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.
Highlights
Translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics
The non-acetylated peptide signal from clone 3 was much higher than the background signal from the other clones (1, 2, 4, 5, 6, and 7) by ELISA assay (Fig. 1A, supplemental Table S1), the mass spectrometry results clearly highlight the importance of this clone in the final formulation
We find that Basic Reversed Phase (bRP) fractionation of isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tag (TMT) labeled peptides leads to a uniform distribution of peptides across four fractions
Summary
A landmark study by Choudhary et al used anti-Kac antibodies to globally map 3600 lysine acetylation sites on 1750 proteins, thereby demonstrating the feasibility of profiling the acetylome [10]. To improve the depth-of-coverage in acetylation profiling experiments there is a clear need for [1] alternative anti-acetyl lysine antibodies with higher specificity, [2] optimized antibody usage parameters, and [3] robust proteomic workflows that permit low to moderate protein input. We describe a newly commercialized mixture of anti-Kac antibodies and detail a complete proteomic workflow for achieving unprecedented coverage of the acetylome from a single stable isotope labeling by amino acids in cell culture (SILAC) labeled sample as well as isobaric tags for relative and absolute quantitation (iTRAQ)- and tandem mass tag (TMT)-labeled samples
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