Introduction: Biomarkers to identify osteoarthritis (OA) patients at risk for disease progression are needed. As part of a proteomic analysis of knee synovial fluid from normal and OA patients, differentially expressed proteins were identified that could represent potential biomarkers for OA. This study aimed to use mass spectrometry assays to identify representative peptides from several proteins in synovial fluid and peripheral blood, and assess their levels as biomarkers of OA progression. Methods: Multiplexed high throughput selected reaction monitoring (SRM) assays were developed to measure tryptic peptides representative of 23 proteins in matched serum and synovial fluid samples from late OA subjects at the time of joint replacement. Subsequently plasma samples from the baseline visit of 173 subjects in an observational OA cohort were tested by SRM for peptides from nine of these proteins: afamin, clusterin, cartilage oligomeric matrix protein, hepatocyte growth factor, kallistatin, insulin-like growth factor binding protein, acid labile subunit, lubricin, lumican, and pigment epithelium-derived factor. Linear regression was used to determine the association between the peptide biomarker level at baseline and change in joint space width (ΔJSW) from baseline to 30 months, adjusting for age and sex. Results: In the matched cohort, 17 proteins could be identified in synovial fluid and 16 proteins were detected in serum. For the progression cohort, the average age was 62 and average ΔJSW over 30 months was 0.68 mm. A high correlation between different peptides from individual proteins was observed, indicating our assays correctly measured their target proteins. Peptides representative of clusterin, lumican and lubricin showed statistically significant associations with joint space narrowing after adjustment for age and sex. Partial R² values showed clusterin FMETVAEK and lubricin LVEVNPK peptide biomarkers explains about 2 to 3% of the variability of ΔJSW, similar to that explained by age. A biomarker score combining normalized data for both lubricin and clusterin peptides increased the model R² to 0.079. Conclusions: Our results suggest that when combined, levels of peptides representative of clusterin and lubricin in plasma are as predictive of OA progression as age. Replication of these findings in other prospective OA cohorts is planned.

In this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle.

The anatomy of PFO suggests that it can allow thrombi and potentially harmful circulatory factors to travel directly from the venous to the arterial circulation – altering circulatory phenotype. Our previous publication using high-resolution LC-MS/MS to profile protein and peptide expression patterns in plasma showed that albumin was relatively increased in donor samples from PFO-related than other types of ischemic strokes. Since albumin binds a host of molecules and acts as a carrier for lipoproteins, small molecules and drugs, we decided to investigate the albumin-bound proteins (in a similar sample cohort) in an effort to unravel biological changes and potentially discover biomarkers related to PFO-related stroke and PFO endovascular closure. The method used in this study combined albumin immuno-enrichment with high resolution LC-MS in order to specifically capture and quantify the albumin-bound proteins. Subsequently, we measured cholesterol and HDL in a larger, separate cohort of PFO stroke patients, pre and post closure. The results demonstrated that a number of proteins were specifically associated with albumin in samples with and without endovascular closure of the PFO, and that the protein profiles were very different. Eight proteins, typically associated with HDL were common to both sample sets and quantitatively differently abundant. Pathway analysis of the MS results suggested that enhanced cholesterol efflux and reduced lipid oxidation were associated with PFO closure. Measurement of total cholesterol and HDL in a larger cohort of PFO closure samples using a colorimetric assay was consistent with the proteomic predictions. The collective data presented in this study demonstrate that analysis of albumin-bound proteins could provide a valuable tool for biomarker discovery on the effects of PFO endovascular closure. In addition, the results suggest that PFO endovascular closure can potentially have effects on HDL, cholesterol and albumin-bound ApoA-I abundance, therefore possibly providing benefits in cardioprotective functions.

Parathyroid hormone is involved in calcium homeostasis through interactions with vitamin D. Because intact and truncated forms of parathyroid hormone (PTH) vary in their biological activity, assays that can accurately quantify the ratio of intact hormone to its fragments are of increasing significance in the diagnosis of endocrine, renal and bone diseases. Vitamin D and its metabolites circulate tightly bound to vitamin D-binding protein (DBP). Because DBP concentrations are altered in pregnancy, liver and renal diseases and also show genetic variations in different ethnic groups, total vitamin D in serum can be misleading. In addition, both calcium and vitamin D metabolites can decrease the secretion of PTH. Previously, we developed multiplexed mass spectrometric immunoassay (MSIA)–SRM assays for PTH that allow quantification of four fully-tryptic monitoring peptides (that span the entire PTH sequence) and two semi-tryptic variant specific peptides¹. Using this approach, it is possible to monitor intact PTH and also the degree of N-terminal fragmentation. In this study, the objective was to develop a multiplexed, MSIA-SRM-based targeted assay for PTH and DBP. We applied this MSIA-SRM assay and a commercially available immunoassay to a cohort of 500 clinical samples from a variety of different patient groups including renal disease, cancer, vitamin D deficiency and other conditions that can affect calcium homeostasis. The results demonstrated excellent assay linearity (R²=0.90–0.99) with sensitivity for analytes in the published clinical ranges and limits of detection in the pg/ml range. Comparison of the PTH MSIA –SRM assay with the commercial ELSA assay demonstrated good correlation in normal subjects but important differences in renal failure. There were also some unusual fragments seen in clinical samples, not previously reported in the literature. References 1. Lopez MF, Rezai T, Sarracino DA, Prakash A, Krastins B, Athanas M, Singh RJ, Barnidge DR, Oran P, Borges C & Nelson RW. Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. Clin Chem 2010 56 281–90. Declaration of funding: Funding of reagents and consumables were from Thermo Scientific US.