title: MS³ fragmentation patterns of monomethylarginine species and the quantification of all methylarginine species in yeast using MRM³.
abstract: Protein arginine methylation is one of the epigenetic modifications to proteins that is studied in yeast and is known to be involved in a number of human diseases. All eukaryotes produce Nη-monomethylarginine (ηMMA), asymmetric Nη1, Nη1-dimethylarginine (aDMA), and most produce symmetric Nη1, Nη2-dimethylarginine (sDMA) on proteins, but only yeast produce Nδ-monomethylarginine (δMMA). It has proven difficult to differentiate among all of these methylarginines using mass spectrometry. Accordingly, we demonstrated that the two forms of MMA have indistinguishable primary product ion spectra. However, the secondary product ion spectra of δMMA and ηMMA exhibited distinct patterns of ions. Using incorporation of deuterated methyl-groups in yeast, we determined which secondary product ions were methylated and their structures. Utilizing distinct secondary product ions, a triple quadrupole multiple reaction monitoring cubed (MRM(3)) assay was developed to measure δMMA, ηMMA, sDMA and aDMA derived from hydrolyzed protein. As a proof-of-concept, δMMA and ηMMA were measured using the MRM(3) method in wild type and mutant strains of Saccharomyces cerevisiae and compared to the total MMA measured using an existing assay. The MRM(3) assay represents the only method to directly quantify δMMA and the only method to simultaneously quantify all yeast methylarginines.
authors: Faculty of Pharmacy, The University of Manitoba, Winnipeg, Manitoba, Canada. Electronic address: ted.lakowski@ad.umanitoba.ca.
authors: Ted M
authors: TM
authors: Lakowski
authors: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
authors: András
authors: A
authors: Szeitz
authors: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
authors: Magnolia L
authors: ML
authors: Pak
authors: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
authors: Dylan
authors: D
authors: Thomas
authors: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
authors: Mynol I
authors: MI
authors: Vhuiyan
authors: Pharmaceutical Institute, Gutenbergstr. 76, 24118 Kiel, Germany.
authors: Joscha
authors: J
authors: Kotthaus
authors: Pharmaceutical Institute, Gutenbergstr. 76, 24118 Kiel, Germany.
authors: Bernd
authors: B
authors: Clement
authors: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada. Electronic address: afrankel@mail.ubc.ca.
authors: Adam
authors: A
authors: Frankel
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry*; D002453:Cell Cycle; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D009154:Mutation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007477:Ions; C487735:dimethylarginine; C018524:N,N-dimethylarginine; D001120:Arginine