In-depth Analytical Platforms Facilitating Serum Glycome, Proteome and Glycoproteome Investigation for Biomarker Discovery

Abstract

Human serum is a great source of potential protein disease biomarkers because of how blood interacts with all parts of the body, allowing it to contain proteins secreted from specific tissues of interest, and due the non-invasive method for which it is procured. However, tissue specific proteins found in serum are generally of low abundance, making them difficult to identify as the more readily present serum proteins mask their detection. Tissue specific glycoproteins and their associated glycans are of particular interest for study as potential biomarkers because of how glycosylation impacts protein function. To investigate the depths of the human serum N-glycome, a 2-dimensional (2D) ultra-high performance liquid chromatography (UHPLC) platform was developed using weak anion exchange (WAX) in the first dimension followed by subsequent offline hydrophilic interaction liquid chromatography (HILIC) fractionation in the second dimension to separate released serum N-glycans by charge and size respectively. Sialic acid derivatization using 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) enables the detection of sialic acid linkages to enable greater understanding of glycan composition when fractionated HILIC peaks are analysed using UHPLC coupled to elevated collision energy tandem mass spectrometry (UHPLC-MSE). The impact of DMT-MM on N-glycan retention to HILIC was studied using the described 2D separations platform and to further investigate the how DMT-MM derivatization can be used to improve chromatographic separations on HILIC. The use of DMT-MM derivatization coupled with isotopic labelling strategies was also investigated to study how DMT-MM derivatization can enable quantitative glycomic analysis of sialic acid linkage isoforms. Finally, an off-gel isoelectric focusing platform was developed to mine the human serum glycoproteome. The depletion of the fourteen most abundant serum proteins (14P depletion) was paired with off-gel isoelectric focusing fractionation (OGF), targeted to enrich glycoproteins, to reduce the complexity of the serum matrix and detect tissue leakage glycoproteins and their associated glycans. The developed platform allows for parallel N-glycomic and proteomic study after OGF, the results of which are then combined to facilitate site-specific glycopeptide identification on a global scale. The developed platforms were applied to the analysis of gastric cancer (GC) and colorectal cancer (CRC) serum samples, to investigate how glycoprotein and glycan expression is impacted by changes in pathology. The depths of these platforms enable the detection of low level cellular glycoproteins and their associated glycans, providing valuable opportunities to investigate potential disease biomarkers without invasive patient procedures.