In vitro methylation by methanol: proteomic screening and prevalence investigation.
It is assumed that much more functional importance for protein activity than expected may be granted by methylation that occurs at the side-chain of aspartate or glutamate residue. In vitro methylation mainly comes from the use of methanol in sample preparation prior to MS analysis. In this study, we first performed the methylation site-directed proteomic screening of bovine serum albumin, ovalbumin and 20S proteasome for gel staining using a meaningfully indicative MS-pattern of peak tag (termed as 4P tag) and manual inspection for mass spectral data. As a result, there were 17 proteolytic peptides with 20 modified sites confirmed to be in vitro methylated. Subsequently, the prevalence investigation was performed, focusing on the reaction kinetic behavior of in vitro methylation. This study provided a simple and robust approach for confirmation of in vitro methylation by methanol, as well as the precautious guide for the use of methanol in proteomic study.
Chen
Guoqiang
G
Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China.
Liu
Hui
H
Wang
Xiaodong
X
Li
Zhili
Z
D000595:Amino Acid Sequence; D000818:Animals; D057230:Limit of Detection; D000432:Methanol / Q000737:chemistry*; D008745:Methylation; D008969:Molecular Sequence Data; D040901:Proteomics; D020539:Sequence Analysis, Protein; D012710:Serum Albumin, Bovine; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D012710:Serum Albumin, Bovine; D000432:Methanol