Table 3.

Proteomic analysis of saliva and/or serum specifically related to autoantibodies in patients with SS and control subjects (1.3).

Author (year)	No. of subjects	Materials and methods	Findings and conclusion
Ottosson L, et al., 2006 (27)	10 pSS	Serum MALDI-TOF-MS	Two structured parts of Ro52 were identified, corresponding to the RING-B-box and the coiled-coil regions. The two subregions were independently structured. Analysis indicated Zn2+-dependent stabilization against proteolysis and functional Zn2+-binding sites in both the RING and the B-box. Oligomerization of the coiled-coil was investigated showing weak homodimer affinity, in parity with other coiled-coil domains involved in regulatory interactions. The findings form a basis for further Ro52 functional studies on the proteome level.
Van den Bergh K, et al., 2009 (46)	93 pSS 6 sSS 147 SLE, RA, scleroderma, polymysitis, MCTD and dermatomyositis	Serum Immunofluorescence, SDS-PAGE, WB, MALDI-TOF/TOF	Recombinant rat heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) reacted with 48% of sera from pSS patients and 5.2% of 153 sera from patients with other connective tissue diseases. Five of 11 pSS patients with no anti-SSA or -SSB antibodies had anti-hnRNP H1 antibodies. hnRNP H1 was suggested as novel, potential and additional diagnostic marker in pSS.
Hu S, et al., 2011 (32)	48 pSS 47 SLE 47 HC	Whole saliva PhotoArray, ELISA	Identification of 24 potential autoantibody biomarkers that could discriminate patients with pSS from both patients with SLE and healthy subjects. Four saliva autoantibody biomarkers, anti-transglutaminase, anti-histone, anti-SSA, and anti-SSB, were further tested in independent groups of pSS and SLE patients and healthy control subjects and all were successfully validated with ELISA.
Lindop R, et al., 2011 (47)	7 pSS	Serum High-resolution Orbitrap mass spectrometry (MS), 2-DE, ELISA	Proteomic analysis revealed a Ro60-peg-specific IgG1κ-restricted monoclonal autoantibody that was present in the sera of all patients and specified by a V(H)3–23 heavy chain paired with a V(κ)3–20 light chain. The public anti–Ro 60–peg clonotype was specified further by common mutations in the heavy-chain CDR1 and light-chain complementarity-determining regions. Titers and relative affinities of clonotypic IgG did not vary over the course of the disease. The expression of a Ro 60-reactive public B cell clonotype in a subset of patients with pSS as a long-lived, class-switched circulating autoantibody implies a common breach of B cell tolerance checkpoints in these patients.
Arentz G, et al., 2012 (48)	24 pSS 14 HC SLE Polymyositis SSc	Serum 2-DGE, MS	De novo sequencing was used to determine the clonality and V region structures of human autoantibodies directed against Ro52 (TRIM21). Anti-Ro52 autoantibodies from patients with pSS, SLE, SSc or polymyositis were restricted to two IgG1 kappa clonotypes that migrated as a single species on isoelectric focusing; shared a common light chain (VK3-20) paired with one of two closely-related heavy chains (VH3-7, VH3-23); and were public in unrelated patients. Targeted mass spectrometry using these uniquely mutated V rion peptides as surrogates detected anti-Ro52 autoantibodies in sera with high sensitivity (87.5%) and specificity (92.9%).
Thurgood LA, et al., 2013 (49)	7 pSS, anti-SSA- and -SSB pos. 2 pSS, anti-SSA pos. and anti-SSB neg. (controls) 1 asymptomic, anti-SSA and - SSB pos. (control) 4 HC	Serum High-resolution Orbitrap mass spectrometry (MS), 2-DE, ELISA	A majority of patients with linked anti-Ro60/Ro52/La responses target an NH2-terminal epitope designated LaA, which is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. Autoantibody responses comprised two heavily mutated IgG1 kappa-restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3-30 heavy chain paired with IGKV3-15 light chain, and the second by an IGHV3- 43/IGKV3-20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity-determining regions, consistent with a common breach of B cell tolerance followed by antigen-driven clonal selection.
