Structural analysis of N- and O-glycans released from glycoproteins.
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.
Faculty of Science, Biomolecular Frontiers Research Centre, Macquarie University, Sydney, New South Wales, Australia.
Pia H
PH
Jensen
Niclas G
NG
Karlsson
Daniel
D
Kolarich
Nicolle H
NH
Packer
D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D054794:Glycomics / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008567:Membranes, Artificial; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011145:Polyvinyls; D053719:Tandem Mass Spectrometry; D005465:Fluorocarbon Polymers
D006023:Glycoproteins; D007074:Immunoglobulin G; D008567:Membranes, Artificial; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers