Protein citrullination is a posttranslational modification that has attracted 
increased attention, especially for its involvement in rheumatoid arthritis 
(RA). Here, we assess the citrullinome in RA synovial fluid by direct LC-MS/MS 
analysis and by the use of an enrichment strategy based on citrulline specific 
biotinylation. RA synovial fluid was depleted for abundant proteins, and total 
and depleted fractions were analyzed. Frequency of citrullinated peptides and 
their degree of citrullination could be determined for four known RA 
autoantigens, as well as a novel in vivo autocitrullination site of 
peptidylarginine deiminase 4. From the analysis of total and depleted synovial 
fluid after enrichment we could estimate the numbers of citrullinated peptides 
to be approximately 3600 and 2100, respectively. However, identification of 
these biotinylated peptides by MS/MS turned out to be very difficult due to 
fragmentation of the biotin moiety. By direct MS analysis of the total and 
depleted synovial fluid without enrichment, 119 and 157 citrullinated peptides 
were identified, respectively. This indicates that direct analysis allows 
identification of only a fraction of the citrullinated proteins present in 
synovial fluid and that specific enrichment is still needed for a comprehensive 
in-depth elucidation of the citrullinome.