Abstract
Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies.
Highlights
Lysine acetylation is a post-translational modification that has been extensively studied for their roles in cell signaling, transcription and metabolism through its effects on protein activity, subcellular localization and interaction with protein or DNA [1, 2]
Various work have found more than a thousand acetylated proteins in the proteome, belonging to large macromolecular complexes involved in many cellular functions such as chromatin remodeling, nuclear transport and mitochondrial metabolism [6,7,8]
To improve the coverage of acetyl-lysine peptides that are identified in acetylome studies, it is important to develop acetyl-lysine antibodies that are more diverse in their epitope recognition
Summary
Lysine acetylation is a post-translational modification that has been extensively studied for their roles in cell signaling, transcription and metabolism through its effects on protein activity, subcellular localization and interaction with protein or DNA [1, 2]. Initial studies of acetylation have focused on single proteins such as tubulin, histones and p53, relying often on the use of radioactively labeled acetyl-CoA for selective acetylation of these proteins to study their biological functions [3,4,5]. Since the advent of proteomic approaches, scientists have advanced on studying entire acetylome. Various work have found more than a thousand acetylated proteins in the proteome, belonging to large macromolecular complexes involved in many cellular functions such as chromatin remodeling, nuclear transport and mitochondrial metabolism [6,7,8].
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