1. Rapid LC-MS/MS Evaluation of Collagen and Elastin Crosslinks in Human and Mouse Lung Tissue with a Novel Bioanalytical Surrogate Matrix Approach.
- Author
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Lloyd SM, Sande EJ, Ruterbories K, O'Brien SP, Wang YT, Phillips LA, Carr TL, Clements M, Hazelwood LA, Tian Y, He Y, and Ji QC
- Subjects
- Animals, Humans, Mice, Chromatography, Liquid methods, Idiopathic Pulmonary Fibrosis metabolism, Idiopathic Pulmonary Fibrosis pathology, Extracellular Matrix metabolism, Cross-Linking Reagents chemistry, Male, Mice, Inbred C57BL, Liquid Chromatography-Mass Spectrometry, Tandem Mass Spectrometry methods, Elastin metabolism, Elastin chemistry, Lung metabolism, Lung pathology, Collagen metabolism, Collagen chemistry, Collagen analysis
- Abstract
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.
- Published
- 2024
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