Background/Purpose:  

The current diagnosis of osteoarthritis (OA) relies on the description of pain symptoms, affected joint stiffness, and radiography used as the reference technique for determining the grade of joint destruction. Limitations of the presently available diagnostic tests have provided an impetus for the substantial increase in interest in finding new specific biological markers for OA to facilitate the early diagnosis, evaluate disease progression and improve disease prognosis. Recently, there has been a remarkable development in mass spectrometry-based methodologies for the verification and validation of biomarkers with high specificity and sensitivity, since the multiple reaction monitoring (MRM) assay can measure several targets (peptides) with high sensitivity and throughput in biological samples. The aim of this work was to verify a panel of OA biomarker candidates in serum samples using the MRM technology.

Methods:  

Serum samples were obtained from OA patients at different stages of the disease (K/L grade II=38, K/L grade IV=39 and control donors=39). Proteins were quantified and digested with trypsin. The peptide mixture was separated by nano-LC coupled to a 5500 QTRAP mass spectrometer. A single multiplexed MRM assay was used to quantify the levels of seven proteins (identified as putative OA biomarker candidates in shotgun proteomics experiments performed previously by our group) at different stages of OA, and compared to control donors: Pigment epithelium derived factor, Haptoglobin, Von Willebrand factor, Fructose-bisphosphate aldolase A, Insulin-like growth factor-binding protein complex acid labile subunit, C-type lectin domain family 3 member A and matrix metallopeptidase 2. Von Willebrand factor and Haptoglobin were found quantitatively altered in the serum of OA patients compared to control donors. The relative quantification values of peptides were determined by calculating the ratio of peak areas from transitions of target peptides in OA and control samples, normalized to the peak area of the internal standard. Data analysis was performed using the Skyline software. Kruskal–Wallis and Mann–Whitney U test were used for the statistical analysis.

Results:  

Haptoglobin was found to discriminate between the different K/L grades of OA, and it was found to be altered between early OA compared to controls with a significant p-value. Therefore, this protein could be a putative diagnostic biomarker. On the other hand, Von willebrand factor was found altered in OA vs control samples with a significant p-value (p<0,05), but showed no differences between the different K/L grades of OA.

Conclusion:  

A multiplexed method for the simultaneous quantification of a panel of seven proteins in serum has been developed, which is based on liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometry. By these means, two novel putative OA serum protein biomarkers, Von Willebrand factor and Haptoglobin, were verified in a cohort of individual serum samples. Further qualification studies will be necessary to establish their usefulness for OA diagnosis and progression studies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/verification-of-haptoglobin-and-von-willebrand-factor-as-potential-biomarkers-of-knee-osteoarthritis-using-a-targeted-proteomics-approach-in-serum/