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.