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Structural analysis of N- and O-glycans released from glycoproteins.

Nature protocols (2012-06-09)
Pia H Jensen, Niclas G Karlsson, Daniel Kolarich, Nicolle H Packer
ABSTRACT

This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glycopeptidase A from almonds, buffered aqueous glycerol solution, ≥0.05 unit/mL
Sigma-Aldrich
PNGase F from Elizabethkingia meningoseptica, BioReagent, ≥95% (SDS-PAGE), for proteomics
Sigma-Aldrich
PNGase F from Elizabethkingia meningoseptica, recombinant, expressed in E. coli, set of 100 units nanomolar unit
Sigma-Aldrich
PNGase F from Elizabethkingia miricola, buffered aqueous solution
Sigma-Aldrich
PNGase F from Elizabethkingia meningoseptica, lyophilized powder, recombinant, expressed in E. coli
Sigma-Aldrich
PNGase F from Elizabethkingia meningoseptica, ready-to-use solution, recombinant, expressed in E. coli