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

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.