from estrogen receptor (ER) positive and negative tumors. Comparing with our previously identified ADMA sites from breast cancer cell lines, 75 ADMA sites overlapped between cell lines and PDX tumors. Collectively, this study provides a useful resource to PRMT and breast cancer communities to exploit the functions of PRMT dysregulation during breast cancer progression.
2024-01-26 | PXD038203 | Pride
Project description:Nitrotyrosine is a modification in proteins caused by oxidative reactions with reactive nitrogen species. It's commonly associated with oxidative stress and linked to various health conditions like inflammation, neurodegeneration, cardiovascular disease, and cancer. However, detecting nitrotyrosine-modified proteins is challenging due to their low levels in biological samples. To make this detection more comprehensive, we optimized an immunoprecipitation-based method for both proteins and peptides using a cell line model. We tested the efficiency of four different commercially available monoclonal antibodies for enriching nitrotyrosine-modified proteins and peptides. Through LC-MS/MS analysis, we identified 1,377 nitrotyrosine-containing peptides from protein-based enrichment and 1,624 from peptide-based enrichment. However, we noticed a bias in the peptides enriched; a significant portion (37-65%) had nitrotyrosine at the beginning of the peptide when using the peptide-based approach, whereas this bias was minimal (<9%) with the protein-based immunoprecipitation. In total, we found 2,605 nitrotyrosine-containing peptides, with over 2,000 of them not previously reported. To validate our findings, we synthesized 110 of these peptides and analyzed them with LC-MS/MS. Additionally, we confirmed the presence of nitrotyrosine modifications in PKM and EF2 proteins in peroxynitrite-treated samples through immunoblot analysis. This extensive dataset and workflow will aid further exploration of nitrotyrosine as an oxidative modification in various contexts.
2024-08-29 | PXD046168 | Pride
Project description:Nitrotyrosine is a modification in proteins caused by oxidative reactions with reactive nitrogen species. It's commonly associated with oxidative stress and linked to various health conditions like inflammation, neurodegeneration, cardiovascular disease, and cancer. However, detecting nitrotyrosine-modified proteins is challenging due to their low levels in biological samples. To make this detection more comprehensive, we optimized an immunoprecipitation-based method for both proteins and peptides using a cell line model. We tested the efficiency of four different commercially available monoclonal antibodies for enriching nitrotyrosine-modified proteins and peptides. Through LC-MS/MS analysis, we identified 1,377 nitrotyrosine-containing peptides from protein-based enrichment and 1,624 from peptide-based enrichment. However, we noticed a bias in the peptides enriched; a significant portion (37-65%) had nitrotyrosine at the beginning of the peptide when using the peptide-based approach, whereas this bias was minimal (<9%) with the protein-based immunoprecipitation. In total, we found 2,605 nitrotyrosine-containing peptides, with over 2,000 of them not previously reported. To validate our findings, we synthesized 110 of these peptides and analyzed them with LC-MS/MS. Additionally, we confirmed the presence of nitrotyrosine modifications in PKM and EF2 proteins in peroxynitrite-treated samples through immunoblot analysis. This extensive dataset and workflow will aid further exploration of nitrotyrosine as an oxidative modification in various contexts.
2024-03-12 | PXD048204 | Pride
Project description:We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine crotonylation (Kcr), confirming that it represents an evolutionarily-conserved histone posttranslational modification. The unique structure and genomic localization of histone Kcr suggest that it is mechanistically and functionally different from histone lysine acetylation (Kac). Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in postmeiotic male germ cells. 2 histone marks (pan-lysine acetylation and pan-lysine crotonylation) were studied in 1 human cell type and 2 mouse cell types using ChIP-Seq. Input was sequenced for each cell type as a control. Pan-anti_Kac and pan-anti_Kcr antibodies were custom developed with PTM BioLab, Co., Ltd (Chicago, IL).
2011-10-06 | E-GEOD-32663 | biostudies-arrayexpress
Project description:A computer program was used to create random amino acid sequences based on and restricted by physical shadow masks which will be used for lithography-based synthesis of peptides. The output from this algorithm was used to create peptides that were synthesized by Sigma Aldrich, and printed onto glass slides. The arrays contained 384 peptides printed in duplicate for each of 4 different mask designs. 52 different monoclonal antibodies were incubated on these microarrays and analyzed for their propensity to bind the peptides created from each mask set. The diversity of binding served as a proxy for the 'randomness' of these peptides, and provided information about how many masks are needed to truly generate random sequence peptides.
2014-01-16 | GSE49217 | GEO