Hydrophobic Derivatization Strategy Facilitates Comprehensive Profiling of Protein Methylation

Protein methylation is receiving more and more attention
due to
its essential role in diverse biological processes. Large-scale analysis
of protein methylation requires the efficient identification of methylated
peptides at the proteome level; unfortunately, a significant number
of methylated peptides are highly hydrophilic and hardly retained
during reversed-phase chromatography, making it difficult to be identified
by conventional approaches. Herein, we report the development of a
novel strategy by combining hydrophobic derivatization and high pH
strong cation exchange enrichment, which significantly expands the
identification coverage of the methylproteome. Noteworthily, the total
number of identified methylated short peptides was improved by more
than 2-fold. By this strategy, we identified 492 methylation sites
from NCI-H460 cells compared to only 356 sites identified in native
forms. The identification of methylation sites before and after derivatization
was highly complementary. Approximately 2-fold the methylation sites
were obtained by combining the results identified in both approaches
(native and derivatized) as compared with the only analysis in native
forms. Therefore, this novel chemical derivatization strategy is a
promising approach for the comprehensive identification of protein
methylation by improving the identification of methylated short peptides.