Assessing the citrullinome in rheumatoid arthritis synovial fluid with and without enrichment of citrullinated peptides.
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.
Centre for Immune Regulation, Department of Immunology, University of Oslo, Oslo University Hospital-Rikshospitalet , Oslo 0372, Norway.
Astrid E V
AE
Tutturen
Burkhard
B
Fleckenstein
Gustavo A
GA
de Souza
D000328:Adult; D000595:Amino Acid Sequence; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D002956:Citrulline / Q000378:metabolism*; D005260:Female; D006801:Humans; D007719:Knee Joint / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013582:Synovial Fluid / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D010446:Peptide Fragments; D020543:Proteome; D002956:Citrulline