Glycome mapping on DNA sequencing equipment.
Here we provide a detailed protocol for the analysis of protein-linked glycans on DNA sequencing equipment. This protocol satisfies the glyco-analytical needs of many projects and can form the basis of 'glycomics' studies, in which robustness, high throughput, high sensitivity and reliable quantification are of paramount importance. The protocol routinely resolves isobaric glycan stereoisomers, which is much more difficult by mass spectrometry (MS). Earlier methods made use of polyacrylamide gel-based sequencers, but we have now adapted the technique to multicapillary DNA sequencers, which represent the state of the art today. In addition, we have integrated an option for HPLC-based fractionation of highly anionic 8-amino-1,3,6-pyrenetrisulfonic acid (APTS)-labeled glycans before rapid capillary electrophoretic profiling. This option facilitates either two-dimensional profiling of complex glycan mixtures and exoglycosidase sequencing, or MS analysis of particular compounds of interest rather than of the total pool of glycans in a sample.
Laroy
Wouter
W
Unit for Molecular Glycobiology, Department for Molecular Biomedical Research, Ghent University, and VIB, Technologiepark 927, B-9052 Gent-Zwijnaarde, Belgium. natprotcontact@dmbr.ugent.be
Contreras
Roland
R
Callewaert
Nico
N
D005591:Chemical Fractionation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000378:metabolism; D011721:Pyrenes / Q000032:analysis; D017422:Sequence Analysis, DNA / Q000295:instrumentation
C098210:1-aminopyrene-3,6,8-trisulfonic acid; D006023:Glycoproteins; D011134:Polysaccharides; D011721:Pyrenes