Negative Ion Mode Collision-Induced Dissociation for Analysis of Protein Arginine Methylation

Abstract

Arginine methylation is a common protein post-translational modification (PTM) that plays a key role in eukaryotic cells. Three distinct types of this modification are found in mammals: asymmetric N η1 N η1 -dimethylarginine (aDMA), symmetric N η1 N η2 -dimethylarginine (sDMA), and an intermediate N η1 -monomethylarginine (MMA). Elucidation of regulatory mechanisms of arginine methylation in living organisms requires precise information on both the type of the modified residues and their location inside the protein amino acid sequences. Despite mass spectrometry (MS) being the method of choice for analysis of multiple protein PTMs, unambiguous characterization of protein arginine methylation may not be always straightforward. Indeed, frequent internal basic residues of Arg methylated tryptic peptides hamper their sequencing under positive ion mode collision-induced dissociation (CID), the standardly used tandem mass spectrometry method, while the relative stability of the aDMA and sDMA side chains under alternative non-ergodic electron-based fragmentation techniques, electron-capture and electron transfer dissociations (ECD and ETD), may impede differentiation between the isobaric residues. Here, for the first time, we demonstrate the potential of the negative ion mode collision-induced dissociation MS for analysis of protein arginine methylation and present data revealing that the negative polarity approach can deliver both an unambiguous identification of the arginine methylation type and extensive information on the modified peptide sequences.

Non-Histone Protein Methylation: Biological Significance and Bioengineering Potential

“…Standard MS workflows applied to studying protein methylation results in intolerably high false discovery rates due to the number of possible isobaric nonmethylated and methylated peptides, and the methylation of acidic residues arising when methanol/ethanol is present in sample preparation buffers . While it is possible to adjust some mass spectrometers to provide signatures indicative of arginine methylation (e.g., refs and ), these are not appropriate for global proteomics studies. The most robust approach is to use isomethionine methyl-stable isotope labeling of amino acids in cell culture (iMethyl-SILAC), a progression of heavy-methyl SILAC (hmSILAC) .…”

Current Methods of Post-Translational Modification Analysis and Their Applications in Blood Cancers

“…Recently, Wang et al [ 175 ] described a new chromatography-based method of methylome analysis combining SCX, IMAC and high-pH reversed-phase chromatography which led to the identification of 765 methylation sites [ 175 ]. Positive- and negative-mode CID, HCD and ETD fragmentation techniques have been applied for methylation analysis [ 176 ]. For quantitation, label-based techniques such as heavy-methyl SILAC and isomethionine methyl-SILAC; or label-free techniques are often used [ 171 , 177 , 178 , 179 ].…”

mNeuCode Empowers Targeted Proteome Analysis of Arginine Dimethylation

“…Protein arginine methylation is a widely occurring and relatively conserved protein post-translational modification in eukaryotes that is involved in a variety of biological processes, including RNA processing, DNA repair, chromosome organization, protein folding, and gene expression. − These modifications are primarily catalyzed by nine members of the protein arginine methyltransferases (PRMTs) that transfer the methyl groups from S -adenosyl- l -methionine (SAM) to the guanidinium group of arginine. Monomethylated arginine is formed as a first step and serves as an intermediate, followed by asymmetrical and symmetrical dimethylation.…”