Alterations to post-translational crosslinking modifications in the extracellular matrix (ECM) are known to drive the pathogenesis of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Thus, the methodology for measuring crosslinking dynamics is valuable for understanding disease progression. The existing crosslinking analysis sample preparation and liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are typically labor-intensive and time-consuming which limits throughput. We, therefore, developed a rapid approach minimizing specialized equipment and hands-on time. The LC-MS/MS sample analysis time was reduced to two minutes per sample. We then improved the analytical integrity of the method by developing a novel surrogate matrix approach for the dihydroxylysinonorleucine (DHLNL) crosslink. By modifying sample preparation, we prepared a tissue-based surrogate matrix with undetectable levels of endogenous DHLNL, providing a strategy for quantifying this crosslink with a more relevant standard matrix. We then applied this rapid methodology to evaluating crosslinking in lung fibrosis. We showed an increase in DHLNL in human IPF lung relative to healthy donors, as well as in a fibrotic mouse model. Finally, we demonstrated that this increase in DHLNL could be mitigated with an anti-fibrotic compound, suggesting that this assay has potential for evaluating pharmaceutical compound efficacy.
Keywords: LC-MS/MS; bioanalysis; collagen; crosslinking; elastin; fibrosis; lung; surrogate matrix.