Mass spectrometry-based identification and characterisation of lysine and arginine methylation in the human proteome

Abstract

Protein methylation is a post-translational modification (PTM) by which a variable number of methyl groups are transferred to lysine and arginine residues within proteins. Despite increased interest in this modification due to its reversible nature and its emerging role in a diverse set of biological pathways beyond chromatin, global identification of protein methylation has remained an unachieved goal. To characterise sites of lysine and arginine methylation beyond histones, we employed an approach that combines heavy methyl stable isotope labelling by amino acids in cell culture (hmSILAC) with high-resolution mass spectrometry-based proteomics. Through a broad evaluation of immuno-affinity enrichment and the application of two classical protein separation techniques prior to mass spectrometry, to nucleosolic and cytosolic fractions separately, we identified a total of 501 different methylation types, on 397 distinct lysine and arginine sites, present on 139 unique proteins. Our results considerably extend the number of known in vivo methylation sites and indicate their significant presence on several protein complexes involved at all stages of gene expression, from chromatin remodelling and transcription to splicing and translation. In addition, we describe the potential of the hmSILAC approach for accurate relative quantification of methylation levels between distinct functional states.

Proteomic Analysis of Arginine Methylation Sites in Human Cells Reveals Dynamic Regulation During Transcriptional Arrest

“…Proteins modified by MMA were grouped using associated GO biological processes, and reveals that methylated proteins prominently are involved in RNA processing, RNA transportation, chromatin remodeling and transcription. based proteomics, including 10-fold lower amounts of antibody (12-24 g) for the immunoenrichment steps (10,62,72). Notably, our described methodology does not entail use of any methanol-based sample preparation, which previously has been described to induce artificial mono-methylations on glutamic acids (67), and potentially could give rise to incorrect identifications of protein arginine methylation sites during database searches.…”

“…S3). Both DDX5 and DDX17 were recently confirmed by Western blot to harbor arginine methylation (62), yet the exact amino acid location of these modified sites was not mapped. As an example of a protein harboring regulated MMA sites that do not conform to our cluster analysis, we find the RNA-processing factor THRAP3/THRAP150 to be modified with MMA at position R66.…”

A Chemical Proteomics Approach for Global Analysis of Lysine Monomethylome Profiling *

“…reported previously (24,25,45,46). A complete list of the methyllysine peptides and proteins identified in this study is provided in supplemental Table S1 and supplemental Fig.…”

“…Thus, it is still a daunting challenge to develop sequence-independent (or pan) anti-Kme antibodies with adequate affinity and specificity. As a consequence, progress in identifying Kme substrates has been slow, largely because of a lack of suitable affinity enrichment technology for isolation of Kme peptides that can be used for mass spectrometry-based proteomic screening (23)(24)(25)(26).…”

Modulation of the cytoplasmic functions of mammalian post-transcriptional regulatory proteins by methylation and acetylation: a key layer of regulation waiting to be uncovered?

“…It seems highly unlikely that the currently documented numbers of meKs is an accurate reflection of this PTM in the proteome, since more than 50 putative lysine methyltransferases (KMTs) and approximately 25 lysine demethylases (KDMTs) are encoded within the human genome, although very few have annotated substrates. Consistent with this view, recent proteomic studies collectively document several hundred novel meK residues (still relatively few compared with the >10 000 acK residues documented) across a broad range of protein classes [66][67][68][69] (see Table 1 for examples relating to post-transcriptional control).…”