Isotopic Labeling-Enabled Chemical Proteomics Analysis Revealed Structural and Functional Features of Allysine Modifications in Mammalian Cells and Tissues.

Allysine is a pivotal protein post-translational modification that regulates protein interaction and activities. It is also recognized as a marker of oxidative stress under certain metabolic and physiological conditions. In this study, we developed a capture-and-release chemical proteomics workflow with heavy isotopic labeling that enables system-wide enrichment and site-specific identification of allysine as well as other carbonylated peptides, such as peptides containing glutamic semialdehyde derived from the oxidative damage of arginine and proline, with high confidence. The streamlined workflow led to the identification of 434 allysine sites on 349 proteins in human 293T and HCT116 cells and 317 allysine sites on 157 proteins in mouse muscle tissues without any treatment with an oxidative stress-inducing chemical reagent. We identified 48 histone allysine sites, including 38 sites on core histones in human 293T cells, many of which overlapped with well-characterized histone acetylation and methylation epigenetic marks. Bioinformatic analysis revealed notable characteristics of the amino acid preferences of allysine flanking sequences and the significant depletion of allysine sites in the protein secondary structure in cultured human cells. Pathway analysis showed that allysine substrates were involved in diverse cellular processes including translation, protein folding, and RNA processing in human cells and were enriched with muscle contractile fiber proteins and metabolic enzymes in mouse muscle tissue. Thus, our integrated chemical proteomics analysis revealed the structural and functional features of allysine targets under regular growth conditions in cultured human cells and mouse tissues.