Determination of sites citrullinated by peptidylarginine deiminase using 18O stable isotope labeling and mass spectrometry.
Peptidylarginine deiminase (PADI) is an enzyme which catalyzes conversion of arginine residues into citrulline residues in proteins. Citrullination is known to be related to autoimmune diseases including rheumatoid arthritis. Previous work in this laboratory succeeded in identifying citrullinated sites of human fibrinogen by mass spectrometry, but discrimination between citrullination and deamidation of asparagines and glutamine required time-consuming and labor-intensive inspection of tandem mass spectra. In this work a stable isotope is utilized to improve on a previous method for the determination of citrullinated sites by mass spectrometry. Since an oxygen atom is incorporated into the citrulline residue from H(2)O in citrullination by PADI, peptides citrullinated in 50% H(2)(18)O would show a characteristic isotope distribution different from natural abundance, and thus determination of citrullinated sites is expected to be much easier. To verify the utility of this new method, the sites of citrullination of human fibrinogen by human PADI4 were investigated using 50% H(2)(18)O. Compared with the previous method, this new method identified citrullinated sites more easily and effectively, while both the determined citrullinated sites and protein sequence coverage were unaltered.
Kubota
Kazuishi
K
Biomedical Research Laboratories, Sankyo Co., Ltd., Shinagawa-ku, Tokyo 140-8710, Japan. kkubot@sankyo.co.jp
Yoneyama-Takazawa
Tomoko
T
Ichikawa
Kimihisa
K
D000595:Amino Acid Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid / Q000379:methods*; D002956:Citrulline / Q000032:analysis / Q000737:chemistry*; D004789:Enzyme Activation; D005340:Fibrinogen / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D006867:Hydrolases / Q000032:analysis / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods; D008969:Molecular Sequence Data; D010104:Oxygen Radioisotopes / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010104:Oxygen Radioisotopes; D002956:Citrulline; D005340:Fibrinogen; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases