Pitfalls in the detection of citrullination and carbamylation

Abstract

Carbamylation and citrullination are both post-translational modifications against which (auto)antibodies can be detected in sera of rheumatoid arthritis (RA) patients. Carbamylation is the chemical modification of a lysine into a homocitrulline, whereas citrullination is an enzymatic conversion of an arginine into a citrulline. It is difficult to distinguish between the two resulting amino acids due to similarities in structure. However, differentiation between citrulline and homocitrulline is important to understand the antigens that induce antibody production and to determine which modified antigens are present in target tissues. We have observed in literature that conclusions are frequently drawn regarding the citrullination or carbamylation of proteins based on reagents that are not able to distinguish between these two modifications. Therefore, we have analyzed a wide spectrum of methods and describe here which method we consider most optimal to distinguish between citrulline and homocitrulline. We have produced several carbamylated and citrullinated proteins and investigated the specificity of (commercial) antibodies by both ELISA and western blot. Furthermore, detection methods based on chemical modifications, such as the anti-modified citrulline-"Senshu" method and also mass spectrometry were investigated for their capacity to distinguish between carbamylation and citrullination. We observed that some antibodies are able to distinguish between carbamylation and citrullination, but an overlap in reactivity is often present in the commercially available anti-citrulline antibodies. Finally, we conclude that the use of mass spectrometry is currently essential to differentiate between citrullinated and carbamylated proteins present in complex biological samples.

Clinical and etiological meaning of anti-carbamylated protein antibodies in rheumatoid arthritis

“…However, a difficulty is associated with distinguishing citrullinated proteins from carbamylated proteins: not only the cross-reaction of antibodies in patient serum, but also that of commercially available antibodies for experiments. Although the antimodified citrulline (AMC)-Senshu antibody [56] and antibody against the deca-citrulline peptide (F95) [57] have been used in many studies on citrullinated antigens, both were shown to bind to carbamylated antigens [9,58,59]. These findings indicate the potential misinterpretation of carbamylated antigens as citrullinated antigens in many previous studies because citrullinated proteins and carbamylated proteins may both be produced by activated neutrophils, including NETs-forming neutrophils, as discussed above.…”

“…While several other methods for detecting citrullination have been reported [60], the most reliable is mass spectrometry (MS); however, this involves a detailed procedure to identify citrullinated proteins from many other proteins (reviewed by Verheul et al [59]). High-performance liquid chromatography (HPLC) is also useful for focusing on one specific protein and discriminating citrullination from carbamylation [61].…”

Autoantibodies and B Cells: The ABC of rheumatoid arthritis pathophysiology

“…This cross‐reactivity is also detected in patients in which anti‐CarP and ACPA responses show cross‐reactivity to varying degrees . Furthermore, preliminary data illustrated that an ACPA/AAPA monoclonal antibody can be cross‐reactive, and commercially available (polyclonal) antibodies are often unable to distinguish between different citrullinated and carbamylated residues . Together, these observations hint toward a cross‐reactive antibody response targeting citrullinated, carbamylated, and acetylated proteins.…”

Insights into the study and origin of the citrullinome in rheumatoid arthritis

“…This exact mass increment also occurs on one of the most frequent modifications, the deamidation of asparagine (Asn) or glutamine (Gln), which is a spontaneous non‐enzymatic conversion of an uncharged side‐chain residue (Figure ). Occurrence of deamidated and citrullinated residues in the same peptide leads to interference during precursor ion isolation and can result in incorrect assignments of modified amino acids (ie, co‐isolation of other species), which leads to mixed or contaminated MS/MS spectra . In fact, deamidation of Asn/Gln occurs much more frequently than citrullination.…”

“…Occurrence of deamidated and citrullinated residues in the same peptide leads to interference during precursor ion isolation and can result in incorrect assignments of modified amino acids (ie, coisolation of other species), which leads to mixed or contaminated MS/ MS spectra. 35 In fact, deamidation of Asn/Gln occurs much more frequently than citrullination. Thus, this is the major problem leading to a high degree of ambiguity during data interpretation.…”