Characterization of Proteoglycanomes by Mass Spectrometry

Abstract

As composites of a core protein and several chemically distinct types of glycosaminoglycan (GAG) chains, proteoglycans are diverse molecules that occupy a unique niche in biology. They have varied and essential roles as structural and regulatory molecules in numerous physiological processes and disease pathology. In regard to cellular context, some link the interior of the cell to the extracellular matrix (ECM) as transmembrane or membrane-anchored molecules with a major role in cell adhesion and signal transduction. Others reside in pericellular matrix, where they influence crucial aspects of cell behavior, and several reside in interstitial ECM as components of structural macromolecular networks. Because of their unique composition, they can be challenging to identify and characterize using conventional biochemical or antibody-based methods. In contrast, the GAG component, despite its immense chemical diversity, typically carries a strong net negative charge which can be exploited to advantage for affinity-isolation and enrichment of proteoglycans from any biological system in a core protein-, GAG-, tissue-, and species-agnostic manner by anion exchange chromatography. This method, when coupled with high resolution liquid-chromatography tandem mass spectrometry (LC-MS/MS) can be used to define the proteoglycanome of any cell type, tissue or organism. A proteoglycanomics strategy can be further refined by inclusion of additional orthogonal affinity steps or fractionation for greater specificity and to deliver proteoglycans with distinct specified characteristics. Moreover, elimination of the GAG chain chemically and/or obliteration of the core protein enables glycomics characterization of GAG structure. Enzymatic digestion of GAGs on tryptic peptides allows mapping of glycopeptides, which has been used for identification of novel proteoglycans and to precisely define sites of GAG attachment. Recent application of proteoglycanomics to human aorta and human aortic aneurysms demonstrated its potential to identify tissue and disease proteoglycanomes and the detailed method that was used is provided here for application to other tissues or biological systems.