Human disease glycomics: technology advances enabling protein glycosylation analysis – part 1

Abstract

Protein glycosylation is recognized as an important post-translational modification, with specific substructures having significant effects on protein folding, conformation, distribution, stability and activity. However, due to the structural complexity of glycans, elucidating glycan structure-function relationships is demanding. The fine detail of glycan structures attached to proteins (including sequence, branching, linkage and anomericity) is still best analysed after the glycans are released from the purified or mixture of glycoproteins (glycomics). The technologies currently available for glycomics are becoming streamlined and standardized and many features of protein glycosylation can now be determined using instruments available in most protein analytical laboratories. Areas covered: This review focuses on the current glycomics technologies being commonly used for the analysis of the microheterogeneity of monosaccharide composition, sequence, branching and linkage of released N- and O-linked glycans that enable the determination of precise glycan structural determinants presented on secreted proteins and on the surface of all cells. Expert commentary: Several emerging advances in these technologies enabling glycomics analysis are discussed. The technological and bioinformatics requirements to be able to accurately assign these precise glycan features at biological levels in a disease context are assessed.

Finding the Keys to the CAR: Identifying Novel Target Antigens for T Cell Redirection Immunotherapies

“…Glycoproteins are another class of cell surface molecules which may be suitable therapeutic targets. As reviewed by Arub Everest-Dass, there are three main approaches to studying glycosylation [50]. The first is characterisation of intact glycoproteins.…”

“…Characterisation of protease-digested glycopeptides is a second approach. The enzymatic digestion of glycoproteins to peptides overcomes the issue of mass limited resolution of intact glycoprotein analysis by MS, however, glycopeptides are detected with a low signal by MS and therefore require enrichment before or during analysis [50]. The structural analysis of N-and O-glycans released from proteins is currently the best approach in investigating the structure, sequence, branching and linkage of glycans.…”

“…The structural analysis of N-and O-glycans released from proteins is currently the best approach in investigating the structure, sequence, branching and linkage of glycans. The recent advances in technology to determine these structures is detailed [50], though two key methodologies include glycan MS fragmentation spectra to determine glycan structure and Ion mobility to identify glycans from complex mixtures.…”

Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping

“…Given the high degree of complexity and similarity among glycan structures found within cells and tissues, the ability to discriminate among isomeric glycan structures is often necessary to fully define their glycome. Capillary electrophoresis and liquid chromatography (LC) are separation techniques capable of resolving isomeric glycan structures 2 . Of the LC methods available, hydrophilic interaction chromatography (HILIC) can provide robust separations of glycan structures 3 while PGC is capable of resolving these glycan isomers further, including those not typically resolved with HILIC 4 .…”

Utilization of a Dextran Ladder to Standardize PGC-LC-MS-Based Glycomics

“…Techniques such as capillary electrophoresis and liquid chromatography (LC) are able to separate isomeric glycan structures 2 . For glycan analysis, hydrophilic interaction chromatography (HILIC) can obtain robust separations 3 whereas PGC is a alternative stationary phase capable of high resolving power for separating glycan isomers 4 .…”