Determination of Citrullinated Sites of Alpha-fibrinogen in Rheumatoid Arthritis Synovial Fluid Using Immunocapture and Two Dimensional Liquid Chromatography Mass Spectrometry

摘要

Citrullination is known to happen in many autoimmune diseases including rheumatoid arthritis (RA). Citrullination is catalyzed by a family of enzymes called peptidylarginine deiminase (PADI). Conversion of arginine (Arg) to citrulline is a deimination process where the imino-group on the side chain of arginine is changed into a carbonyl group. This change causes a small mass change of 1 Dalton and loss of positive charge. Due to the changes, citrullinated proteins can have different conformations and protein/protein interactions that can affect their function. Characterization of citrulline proteins as autoantigens is a highly challenging process. The modification is known to be in low abundance makes its difficult to be detected. The subtle 1 mass unit difference also makes it difficult to distinguish it from commonly observed deamidations of asparagine (Asn) and glutamine (Gln) residues. Previously we described a general methodology for the identification of autoantigens from RA Patient synovial fluid using two dimensional liquid chromatography followed by LC-MS/MS analysis of the isolated fractions after digestion with trypsin.