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.