Fragmentation Pathways of NG-Methylated and Unmodified Arginine Residues in Peptides Studied by ESI-MS/MS and MALDI-MS

Protein methylation at arginine residues is a prevalent posttranslational modification in eukaryotic cells that has been implicated in processes from RNA-binding and transporting to protein sorting and transcription activation. Three main forms of methylarginine have been identified: NG-monomethylarginine (MMA), asymmetric NG,NG-dimethylarginine (aDMA), and symmetric NG,NG-dimethylarginine (sDMA). To investigate gas-phase fragmentations and characteristic ions arising from methylated and unmodified arginine residues in detail, we subjected peptides containing these residues to electrospray triple-quadrupole tandem mass spectrometry. A variety of low mass ions including (methylated) ammonium, carbodiimidium, and guanidinium ions were observed. Fragment ions resulting from the loss of the correspond-ing neutral fragments (amines, carbodiimide, and guanidine) from intact molecular ions as well as from N- and C-terminal fragment ions were also identified. Furthermore, the peptides containing either methylated or unmodified arginines gave rise to abundant fragment ions at m/z 70, 112, and 115, for which cyclic ion structures are proposed. Electrospray ionization tandem mass spectra revealed that dimethylammonium (m/z 46) is a specific marker ion for aDMA. A precursor ion scanning method utilizing this fragment ion was developed, which allowed sensitive and specific detection of aDMA-containing peptides even in the presence of a five-fold excess of phosphorylase B digest. Interestingly, regular matrix-assisted laser desorption/ionization mass spectra recorded from aDMA- or sDMA-containing peptides showed metastable fragment ions resulting from cleavages of the arginine side chains. The neutral losses of mono- and dimethylamines permit the differentiation between aDMA an