The Study Of Protein Components And Carbamoylation And Citrullination Of Rheumatoid Arthritis Synovial Fluid

Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation and damage of joint tissues.The presence of autoantibodies is the most significant serological feature of RA.Clinical detection of anti-cyclic citrullinated peptide antibodies is not only a crucial diagnostic index for RA,but also has important reference value for judging the process,development and prognosis of the disease.The anti-carbamylated proteins discovered in recent years are also valuable for the specificity of RA diagnosis.However,the target antigen recognized and bound by ACPA and Anti-carp remains to be elucidated.Synovial fluid may contain target antigens that induce ACPA and anti carp production.The study of the protein composition of synovial fluid and the carbamylation of lysine and the citrullination modification of arginine are of great value for revealing the target antigens of ACPA and Anti-carp.In this article,the supernatant and precipitation of the synovial fluid of rheumatoid arthritis patients were separated by polyacrylamide gel electrophoresis(SDS-PAGE)and size exclusion chromatography(SEC).The protein components were identified by high-resolution liquid chromatography-electrospray tandem mass spectrometry(LC-ESI-MS/MS),as well as the identification of carbamylation of lysine and citrullination modification sites of arginine.The main findings are as follows:(1)We identified 951 proteins in the supernatant in the synovial fluid of RA patients with,and 1217 proteins in the precipitate.We found that 525 proteins were common in the supernatant and the precipitate,426 proteins were unique to the supernatant,692 proteins were specific to precipitation.In total,1643 proteins were identified in the synovial fluid of RA patients.(2)The proteins identified in supernatant and precipitation were analyzed by Gene Ontology with cell location and function.Analysis based on cell location showed that 29.8%of the supernatant was extracellular.Compared with the supernatant,the amount of protein in the cytoplasm and mitochondria of the precipitate increased significantly,accounting for 12.8%and 7.8%of the total protein,respectively.Based on the classification of biological processes,most of the proteins in the supernatant were involved in cell growth and maintenance(18.8%),signal transduction(12.4%),cell communication(11.3%)and neutrophil degranulation(10.6%),but the number of proteins involved in neutrophil degranulation(15.0%)in the precipitation was significantly up-regulated.(3)The proteins identified in the supernatant and precipitate were quantified by MaxQuent.The results showed that the high-abundance proteins in the supernatant were albumin,immunoglobulin,apolipoprotein AI,transferrin,etc.,and a-1 The content of acid glycoprotein and calcium binding protein S100 are also relatively high,accounting for 4.18%and 3.32%of the total protein respectively.The proteins in the precipitate are mostly cellular components,and the proteins in the precipitate are mostly proteins related to the degranulation of neutrophils.Compared with the supernatant,the content of calcium-binding protein S100 and neutrophil defensins in the total protein increased.They were 13.07%and 4.3%respectively.(4)Studies on the carbamylation modification of lysine have found that carbamylation modification is induced in vitro.The use of urea and polyacrylamide is avoided in sample preparation.We identified 169 lysine sites of 90 proteins that were carbamylated in the joint effusion of patients with rheumatoid arthritis.Among them,the more modified sites include albumin,fibrinogen,immunoglobulin,complement C3,GRIP1 related protein,α1-antitrypsin and so on.(5)For citrullinated arginine residues,the mass number of peptide fragment ions detected by mass spectrometry increased by 0.98 Da,usually accompanied by neutral loss.Excluding the mismatch data of other protein modifications that produced the same mass transfer,we identified a total of 218 peptides of 112 proteins that were citrullinated.Among them,there are more citrullinated sites including fibrinogen,albumin,fibronectin,complement factor H,vimentin,transferrin,and nuclear differentiation antigen of myeloid cells.These results suggest that the high abundance proteins of joint effusion components coincide with high abundance proteins of plasma,suggesting that joint effusion mainly comes from plasma ultrafiltration,and some proteins involved in acute inflammatory response,such as a-The proportion of acid glycoprotein and calcium binding protein S100 is also very considerable.For the identification and classification of the precipitated proteins in joint effusion,we found that the high-abundance proteins in the precipitate are mainly some neutrophil-specific proteins.It is speculated that the infiltrating cells of joint effusion are mainly neutrophils.Our study found that the proteins that undergo carbamylation and citrullination modification mostly occur on high-abundance proteins,suggesting that these two modifications are more abundant in RA patients.Carbamylation and citrullination modification have similar properties,and it is difficult to distinguish between ordinary detection methods.