title: A technique for the specific enrichment of citrulline-containing peptides.
abstract: Protein citrullination results from enzymatic deimination of peptidylarginine and plays an important role in health and disease. Despite increasing scientific interest, the identity and function of citrullinated proteins in vivo remain widely unknown. Thorough proteomic studies could contribute to a better understanding of the role of this posttranslational modification but will require tools for enrichment of citrullinated polypeptides. This study presents a simple technique for a highly specific enrichment of citrullinated peptides that is based on the specific reaction of glyoxal derivatives with the citrulline ureido group under acidic conditions. Beads were functionalized with 4-hydroxyphenylglyoxal attached via a base-labile linker. Incubation of these "citrulline reactive beads" with peptide mixtures at low pH resulted in selective immobilization of citrullinated peptides. Unbound noncitrullinated peptides were removed by extensive washing. Finally, citrullinated peptides carrying a modified ureido group were cleaved off at high pH and were analyzed by mass spectrometry. The procedure was validated by enrichment of synthetic citrulline-containing peptides from a tryptic digest of bovine serum albumin and from an endoproteinase LysC digest of a cytosolic fraction of a cell line. The technique was further applied to enrich citrullinated peptides from a digest of deiminated myelin basic protein.
authors.affiliation: Centre for Immune Regulation, Institute of Immunology, University of Oslo, N-0027 Oslo, Norway. astrid.tutturen@rr-research.no
authors.forename: Astrid E V
authors.initials: AE
authors.lastname: Tutturen
authors.forename: Anders
authors.initials: A
authors.lastname: Holm
authors.forename: Marit
authors.initials: M
authors.lastname: Jørgensen
authors.forename: Patricia
authors.initials: P
authors.lastname: Stadtmüller
authors.forename: Frode
authors.initials: F
authors.lastname: Rise
authors.forename: Burkhard
authors.initials: B
authors.lastname: Fleckenstein
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002956:Citrulline / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D004676:Myelin Basic Protein / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010658:Phenylglyoxal / Q000031:analogs & derivatives* / Q000737:chemistry; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004676:Myelin Basic Protein; D010455:Peptides; C028465:4-hydroxyphenylglyoxal; D002956:Citrulline; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D010658:Phenylglyoxal