Discovery of Candidate Serum Proteomic and Metabolomic Biomarkers in Ankylosing Spondylitis

Abstract

Ankylosing Spondylitis (AS) is a common inflammatory rheumatic disease with a predilection for the axial skeleton, affecting 0.2% of the population. Current diagnostic criteria rely on a composite of clinical and radiological changes, with a mean time to diagnosis of 5 to 10 years. In this study we employed nano liquid-chromatography mass spectrometry analysis to detect and quantify proteins and small compounds including endogenous peptides and metabolites in serum from 18 AS patients and nine healthy individuals. We identified a total of 316 proteins in serum, of which 22 showed significant up- or down-regulation (p < 0.05) in AS patients. Receiver operating characteristic analysis of combined levels of serum amyloid P component and inter-α-trypsin inhibitor heavy chain 1 revealed high diagnostic value for Ankylosing Spondylitis (area under the curve = 0.98). We also depleted individual sera of proteins to analyze endogenous peptides and metabolic compounds. We detected more than 7000 molecular features in patients and healthy individuals. Quantitative MS analysis revealed compound profiles that correlate with the clinical assessment of disease activity. One molecular feature identified as a Vitamin D3 metabolite—(23S,25R)-25-hydroxyvitamin D3 26,23-peroxylactone—was down-regulated in AS. The ratio of this vitamin D metabolite versus vitamin D binding protein serum levels was also altered in AS as compared with controls. These changes may contribute to pathological skeletal changes in AS. Our study is the first example of an integration of proteomic and metabolomic techniques to find new biomarker candidates for the diagnosis of Ankylosing Spondylitis.

Critical Role of Endoplasmic Reticulum Aminopeptidase 1 in Determining the Length and Sequence of Peptides Bound and Presented by HLA–B27

“…HPLC-purified peptides were analyzed with nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS), using an Acquity LC instrument (C18 column with a 75 m ϫ 250-mm, 1.7-m particle size; Waters) coupled to a Thermo LTQ Orbitrap Velos mass spectrometer (resolution of 60,000 full-width half maximum at mass/charge 400, Top 20, collision-induced dissociation), using a gradient of 1-40% acetonitrile for 60 minutes at a flow rate of 250 nl/minute, as described previously (18). Peak lists containing MS/MS spectra were generated using MSConvert (19), and these lists were then searched, using Mascot version 2.3.01 (http://www.matrixscience.com), against the Swiss-Prot protein database with the taxonomy restriction "mouse" (16, Peptide synthesis and peptide binding assay in T2.B27 cells.…”

Serum Proteomes Distinguish Children Developing Type 1 Diabetes in a Cohort With HLA-Conferred Susceptibility

“…The search criteria were trypsin digestion, MMTS modification of cysteine, deamidation of N/Q, and methionine oxidation, using the aforementioned database. For the quantitative analysis, Progenesis version 4.0 software was used for feature detection, alignment, and calculation of intensitybased abundance measurements for each protein (26). To facilitate comparison of the label-free data with the iTRAQ results, the intensity values of each protein were scaled relative to the median intensity of each protein across the paired case-control sample series.…”

Cell Metabolomics

“…By using a combination of feature selection, pattern recognition, and multivariate data analysis approaches, metabolomic profiling aims to provide a comprehensive assessment of the alterations in the metabolite levels in cells (Lange et al, 2011;Stringari et al, 2011;Turnbaugh et al, 2011). Recent technological advancements in nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) have led to wide use of these technologies for precise measurements of metabolites with improved sensitivity, resolution, and mass accuracy (Fischer et al, 2012;Kleiner et al, 2012). At the same time, data provided by metabolic profiling of cells can be complementary to the whole system results.…”