Detection of arginine dimethylated peptides by parallel precursor ion scanning mass spectrometry in positive ion mode.
Dimethylation at arginine residues has been shown to be central in cellular processes such as signal transduction, transcription activation, and protein sorting. The two methyl groups are either placed symmetric or asymmetric on the zeta standing nitrogen atoms of the arginine side chain. Here, we introduce a novel method that enables the localization of dimethylarginine (DMA) residues in gel-separated proteins at a level of sensitivity of better than 1 pmol and that allows one to distinguish between the isomeric symmetric and asymmetric position of the methyl groups. The method utilizes two side-chain fragments of DMA, the dimethylammonium ion (m/z 46.06) and the dimethylcarbodiimidium ion (m/z 71.06), for positive ion mode precursor ion scanning. Dimethylcarbodiimidium ions (m/z 71.06) are produced by symmetric as well as asymmetric dimethylarginine but are observed more strongly for symmetric DMA. It is utilized here in the precursor of m/z 71 scan to indicate the presence of DMA in a peptide. The dimethylammonium ion (m/z 46.06) is specific for asymmetric DMA and is utilized here in the precursor of m/z 46 scan. The positive ion mode allows for the identification of the protein by peptide sequencing and simultaneous detection and localization of the modified residues. The analysis can be conducted on any mass spectrometer capable of precursor ion scanning. However, the high resolution of a quadrupole TOF instrument is beneficial to assign the accurate charge state of the often highly charged precursors. Using the precursor of m/z 71 scan, we found FUS/TLS and Sam68 to be DMA-containing proteins. We discovered at least 20 DMA sites in FUS/TLS. In MS/MS, we observed neutral loss of dimethylamine (m/z 45.05) from which it follows that the dimethylation in FUS/TLS is asymmetric. Monitoring in parallel the fragments m/z 46.06 and 71.06 in precursor ion scans and peptide sequencing, we identified at least nine asymmetric DMA modifications in Sam68. The parallel monitoring of fragments in precursor ion scans is a versatile tool to specify the nature of protein modifications in cases where a single fragment is not conclusive.
Rappsilber
Juri
J
Center for Experimental Bioinformatics and Department of Biochemistry & Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
Friesen
Westley J
WJ
Paushkin
Sergey
S
Dreyfuss
Gideon
G
Mann
Matthias
M
D000595:Amino Acid Sequence; D001120:Arginine / Q000032:analysis* / Q000737:chemistry*; D002412:Cations; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*
D002412:Cations; D010446:Peptide Fragments; D010455:Peptides; D001120:Arginine