title: Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides.
abstract: Lysine acetylation has emerged as one of the major post-translational modifications, as indicated by its roles in chromatin remodeling, activation of transcription factors and, most recently, regulation of metabolic enzymes. Identification of acetylation sites in a protein is the first essential step for functional characterization of acetylation in physiological regulation. However, the study of the acetylome is hindered by the lack of suitable physical and biochemical properties of the acetyl group and existence of high-abundance acetylated histones in the cell, and needs a robust method to overcome these problems. Here we present protocols for (i) using chemically acetylated ovalbumin and synthetic acetylated peptide to generate a pan-acetyllysine antibody and a site-specific antibody to Lys288-acetylated argininosuccinate lyase, respectively; (ii) using subcellular fractionation to reduce highly abundant acetylated histones; and (iii) using acetyllysine antibody affinity purification and mass spectrometry to characterize acetylome of human liver tissue. The entire characterization procedure takes ∼2-3 d to complete.
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences and Institutes of Biomedical Sciences, Fudan Unversity, Shanghai, China. kuguan@ucsd.edu
authors.forename: Kun-Liang
authors.initials: KL
authors.lastname: Guan
authors.forename: Wei
authors.initials: W
authors.lastname: Yu
authors.forename: Yan
authors.initials: Y
authors.lastname: Lin
authors.forename: Yue
authors.initials: Y
authors.lastname: Xiong
authors.forename: Shimin
authors.initials: S
authors.lastname: Zhao
mesh_terms: D000107:Acetylation; D000906:Antibodies / Q000378:metabolism; D001123:Argininosuccinate Lyase / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007158:Immunologic Techniques; D008099:Liver / Q000737:chemistry; D008239:Lysine / Q000378:metabolism; D010047:Ovalbumin / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D013347:Subcellular Fractions / Q000737:chemistry
chemical_list: D000906:Antibodies; D010455:Peptides; D010047:Ovalbumin; D001123:Argininosuccinate Lyase; D008239:Lysine