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IntroductionRheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients.MethodsIC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining.ResultsCirculating IC in the serum of RA patients and healthy controls contain fibrinogenβ and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogenγ and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogenβ were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients.ConclusionsCitrullinated fibrinogenβ and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA- RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA.

Citrullination is a posttranslational modification of arginine. It plays both a physiological role, for instance during apoptosis and epigenetics, and a pathological role in cancer or diseases of the central nervous system. Most research on citrullination to date focuses on its role in auto-immune diseases such as multiple sclerosis and rheumatoid arthritis. In this context, the exact knowledge of citrullination sites in a protein can provide invaluable information about the etiological importance of these citrullinated proteins. However, few techniques exist that can accurately detect citrullination on the peptide level. This review aims to give an overview of the different methods available to date for the detection of citrullinated proteins and peptides. These include 2D-SDS-PAGE and immunodetection, as well as specific mass spectrometry (MS) approaches, both labeled and unlabeled. These MS approaches have been developed to pinpoint the exact location of citrullination on the peptide level. Improving the currently existing detection strategies while focusing on the role of citrullinated proteins will be invaluable to elucidate the importance of this posttranslational modification in vivo.

Citrullination is a post-translational modification (PTM) that results from the deimination of the amino acid arginine into citrulline by Peptidyl Arginine Deiminase enzymes and occurs in a wide range of proteins in health and disease. This modification causes a 1 Da mass shift, which can be used to identify citrullination sites in proteins by the use of mass spectrometry. However, other PTMs, such as deamidation from asparagine to aspartic acid or from glutamine to glutamic acid, can also cause a 1 Da mass shift, making correct interpretation of the data more difficult. We developed a chemical tagging strategy which, combined with an open source search application, allowed us to selectively pinpoint citrullinated peptides in a complex mixture after liquid chromatography/mass spectrometry (LC/ MS) analysis. After incubation of a peptide mixture with 2,3 butanedione, citrulline residues were covalently modified which resulted in a 50 Da shift in singly charged mass. By comparison of the peptide mass fingerprint from a modified and an unmodified version of the same sample, our in-house search application was able to identify the citrullinated peptides in the mixture. This strategy was optimized on synthetic peptides and validated on a digest of in vitro citrullinated fibrinogen, where different proteolytic enzymes were used to augment the protein coverage. This new method results in easy detection of citrullinated residues, without the need for complex mass spectrometry equipment.

Citrullination is a post-translational modification of arginine, resulting in a loss of positive charge and a 1 Da mass increase. Research on citrullinated proteins is crucial in rheumatoid arthritis, an autoimmune disease characterized by the presence of antibodies against citrullinated proteins. However, little is known about the location or quantity of deiminated arginine residues in these proteins. Since citrullination gives rise to a mass gain of only 1 Da, the isotope pattern of the citrullinated and the noncitrullinated version of a peptide will overlap. However, the difference between the theoretical, or noncitrullinated, and the measured isotope pattern can be used to quantify the amount of citrullination. We developed a method to quantify citrullinated peptides by means of their skewed isotopic distribution pattern. The method was first optimized with synthetic peptides, after both direct infusion and RP-HPLC separation on an ESi-QqTOF mass spectrometer. Additionally, we analyzed synovial fluid samples from rheumatoid arthritis patients and were able to quantify citrullinated peptides originating from citrullinated fibrinogen, a well-known antigen.