Abstract
Collagen cross-links created by the lysyl oxidase and lysyl hydroxylase families of enzymes are a significant contributing factor to the biomechanical strength and rigidity of tissues, which in turn influence cell signaling and ultimately cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach involving chemical digestion for protein solubilization coupled with mass spectrometry allows for the identification of the NTX cross-links in a range of modification states. Based on the specificity of the enzymatic cross-linking reaction we utilized follow-up variable modification searches to facilitate identification with a wider range of analytical workflows. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl-hydroxylase and lysyl-oxidase enzymes.