Complications in the assignment of 14 and 28 Da mass shift detected by mass spectrometry as in vivo methylation from endogenous proteins.
Identification of protein methylation sites typically starts with database searching of MS/MS spectra of proteolytic digest of the target protein by allowing addition of 14 and 28 Da in the selected amino acid residues that can be methylated. Despite the progress in our understanding of lysine and arginine methylation, substrates and functions of protein methylation at other amino acid residues remain unknown. Here we report the analysis of protein methylation for p53, SMC3, iNOS, and MeCP2. We found that a large number of peptides can be modified on the lysine, arginine, histidine, and glutamic acid residues with a mass increase of 14 or 28 Da, consistent with methylation. Surprisingly, a majority of which did not demonstrate a corresponding mass shift when cells were cultured with isotope-labeled methionine, a precursor for the synthesis of S-adenosyl-l-methionine (SAM), which is the most commonly used methyl donor for protein methylation. These results suggest the possibility of either exogenous protein methylation during sample handling and processing for mass spectrometry or the existence of SAM-independent pathways for protein methylation. Our study found a high occurrence of protein methylation from SDS-PAGE isolated endogenous proteins and identified complications for assigning such modifications as in vivo methylation. This study provides a cautionary note for solely relying on mass shift for mass spectrometric identification of protein methylation and highlights the importance of in vivo isotope labeling as a necessary validation method.
Jung
Sung Yun
SY
Center for Molecular Discovery, Verna and Mars McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Li
Yehua
Y
Wang
Yi
Y
Chen
Yue
Y
Zhao
Yingming
Y
Qin
Jun
J
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D013058:Mass Spectrometry; D008745:Methylation; D051379:Mice; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results
D011506:Proteins