Multiplexed Imaging Mass Spectrometry of Histological Staining, N-Glycan and Extracellular Matrix from One Tissue Section: A Tool for Fibrosis Research.

We describe a multiplexed imaging mass spectrometry approach especially suitable for fibrosis research. Fibrosis is a process characterized by excessive extracellular matrix (ECM) secretion. Buildup of ECM impairs tissue and organ function to promote further progression of disease. It is an ongoing analytical challenge to access ECM for diagnosis and therapeutic treatment of fibrosis. Recently, we reported the use of the enzyme collagenase type III to target the ECM proteome in thin histological tissue sections of fibrotic diseases including hepatocellular carcinoma, breast cancer, prostate cancer, lung cancer and aortic valve stenosis. Detection of collagenase type III peptides by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows localization of ECM peptide sequences to specific regions of fibrosis. We have further identified that the ECM proteome accessed by collagenase type III has on average 3.7 sites per protein that may be differentially N-glycosylated. N-glycosylation is a major posttranslational modification of the ECM proteome, influencing protein folding, secretion, and organization. Understanding both N-glycosylation signaling and regulation of ECM expression significantly informs on ECM signaling in fibrosis.