title: Monoclonal antibody cocktail as an enrichment tool for acetylome analysis.
abstract: The availability and robustness of methods to analyze phosphorylated proteins has greatly expanded our knowledge of phosphorylation based cell signaling. A key ingredient to the success of these studies is the ability to enrich phosphopeptides using antibodies or other chemical approaches. Most other post-translational modifications, such as lysine acetylation, are still poorly characterized because of the lack of availability of such enrichment methods. Recently, some groups have reported identification of acetylation sites in a global fashion by enriching acetylated peptides with a polyclonal antibody from a single source that was raised against pan-acetylated lysine. Instead of the use of this polyclonal antibody, we used a cocktail of monoclonal antibodies where each was directed against acetylated lysine in different contexts. Using high resolution Fourier transform mass spectrometry, we observed that the majority of acetylated lysine residues identified using the monoclonal antibody cocktail were distinct from those enriched by the polyclonal antibody used by the other groups. Our study demonstrates that immunoaffinity enrichment of acetylated peptides is somewhat limited by substrate specificity and that an optimal yield of enrichment can be achieved by employing a broader array of affinity reagents.
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Shaw
authors.forename: Raghothama
authors.initials: R
authors.lastname: Chaerkady
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhang
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Davidson
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D045744:Cell Line, Tumor; D005583:Fourier Analysis; D006801:Humans; D008239:Lysine / Q000737:chemistry* / Q000276:immunology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D010748:Phosphopeptides; D008239:Lysine