Relative versus absolute quantitation in disease glycomics

Abstract

The glycome of a diagnostic biological material such as blood, urine, saliva, tissue, or cell cultures comprises of a vast array of structurally distinct glycans attached to the protein complement. Aberrant glycan structures and distributions result from changes in specific glycosyltransferase activities and have different biological significance, making proper quantitation of glycans highly important. In this review, common HPLC/CE and LC-MS/MS-based methods for glycomics, their advantages and disadvantages, will be discussed with respect to the main quantitative strategies. With the increasing interest in absolute quantitation for glycomics, we discuss absolute and relative glycome quantitation and how it affects the resulting conclusions drawn from glycomics studies. We argue that while absolute quantitation of glycomes may be attractive for some areas of clinical glycomics, relative quantitation of glycans remains the most informative and time/cost-effective method to obtain biological insight into the regulation of the cellular glycosylation machinery and the synthesis of the resultant glycan structures in most research questions due to the enzymatic relatedness of the biosynthesized glycans. Recent developments in multiplexing of glycomes by the introduction of stable isotopic labeling of glycans show promise for providing another level of information to the existing benefits of relative quantitation.

Site-SpecificN-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

“…A total abundance of only 23.4% high mannose structures was observed, indicating approximately 17% glycosylation of Asn563, based on a fully occupied Asn402. Compared with the intact glycopeptide/peptide analysis presented here, the study employed an indirect approach of analysis of IgM released glycans, and experimental issues such as protein purity, incomplete deglycosylation, unequal labelling efficiencies, individual biological variation, and sample losses from additional clean-up procedures might result in bias [61].…”

Modification of Asparagine-Linked Glycan Density for the Design of Hepatitis B Virus Virus-Like Particles with Enhanced Immunogenicity

“…GlycoWorkbench was additionally employed (http://www.eurocarbdb.org/applications/ms-tools) to assist in assigning the glycan structure using in silico fragmentation analysis. The distribution of N-glycans of WT and glycosylation variants of HBsAgS was mapped using the MS signal strength of the observed N-glycan structures as an estimate of their relative abundances (58,59). This was performed using the areas of the extracted ion chromatograms (EIC) of all the charge states in which the identified N-glycans appeared.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…This has enabled accurate relative quantitation of single glycoforms on individual protein sites by comparing the precursor (SILAC) or the product (iTRAQ) ion intensities across two or more conditions. However, as we discussed in a recent review (67), comparing the entire glycoform distribution (micro-heterogeneity) at individual glycosylation sites in separate conditions may be more informative in glycobiology. Both isotope-assisted quantitative glycoproteomics (46,105,108,164) and label-free (based on XIC area, precursor intensity or spectral count) quantitative glycoproteomics (21,104,106,159,165) can generate quantitative information of site micro-heterogeneity.…”