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Deep, quantitative coverage of the lysine acetylome using novel anti-acetyl-lysine antibodies

ABSTRACT: Svinkina T, Gu H, Silva JC, Mertins P, Qiao J, Fereshetian S, Jaffe JD, Kuhn E, Udeshi ND,Carr SA. Mol Cell Proteomics, 2015. 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-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 2-fold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline bRP separation, and use of state-of-the art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods presented in this study enabled the quantification of over 10,000 Kac peptides from over 3000 Kac proteins from a single SILAC labeled sample using 7.5 mg of peptide input per state. These methods result in the deepest coverage of acetylation sites from SAHA treated Jurkat cells. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4-plex, TMT6 and TMT10-plex reagents) for quantification. Greater than 6,700 Kac peptides from over 2,300 Kac proteins were quantified using 1 mg of tumor per iTRAQ 4-plex channel.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000079068 | MassIVE | Sun Mar 08 19:59:00 GMT 2015

SECONDARY ACCESSION(S): PXD001887

REPOSITORIES: MassIVE

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