Mass Spectrometry-Based Glycoproteomic Workflows for Cancer Biomarker Discovery

Abstract

Glycosylation has a clear role in cancer initiation and progression, with numerous studies identifying distinct glycan features or specific glycoproteoforms associated with cancer. Common findings include that aggressive cancers tend to have higher expression levels of enzymes that regulate glycosylation as well as glycoproteins with greater levels of complexity, increased branching, and enhanced chain length1. Research in cancer glycoproteomics over the last 50-plus years has mainly focused on technology development used to observe global changes in glycosylation. Efforts have also been made to connect glycans to their protein carriers as well as to delineate the role of these modifications in intracellular signaling and subsequent cell function. This review discusses currently available techniques utilizing mass spectrometry-based technologies used to study glycosylation and highlights areas for future advancement.

Mass Spectrometry Strategies for O-Glycoproteomics

“…Glycans are responsible for mediating cell-cell interactions [ 60 , 61 ], protein folding [ 62 , 63 ], and activating or inhibiting downstream processes within cells [ 64 , 65 , 66 ]. Additionally, glycans are common disease biomarkers but can be difficult to deduce owing to the complexity of combinatorial glycosylation and its role in disease progression [ 67 , 68 , 69 ]. Composed of mono- or oligosaccharides, the latter which are frequently multi-branched, glycans exhibit great microheterogeneity that increases proteome complexity as a single glycosite can host many different glycans [ 70 , 71 ].…”

“…Much effort has been expended to increase the quality of data generated from bottom-up O -glycoproteomics workflows, including refining or combining separation methods (typically various modes of liquid chromatography (LC)) [ 14 , 22 , 68 , 98 ] and integrating ion mobility (IM), a type of gas-phase electrophoretic separation [ 68 , 113 , 114 , 115 ]. Although reversed-phase chromatography is by far the most commonly utilized mode for separation of peptides, other methods like hydrophilic interaction liquid chromatography (HILIC) [ 14 , 69 , 116 , 117 ], porous graphitized carbon [ 22 , 68 , 118 , 119 , 120 ], and strong anion exchange (SAX) [ 19 , 88 , 116 , 117 , 121 ] chromatography have been successfully implemented for the analysis of both O -glycopeptides and O -glycoproteins alike. Ion mobility adds another dimension of separation based on how an ion’s charge and shape influences its migration through a collision cell or drift tube in the gas phase [ 122 , 123 ].…”

Potassium Channels, Glucose Metabolism and Glycosylation in Cancer Cells

“…The glycosylation pattern of cancer cells differs significantly from that of normal cells [163]. Oligosaccharide structures overrepresented in cancer cells include branched high-mannose N-glycans, truncated O-glycans with a large number of O-GLCNAc (Olinked N-Acetyl-D-glucosamine), glycans with abnormal core fucosylation, and oligosaccharides containing terminal sialic acid [164]. The reasons for the changes in the glycosylation pattern are not yet fully understood.…”

Proteome Analysis of Serum Purified Using Solanum tuberosum and Lycopersicon esculentum Lectins

“…Glycosylation is a highly complex post-translational modification that plays a pivotal role in various biological processes, such as cell differentiation, intercellular communication, and immunity [20,21]. In addition, many proteins identified as potential biomarkers for diseases, including cancer, are known to be glycosylated [22][23][24]. Therefore, comparative analysis of glycoproteins using LC-MS has the potential to reveal novel biomarkers that may not be detectable via conventional abundance-based comparisons.…”