Ohyama K, et al., 2015 (51)	14 pSS 14 SLE 11 HC 7 SSc 7 AAV 7 TA 9 MCTD 8 DM	Serum Nano-LC-MS/MS	468 distinct IC-associated antigens were identified, 62 were disease-specific antigens, and least three disease-specific antigens for each of the 7 autoimmune diseases. Coiled-coil domain-containing protein 158 and spectrin were identified as potential autoantigens important to SSc and SS pathogenesis, respectively; notable titin and spectrin autoantibodies were reportedly found in SSc and SS patients, respectively. The sensitivity of each disease-specific antigen was less than 33%. Immune complexome analysis may be generally applicable to the study of the relationship between ICs and autoimmune diseases.
Liao CC et al, 2016 (53)	18 pSS 25 RA-sSS, 18 RA 25 HC	Serum Con A affinity chromatography, 1D SDS-PAGE, in-gel digestion, and protein identification by LC-MS/M	In summary, Con A-bound ITIH3 in serum was identified as a robust diagnostic biomarker enabling discriminating RA-sSS from both HC and from pSS and RA alone. The performance of antibody isotypes against citrullinated peptides was generally better than their non-citrullinated/native counterparts, in discriminating between pSS, RA and RA-sSS and, healthy controls.
Wang JJ, et al., 2016 (55)	8 pSS 3 SLE anti-Ro60 pos. 4 HC 2 asymptomatic anti-Ro60 pos.	Serum High-resolution mass spectrometry (MS)	Monospecific anti-Ro60 Igs comprised dominant public and minor private sets of IgG1 kappa and lambda restricted heavy and light chains. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60/La responses, providing a molecular fingerprint of Ro60/La determinant spreading and suggesting that different forms of Ro60 antigen drive these responses. Sequencing of linked anti-Ro52 proteomes from individual patients and comparison with their anti-Ro60 partners revealed sharing of a dominant IGHV3–23/IGKV3–20 paired clonotype but with divergent IgV mutational signatures. In summary, anti-Ro60 IgV peptide mapping provides insights into Ro/La autoantibody diversification and reveals serum-based molecular markers of humoral Ro60 autoimmunity.
Wang JJ, et al., 2018 (29)	15 RF-positive pSS 5 RF-negative pSS (controls) 30 HC 13 RF-pos + anti-Ro/LA pos. asymptomatic donor	Serum, PBMC Novo mass spectrometric sequencing, IGH repertoire sequencing	RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig VH) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia were distinguishable from non-precipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with mixed cryoglobulinemia. Levels of Ig VH4–34 IgM-RF decreased following immunosuppression and remission of mixed cryoglobulinemia.
Burbelo PD, et al., 2019 (56)	20 SS 20 HC 20 ICIS 11 APECED without sicca 9 APECED with sicca	Unstimulated whole saliva, serum	High percentage of autoantibody seropositivity was detected against Ro52, Ro60, and La in SS, whereas few ICIS patients were seropositive. A few APECED patients had autoantibodies to Ro52 and La, but only Ro60 autoantibodies were weakly associated with a small subset of APECED patients with sicca. Additional testing of the salivary panel failed to detect autoantibodies against any of the salivary-enriched proteins in the SS and ICIS patients. However, APECED patients selectively demonstrated seropositivity against BPI fold containing family A member 1 (BPIFA1), BPI fold containing family A member 2 (BPIFA2)/parotid salivary protein (PSP), and lactoperoxidase, 3 salivary-enriched proteins. Moreover, high levels of serum autoantibodies against BPIFA1 and BPIFA2/PSP occurred in 30% and 67% of the APECED patients with sicca, respectively, and were associated with an earlier age onset of oral dryness. These findings highlight the complexity of humoral responses in different sicca diseases and provide new insights and biomarkers for APECED-associated sicca.