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BackgroundIn up to 20% of patients with systemic sclerosis (SSc) none of the established SSc-specific autoantibodies are present [1]. Notwithstanding, in many of these patients high-titer autoantibodies can be detected on the HEp-2 indirect immunofluorescence assay (HEp-2 IIFA) which suggests the presence of an autoantibody to an intracellular protein expressed by the HEp-2 cell line. Immunoprecipitation of unlabeled cell extract followed by gel-free liquid chromatography tandem mass-spectrometry analysis has the potential to identify new autoantigens in an unbiased manner.ObjectivesTo identify new autoantigens through immunoprecipitation combined with liquid chromatography-tandem mass spectrometry (IP + LC-MS/MS) in HEp-2 IIFA-positive patients with SSc in whom none of the established SSc-specific autoantibody specificities are present.MethodsForty-nine patients from the University Hospitals Leuven that fulfilled the EULAR-ACR 2013 classification criteria for systemic sclerosis or LeRoy and Medsger’s criteria for early systemic sclerosis and who were negative on the EliA CTD Screen (Thermo Fisher Scientific, United States), which includes centromere protein B, topoisomerase I, RNA polymerase III, fibrillarin, PM-Scl and U1 ribonucleoprotein, were identified. Immunoprecipitation was performed by incubation of sera of these patients (1/30 in 300 µl Tris-buffered saline) with Pierce A/G magnetic beads, subsequent cross-linking with bissulfosuccinimidyl suberate (BS3) followed by incubation with nuclear extract of HeLa cells (100-150 µg) overnight at 4°C. The eluted protein was analyzed through liquid chromatography with tandem mass spectrometry. Mass spectrometry data were matched against the Uniprot Homo Sapiens database with the Mascot search engine. Candidate autoantigens were confirmed through immunoprecipitation followed by western blot of the eluate with target-specific polyclonal rabbit antibodies or western blot of recombinant protein incubated with sera of the index patients.ResultsWe identified multiple new autoantigens, including the THO complex subunit 1 (THOC1) and other subunits of the THO complex in 3 patients, nuclear valosin-containing protein like-2 (NVL) in 2 patients, nucleolar and coiled-body phosphoprotein 1 (NOLC1) and multiple interacting proteins in 1 patient, probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase (NOP2) in 1 patient, telomeric repeat-binding factor 2 (TERF2) and TERF2-interacting protein (TERF2IP) in 1 patient and regulator of chromosome condensation 1 (RCC1) in 1 patient. The new targets were confirmed through immunoprecipitation-western blot or western blot of recombinant protein incubated with sera (Figure 1). Furthermore, in 10 patients known SSc-associated autoantigens were strongly immunoprecipitated including multiple Th/To subunits in 5 patients, RuvBL1/2 in 2 patients, multiple PM-Scl subunits in 2 patients (who both were negative on the EliA CTD Screen), and fibrillarin in 1 patient (who was also negative on the EliA CTD .).Figure 1.Immunoprecipitation-western blot with target-specific rabbit polyclonal antibody (1/500-2000 dilution), numbers corresponding to order of description of patients, HC healthy control. NE nuclear extract. RP recombinant protein WB-RP western blot of recombinant protein incubated with patient’s sera.ConclusionMultiple new autoantigens were identified and confirmed in patients with SSc without previously identified autoantibody specificity. Further evaluation of reactivity against the newly identified autoantigens in patients with SSc with known autoantibody specificities and other cohorts is required. IP + LC-MS/MS can identify new and established autoantigens in patients with SSc.References[1]Meier FMP, Frommer KW, Dinser R, et al. Update on the profile of the EUSTAR cohort: an analysis of the EULAR Scleroderma Trials and Research group database. Ann Rheum Dis 2012;71:1355–60. doi:10.1136/annrheumdis-2011-200742Disclosure of InterestsJean-Baptiste Vulsteke: None declared, Daniel Blockmans: None declared, Petra De Haes: None declared, Steven Vanderschueren: None declared, Patrick Verschueren: None declared, Kristl G Claeys: None declared, Wim Wuyts Grant/research support from: Boehringer-Ingelheim, Galapagos, Roche, Jan Leo Lenaerts: None declared, Ellen De Langhe: None declared, Xavier Bossuyt Consultant of: Inova Diagnostics, Thermo Fisher Scientific.