title: Deep, Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic 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.
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, Cambridge Massachusetts 02142;
authors.forename: Tanya
authors.initials: T
authors.lastname: Svinkina
authors.affiliation: §Cell Signaling Technology, Inc. Danvers Massachusetts 01923.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Gu
authors.affiliation: §Cell Signaling Technology, Inc. Danvers Massachusetts 01923.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Silva
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, Cambridge Massachusetts 02142;
authors.forename: Philipp
authors.initials: P
authors.lastname: Mertins
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, Cambridge Massachusetts 02142;
authors.forename: Jana
authors.initials: J
authors.lastname: Qiao
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, Cambridge Massachusetts 02142;
authors.forename: Shaunt
authors.initials: S
authors.lastname: Fereshetian
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, Cambridge Massachusetts 02142;
authors.forename: Jacob D
authors.initials: JD
authors.lastname: Jaffe
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, Cambridge Massachusetts 02142;
authors.forename: Eric
authors.initials: E
authors.lastname: Kuhn
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, Cambridge Massachusetts 02142; udeshi@broadinstitute.org scarr@broad.mit.edu.
authors.forename: Namrata D
authors.initials: ND
authors.lastname: Udeshi
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, Cambridge Massachusetts 02142; udeshi@broadinstitute.org scarr@broad.mit.edu.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Carr
mesh_terms: D000107:Acetylation; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D005260:Female; D006801:Humans; D019169:Jurkat Cells; D008099:Liver / Q000378:metabolism*; D008239:Lysine / Q000276:immunology / Q000378:metabolism*; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D057188:Workflow
chemical_list: D000911:Antibodies, Monoclonal; D008239:Lysine