Electrophoretic sequencing of heparin/heparan sulfate oligosaccharides using a highly sensitive fluorescent end label.
The sequencing of heparan sulfate oligosaccharides has recently become possible using integral Glycan Sequencing, which utilizes a combination of chemical and enzymatic degradation steps followed by polyacrylamide gel electrophoresis. This technique has previously employed the fluorescent label, anthranilic acid, and has been used to sequence low nmol amounts of purified saccharides. Here, we present an improved method, which uses the alternative label, 7-aminonapthalene-1,3-disulfonic acid, the reducing agent sodium triacetoxyborohydride and optimizes the nitrous acid step in heparin/heparan sulfate degradation. These improvements increase the sensitivity at least ten-fold taking the amount of starting material required into the pmol range. We show that this label is compatible with the integral glycan sequencing methodology and demonstrate its application to the sequencing of chemically modified heparin derivatives. Advances in sequencing techniques for heparan sulfate saccharides will permit detailed structure-function studies and will in the future underpin novel proteomics-based approaches aimed at studying their diverse functional roles as protein regulators.
Drummond
K J
KJ
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
Yates
E A
EA
Turnbull
J E
JE
D015081:2-Naphthylamine / Q000031:analogs & derivatives*; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006493:Heparin / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry*; D008969:Molecular Sequence Data; D009608:Nitrous Acid; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D020543:Proteome; D012680:Sensitivity and Specificity
D015081:2-Naphthylamine; D005456:Fluorescent Dyes; D006493:Heparin; D006497:Heparan Sulfate; D009608:Nitrous Acid; D009844:Oligosaccharides; D020543:Proteome; C033153:amido-G-acid