Discovery of Potential Serum Protein Biomarkers in Ankylosing Spondylitis Using Tandem Mass Tag-Based Quantitative Proteomics

Abstract

Ankylosing spondylitis (AS) is a systemic, chronic, and inflammatory rheumatic disease that affects 0.2% of the population. Current diagnostic criteria for disease activity rely on subjective Bath Ankylosing Spondylitis Disease Activity Index scores. Here, we aimed to discover a panel of serum protein biomarkers. First, tandem mass tag (TMT)-based quantitative proteomics was applied to identify differential proteins between 15 pooled active AS and 60 pooled healthy subjects. Second, cohort 1 of 328 humans, including 138 active AS and 190 healthy subjects from two independent centers, was used for biomarker discovery and validation. Finally, biomarker panels were applied to differentiate among active AS, stable AS, and healthy subjects from cohort 2, which enrolled 28 patients with stable AS, 26 with active AS, and 28 healthy subjects. From the proteomics study, a total of 762 proteins were identified and 46 proteins were up-regulated and 59 proteins were down-regulated in active AS patients compared to those in healthy persons. Among them, C-reactive protein (CRP), complement factor H-related protein 3 (CFHR3), α-1-acid glycoprotein 2 (ORM2), serum amyloid A1 (SAA1), fibrinogen γ (FG-γ), and fibrinogen β (FG-β) were the most significantly up-regulated inflammation-related proteins and S100A8, fatty acidbinding protein 5 (FABP5), and thrombospondin 1 (THBS1) were the most significantly down-regulated inflammation-related proteins. From the cohort 1 study, the best panel for the diagnosis of active AS vs healthy subjects is the combination of CRP and SAA1. The area under the receiver operating characteristic (ROC) curve was nearly 0.900, the sensitivity was 0.970%, and the specificity was 0.805% at a 95% confidence interval from 0.811 to 0.977. Using 0.387 as the cutoff value, the predictive values reached 92.00% in the internal validation set (62 with active AS vs 114 healthy subjects) and 97.50% in the external validation phase (40 with active AS vs 40 healthy subjects). From the cohort 2 study, a panel of CRP and SAA1 can differentiate well among active AS, stable AS, and healthy subjects. For active AS vs stable AS, the area under the ROC curve was 0.951, the sensitivity was 96.43%, the specificity was 88.46% at a 95% confidence interval from 0.891 to 1, and the coincidence rate was 92.30%. For stable AS vs healthy humans, the area under the ROC curve was 0.908, the sensitivity was 89.29%, the specificity was 78.57% at a 95% confidence interval from 0.836 to 0.980, and the coincidence rate was 83.93%. For active AS vs healthy subjects, the predictive value was 94.44%. The results indicated that the CRP and SAA1 combination can potentially diagnose disease status, especially for active or stable AS, which will be conducive to treatment recommendation for patients with AS.

Longitudinal Bottom-Up Proteomics of Serum, Serum Extracellular Vesicles, and Cerebrospinal Fluid Reveals Candidate Biomarkers for Early Detection of Glioblastoma in a Murine Model

“…Despite the low amount of serum, immunodepletion of high-abundance proteins and high-pH fractionation allowed the identification of 644 protein groups. This result is comparable with other studies involving higher serum amounts and more extensive immunodepletion and fractionation [ 37 , 38 ] using similar instrumentation. The optimized SP3 workflow allowed the quantification of 274 serum sEV proteins.…”

Fibrinogen-Like Protein 1 Is a Novel Biomarker for Predicting Disease Activity and Prognosis of Rheumatoid Arthritis

“…The comparative proteomic method was described previously ( 14 ). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium ( http://www.proteomexchange.org ) via the PRIDE partner repository with the dataset identifier PXD021689.…”

Tuning Monocytes and Macrophages for Personalized Therapy and Diagnostic Challenge in Rheumatoid Arthritis

“…In addition, SAA proteins were detected by immunohistochemistry (IHC) in RA macrophages [107]. The best known diseases for which SSA may be useful are RA, JIA, ankylosing spondylitis, various types of vasculitis, sarcoidosis, psoriatic arthritis, systemic sclerosis, SLE, familial Mediterranean fever, and, based on recent work, COVID-19 [108][109][110][111][112][113][114][115][116][117][118][119]. It is very important that SAA can be used as a diagnostic marker for RA as its level is elevated compared to osteoarthritis patients not only in serum but also in synovial fluid as a result of local production [120].…”