Methylome Analysis Based On Mass Spectrometry

As one of the important posttranslational modifications,methylation has considerable impacts on physiological processes.Methylation mainly occurs at lysine and arginine residues and the mostly occurred form of methylation is the N-linked methylation on Arg and Lys which is induced by methyltransferases.Methylation on these two amino acids contains six forms,including mono-methylation,di-methylation and tri-methylation on Lys,as well as mono-methylation,symmetric di-methylation and asymmetric di-methylation on Arg.In this work,we mainly focused on Lys and Arg methylation on proteome and peptidome of human cell lines and explored the characteristic of methylation based on high resolution mass spectrometry.We employ high p H reversed phase chromatography and deglyco-assisted methylation site identification method to detect the methylation sites of THP-1 cell line and PMA-induced macrophages in the basis of traditional shotgun proteomic strategies.In total,274 methylation sites were identified in THP-1 cells and 260 methylation sites were in THP-1 derived macrophages.Through further analysis on protein function,we found that the level of methylation before and after induction was different,indicating that methylation was involved and potentially play a regulatory role in the differentiation process of THP-1.In addition,we established a peptidomics-based workflow to discover and characterize the global methylome of endogenous peptides in seven human cells for the first time.In this work,endogenous peptides of human cell lines were extracted by hot water and acidified methanol.Subsequently,the methylated peptides were enriched by deglyco-assisted methylation site identification method.The results showed the excellent complementary use of these two reagents.The reliability of identifications was validated by methyl-SILAC labeling,resulting in 83% true-positive identifications in He La cells.In total,700 methylated forms on 646 putative methylation sites were identified in seven cell lines,with over 61% of the methylation sites being newly identified.This study provides a first methylome draft of endogenous peptides of human cell lines,offering a valuable resource for in-depth studies of biological functions of methylome of endogenous peptides.