ac7b03673_si_003.xlsx (558.16 kB)
Strategy Based on Deglycosylation, Multiprotease, and Hydrophilic Interaction Chromatography for Large-Scale Profiling of Protein Methylation
dataset
posted on 2017-11-01, 00:00 authored by Min Ma, Xinyuan Zhao, Shuo Chen, Yingyi Zhao, Lu yang, Yu Feng, Weijie Qin, Lingjun Li, Chenxi JiaReversible
methylation of proteins regulates the majority of cellular
processes, including signal transduction, mRNA splicing, transcriptional
control, DNA repair, and protein translocation. A fundamental understanding
of these biological processes at the molecular level requires comprehensive
characterization of the methylated proteins. Methylation is often
substoichiometric, and only a very limited number of methylated proteins
and sites have been confidently identified to date. Although the intrinsically
basic/hydrophilic methylated peptides can be enriched by the hydrophilic
interaction liquid chromatography (HILIC), other hydrophilic peptides
can coelute during the enrichment process and suppress the detection
of methylated peptides. In addition, the modified Arg and Lys residues
cannot be efficiently cleaved by trypsin, the most commonly used enzyme
in shotgun proteomics. To overcome these caveats, we develop a novel
de-glyco-assisted methylation site identification (DOMAIN) strategy
which enables straightforward, fast, and reproducible analysis of
protein methylation in a proteome-wide manner. Combining multidimensional
fractionation and multiprotease digestion, our method enabled the
identification of 573 methylated forms in 270 proteins, including
311 new methylation forms, in A549 cells. Combining this technique
with stable isotope labeling quantitative proteomics and RNA interference,
we determined the differential regulation of several putative methylated
sites that are related to the protein arginine N-methyltransferase
3 (PRMT3). Collectively, our integrated proteomics workflow for comprehensive
mapping of methylation sites enables a better understanding of protein
methylation, while providing a rapid and effective approach for global
protein methylation analysis in biomedical research.