title: Identification of Palmitoylated Transitional Endoplasmic Reticulum ATPase by Proteomic Technique and Pan Antipalmitoylation Antibody.
abstract: Protein palmitoylation plays a significant role in a wide range of biological processes such as cell signal transduction, metabolism, apoptosis, and carcinogenesis. For high-throughput analysis of protein palmitoylation, approaches based on the acyl-biotin exchange or metabolic labeling of azide/alkynyl-palmitate analogs are commonly used. No palmitoylation antibody has been reported. Here, the palmitoylated proteome of human colon cancer cell lines SW480 was analyzed via a TS-6B-based method. In total, 151 putative palmitoylated sites on 92 proteins, including 100 novel sites, were identified. Except for 3 known palmitoylated transmembrane proteins, ATP1A1, ZDHHC5, and PLP2, some important proteins including kinases, ion channels, receptors, and cytoskeletal proteins were also identified, such as CLIC1, PGK1, PPIA, FKBP4, exportin-2, etc. More importantly, the pan antipalmitoylation antibody was developed and verified for the first time. Our homemade pan antipalmitoylation antiserum could differentiate well protein palmitoylation from mouse brain membrane fraction and SW480 cells, which affords a new technique for analyzing protein palmitoylation by detecting the palmitic acid moiety directly. Furthermore, the candidate protein transitional endoplasmic reticulum ATPase (VCP) identified in SW480 cells was validated to be palmitoylated by Western blotting with anti-VCP antibody and the homemade pan antipalmitoylation antibody.
authors: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University , Shanghai 200433, China.
authors: Caiyun
authors: C
authors: Fang
authors: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University , Shanghai 200433, China.
authors: Xiaoqin
authors: X
authors: Zhang
authors: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University , Shanghai 200433, China.
authors: Lei
authors: L
authors: Zhang
authors: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University , Shanghai 200433, China.
authors: Xing
authors: X
authors: Gao
authors: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University , Shanghai 200433, China.
authors: Pengyuan
authors: P
authors: Yang
authors: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University , Shanghai 200433, China.
authors: Haojie
authors: H
authors: Lu
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D007106:Immune Sera / Q000737:chemistry / Q000302:isolation & purification; D015151:Immunoblotting; D054878:Lipoylation; D051379:Mice; D019308:Palmitic Acid / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011817:Rabbits
chemical_list: D007106:Immune Sera; D019308:Palmitic Acid; D000251:Adenosine Triphosphatases
keywords: palmitoylation; pan antipalmitoylation antibody; proteomic analysis; transitional endoplasmic reticulum ATPase