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Mol Cell Proteomics DOI:10.1074/mcp.O114.047555

Deep, Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow.

Publication TypeJournal Article
Year of Publication2015
AuthorsSvinkina, T, Gu, H, Silva, JC, Mertins, P, Qiao, J, Fereshetian, S, Jaffe, JD, Kuhn, E, Udeshi, ND, Carr, SA
JournalMol Cell Proteomics
Volume14
Issue9
Pages2429-40
Date Published2015 Sep
ISSN1535-9484
KeywordsAcetylation, Animals, Antibodies, Monoclonal, Female, Humans, Jurkat Cells, Liver, Lysine, Mammary Neoplasms, Experimental, Mass Spectrometry, Mice, Protein Processing, Post-Translational, Proteomics, Workflow
Abstract

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.

URLhttp://www.mcponline.org/cgi/pmidlookup?view=long&pmid=25953088
DOI10.1074/mcp.O114.047555
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/25953088?dopt=Abstract

Alternate JournalMol. Cell Proteomics
PubMed ID25953088
PubMed Central IDPMC4563726
Grant ListU24 CA160034 / CA / NCI NIH HHS / United States
R01HL096738 / HL / NHLBI NIH HHS / United States
R01 HL096738 / HL / NHLBI NIH HHS / United States
HHSN268201000033C / / PHS HHS / United States
HHSN268201000033C / HL / NHLBI NIH HHS / United States
U24CA160034 / CA / NCI NIH HHS / United States