An analytical workflow enabling selective enrichment of proteoglycans and heparin ligands from the human aorta

Proteoglycans, extracellular matrix (ECM) regulators and secreted factors present distinct challenges to their detection and analysis by mass spectrometry and downstream biochemical characterization. These include the presence of glycosaminoglycans in proteoglycans and low abundance/complex post-translational regulation in the case of ECM regulators and secreted factors. As a result, our understanding of diseases where dysregulated ECM remodeling is a major pathogenic driver, like thoracic aortic aneurysms (TAA), remains limited. Characterization of aortic proteoglycans and matrisome-associated proteins is fundamental for the identification of candidate biomarkers and therapeutic targets in TAA. Here, we present an analytical workflow enabling selective enrichment of proteoglycans and ECM regulators/secreted factors from human TAA aorta, based on their different isoelectric points. Anion exchange (AEX) and cation exchange (CEX) chromatography were sequentially employed using diethylaminoethyl and heparin Sepharose, respectively, before analysis by liquid chromatography-tandem mass spectrometry to selectively enrich in proteoglycans and ECM regulators/secreted factors, respectively. We have identified 1202 proteins in protein extracts, 1138 in AEX early peaks (eluted at 250–600 mM NaCl) and 1146 in AEX late peaks (eluted at 650–900 mM NaCl). When ranked by overall abundance, 4 out of the top 10 most abundant proteins in AEX late peaks were proteoglycans compared to only one proteoglycan in protein extracts. In CEX peaks, 187 matrisome proteins were identified, including 53 ECM regulators and 23 secreted factors, as well as 1016 non-ECM proteins. Selective enrichment was orthogonally validated by immunoblot analysis. Our workflow is readily translatable to other connective tissues for in-depth proteoglycanome/heparin ligandome profiling.