Accurate localization and relative quantification of arginine methylation using nanoflow liquid chromatography coupled to electron transfer dissociation and orbitrap mass spectrometry.
Protein arginine (Arg) methylation serves an important functional role in eucaryotic cells, and typically occurs in domains consisting of multiple Arg in close proximity. Localization of methylarginine (MA) within Arg-rich domains poses a challenge for mass spectrometry (MS)-based methods; the peptides are highly charged under electrospray ionization (ESI), which limits the number of sequence-informative products produced by collision induced dissociation (CID), and loss of the labile methylation moieties during CID precludes effective fragmentation of the peptide backbone. Here the fragmentation behavior of Arg-rich peptides was investigated comprehensively using electron-transfer dissociation (ETD) and CID for both methylated and unmodified glycine-/Arg-rich peptides (GAR), derived from residues 679-695 of human nucleolin, which contains methylation motifs that are widely-represented in biological systems. ETD produced abundant information for sequencing and MA localization, whereas CID failed to provide credible identification for any available charge state (z = 2-4). Nevertheless, CID produced characteristic neutral losses that can be employed to distinguish among different types of MA, as suggested by previous works and confirmed here with product ion scans of high accuracy/resolution by an LTQ/Orbitrap. To analyze MA-peptides in relatively complex mixtures, a method was developed that employs nano-LC coupled to alternating CID/ETD for peptide sequencing and MA localization/characterization, and an Orbitrap for accurate precursor measurement and relative quantification of MA-peptide stoichiometries. As proof of concept, GAR-peptides methylated in vitro by protein arginine N-methyltransferases PRMT1 and PRMT7 were analyzed. It was observed that PRMT1 generated a number of monomethylated (MMA) and asymmetric-dimethylated peptides, while PRMT7 produced predominantly MMA peptides and some symmetric-dimethylated peptides. This approach and the results may advance understanding of the actions of PRMTs and the functional significance of Arg methylation patterns.
Wang
Hao
H
Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, New York, USA.
Straubinger
Robert M
RM
Aletta
John M
JM
Cao
Jin
J
Duan
Xiaotao
X
Yu
Haoying
H
Qu
Jun
J
D019540:Area Under Curve; D001120:Arginine / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D005998:Glycine / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D000097796:Nucleolin
D001120:Arginine; D005998:Glycine; D008780:Methyltransferases; D010446:Peptide Fragments; D010455:Peptides; D010750:Phosphoproteins; D011484:Protein-Arginine N-Methyltransferases; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D000097796:Nucleolin; C099988:PRMT1 protein, human; C487994:PRMT7 protein, human