A technique for the specific enrichment of citrulline-containing peptides.
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.
Tutturen
Astrid E V
AE
Centre for Immune Regulation, Institute of Immunology, University of Oslo, N-0027 Oslo, Norway. astrid.tutturen@rr-research.no
Holm
Anders
A
Jørgensen
Marit
M
Stadtmüller
Patricia
P
Rise
Frode
F
Fleckenstein
Burkhard
B
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
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