A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands

The majority of biologically active proteins are glycosylated, therefore an y approach to proteomics which fails to address the analysis of oligosaccha rides is necessarily incomplete. To appreciate the structure of a glycoprot ein fully, to understand the roles for the attached oligosaccharides and to monitor disease associated changes it is necessary to visualise the sugars as well as the protein. To achieve this aim when biological samples are av ailable at the low microgram level or less has involved increasing the sens itivity of the technology for glycan analysis. Since one protein may have m any different oligosaccharides attached to it (glycoforms) this is a major technical challenge. CD59, for example, has over 100 different sugars at on e N-linked glycosylation site. Applications of recently developed technolog y suggest that it is now becoming realistic to extend the proteomics analys is of glycoproteins to include details of glycosylation. This is achieved b y releasing the N-glycans from the protein in a gel by optimised peptide-N- glycosidase F digestion. The released glycans are then tagged with the fuor ophore, 2-amino benzamide. The labelled glycan pools (containing 50-100 fem tomoles of glycans) are resolved by predictive normal phase high performanc e liquid chromatography (HPLC) on an amide based column or by reverse phase HPLC on a C18 column. Preliminary structural assignments are confirmed by exoglycosidase array digestions of the entire glycan pool. Complementary ma trix-assisted laser desorption/ionization-mass spectrometry, which requires 10-20 times as much sugar for a single run, can be used where there is suf ficient material. This provides a composition analysis but not linkage info rmation.