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Tryptic Peptides Bearing C-Terminal Dimethyllysine Need to Be Considered during the Analysis of Lysine Dimethylation in Proteomic Study.

Abstract：

:Lysine methylation plays important roles in structural and functional regulation of chromatin. Although trypsin is the most widely used protease in mass spectrometry-based proteomic analysis for lysine methylation substrates, the proteolytic activity of trypsin on dimethylated lysine residues remains an arguable issue. In this study, we tested the ability of trypsin to cleave dimethylated lysine residues in synthetic peptides, purified albumin, and whole cell lysate, and found that the C-terminal of dimethylated lysine residue could be cleaved in a protein sequence-dependent manner. Kinetic studies revealed that the optimal digestion time and enzyme-to-substrate ratio for the cleavage of dimethylated lysine by trypsin was around 16 h and 1:50, respectively. We further showed the tryptic C-terminal lysine-dimethylated (C-Kme2) peptides could contribute to a significant portion of substrate identification in the proteomic study, which utilizes the chemical dimethylation labeling approach. More than 120 tryptic C-Kme2 peptides (7% of total peptides identified) were identified in chemically lysine-dimethyl-labeled HeLa whole cell lysate by a single-shot nanoflow high performance liquid chromatography with tandem mass spectrometry (nano-HPLC-MS/MS) analysis. Moreover, in an assay for substrate identification of protease Glu-C using stable isotope dimethyl labeling approach, our data showed the tryptic C-Kme2 peptides accounted for more than 13% of total tryptic peptides. Additionally, our in vivo methylome profiling data revealed some C-Kme2 peptides, which is of importance to identification and quantification of biologically relevant protein and lysine-methylated site. Therefore, we reason that the tryptic peptides bearing C-terminal dimethylated lysine need to be considered in the mass spectrometric analysis of lysine dimethylation.

journal_name

J Proteome Res

journal_title

Journal of proteome research

authors

Chen M,Zhang M,Zhai L,Hu H,Liu P,Tan M

doi

10.1021/acs.jproteome.7b00373

subject

Has Abstract

pub_date

2017-09-01 00:00:00

pages

3460-3469

issue

eissn

1535-3893

issn

1535-3907

journal_volume

pub_type

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Tryptic Peptides Bearing C-Terminal Dimethyllysine Need to Be Considered during the Analysis of Lysine Dimethylation in Proteomic Study.