Using targeted proteomics-based detection of collagen propeptides to quantify fibrillar collagen biogenesis in vitro.

Abstract

The collagen superfamily, as the major structural component of the extracellular matrix, encompasses 28 distinct subtypes, with type-I and -III forming fibrils crucial for the matrix scaffold. During collagen biogenesis, trimers of type-I and -III procollagen are secreted into the extracellular matrix. The N- and C-terminal propeptides of these trimers are proteolytically cleaved from procollagen during secretion, initiating collagen fibril formation. The propeptides are released into extracellular space and, therefore, have been used to quantify collagen biogenesis. But high-throughput methods for the quantification of these biomarkers are still lacking. This study presents a state-of-the-art multiplexed approach for the simultaneous quantification of PINP, PICP, PIIINP and PIIICP from cell culture supernatants. The ability of targeted proteomics to quantify these propeptides from cell culture samples was assessed in this study. Using tryptic digestion and solid phase extraction, we were able to accurately quantify precollagen propeptides in a range of 3-1000 ng/mL. The assay showed an average inter-assay variance of 6.86 % with an overall recovery ranging from 92 to 98 %. The assay was validated using recombinant protein standards diluted in surrogate matrix and tested using transforming growth factor β1 mediated induction of normal human dermal fibroblasts. In summary, the assay presented in this paper offers a novel, robust, and precise high-throughput method for measuring human collagen propeptides in cell culture supernatants, empowering researchers to assess collagen biogenesis effectively in in vitro experiments.