An immunoaffinity liquid chromatography-tandem mass spectrometry assay for detection of endogenous aggrecan fragments in biological fluids: Use as a biomarker for aggrecanase activity and cartilage degradation

作者：Dufield, D R^*; Nemirovskiy, O V; Jennings, M G; Tortorella, M D; Malfait, A M; Mathews, W R

来源：Analytical Biochemistry, 2010, 406(2): 113-123.

DOI：10.1016/j.ab.2010.06.044

• Aggrecan
• Aggrecanase
• Biomarker
• Osteoarthritis
• Immunoaffinity
• LC-MS/MS
• Antibody capture
• SISCAPA
• Peptide quantitation

The degradation of articular cartilage by aggrecanases (ADAMTS-4 and ADAMTS-5) plays a significant role in the pathology of osteoarthritis (OA). To monitor aggrecanase activity in OA, we have developed a sensitive, accurate, and versatile assay for detection of two specific cleavage sites on aggrecan. The assay uses an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect cleavage at the (374)ARGS site and the (1820)AGEG site. The dynamic range of the assay is more than three orders of magnitude, with interassay precision less than 15%. It has been successfully applied to various biological fluids and species, including rat, bovine, dog, and human. The assay has been analytically qualified for use in human urine and synovial fluid (SF). The limits of detection (LODs) for ARCS in urine and SF are 2.5 and 10 pg/ml, respectively, whereas the LOD for AGEG is 20 pg/ml in SF. Analysis of these biomarkers from OA subjects and normal healthy volunteers revealed a significant elevation of both markers in OA. Similarly, in a rat model of cartilage degradation, both ARCS and AGEG were elevated, demonstrating the utility of these biomarkers for translational research. These data suggest that the ARCS and AGEG biomarkers developed have potential as measures of aggrecanase activity in OA and may contribute to our understanding of OA pathology.

• 出版日期2010-11-15
• 单位中国科学院广州生物医药与健康研究院