Building a PGC-LC-MS N-glycan retention library and elution mapping resource

Abstract

Porous graphitised carbon-liquid chromatography (PGC-LC) has been proven to be a powerful technique for the analysis and characterisation of complex mixtures of isomeric and isobaric glycan structures. Here we evaluate the elution behaviour of N-glycans on PGC-LC and thereby provide the potential of using chromatographic separation properties, together with mass spectrometry (MS) fragmentation, to determine glycan structure assignments more easily. We used previously reported N-glycan structures released from the purified glycoproteins Immunoglobulin G (IgG), Immunoglobulin A (IgA), lactoferrin, α1-acid glycoprotein, Ribonuclease B (RNase B), fetuin and ovalbumin to profile their behaviour on capillary PGC-LC-MS. Over 100 glycan structures were determined by MS/MS, and together with targeted exoglycosidase digestions, created a N-glycan PGC retention library covering a full spectrum of biologically significant N-glycans from pauci mannose to sialylated tetra-antennary classes. The resultant PGC retention library ( http://www.glycostore.org/showPgc ) incorporates retention times and supporting fragmentation spectra including exoglycosidase digestion products, and provides detailed knowledge on the elution properties of N-glycans by PGC-LC. Consequently, this platform should serve as a valuable resource for facilitating the detailed analysis of the glycosylation of both purified recombinant, and complex mixtures of, glycoproteins using established workflows.

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

“…Further support for these six PMGs was obtained by the fact that the CID‐MS/MS data matched previously published spectra of human PMG standards, that is, M1F, M2a, M3, and M3F (spectral comparisons not shown) . The relative PGC‐LC retention times of the reported PMGs also agreed with published data . The α1,3‐terminating bi‐mannosylated PMG isomers (i.e., M2b and M2Fb) were absent or negligible in most datasets whereas the corresponding α1,6‐terminating M2a and M2Fa isomers were identified in most samples.…”

“…[29] The relative PGC-LC retention times of the reported PMGs also agreed with published data. [52] The 1,3-terminating bi-mannosylated PMG isomers (i.e., M2b and M2Fb) were absent or negligible in most datasets whereas the corresponding 1,6-terminating M2a and M2Fa isomers were identified in most samples. The relative level of paucimannosylation and the distribution of the individual PMG species within the N-glycome profiles were determined using EIC-based quantitation as is common practice in glycomics [48] (see Data S1 and S2, Supporting Information, for tabulated N-glycome profile data of all samples).…”

“…High confidence in the reported PMG structures including their monosaccharide composition and linkage/branching patterns was achieved by thorough de novo characterization based on PGC‐LC‐MS/MS data and via spectral and PGC‐LC retention time matching to previously characterized PMGs and N ‐glycan standards …”

“…High confidence in the reported PMG structures including their monosaccharide composition and linkage/branching patterns was achieved by thorough de novo characterization based on PGC-LC-MS/MS data and via spectral and PGC-LC retention time matching to previously characterized PMGs and N-glycan standards. [29,52] In contrast to other glycomics methods including MALDI-MS profiling of underivatized glycans, in source glycan fragmentation can be ruled out in this study since PGC-LC-MS/MS separates glycan analytes prior to ionization and detection. Furthermore, the employed PGC-LC-MS/MS method detects reduced, but otherwise native (non-derivatized) glycans, which limits the sample handling steps and, in turn, reduces the risk of perturbing individual N-glycan analytes and skewing their composition in mixtures.…”

N‐Glycan Analysis by Ultra‐Performance Liquid Chromatography and Capillary Gel Electrophoresis with Fluorescent Labeling

“…Specific "glycan" chromatographic columns are used for fluorescently labeled glycan separation based on hydrophilic interactions and larger, charged glycans are retained longer on the column, compared to smaller neutral ones. With the use of a glucose ladder (dextran), retention times of individual glycan peaks can be translated into glucose units (GU) and unknown glycan structures assigned with a search through a database like Glyco-Store (www.glycostore.org; Abrahams, Campbell, & Packer, 2018;Campbell, Royle, Radcliffe, Dwek, & Rudd, 2008).…”