Electrophoretic sequencing of heparin/heparan sulfate oligosaccharides using a highly sensitive fluorescent end label

The sequencing of heparan sulfate oligosaccharides has recently become poss ible using Integral Glycan Sequencing, which utilizes a combination of chem ical and enzymatic degradation steps followed by polyacrylamide gel electro phoresis. This technique has previously employed the fluorescent label, ant hranilic acid, and has been used to sequence low nmol amounts of purified s accharides. Here, we present an improved method, which uses the alternative label, 7-aminonapthalene-1,3-disulfonic acid, the reducing agent sodium tr iacetoxyborohydride and optimizes the nitrous acid step in heparin/heparan sulfate degradation. These improvements increase the sensitivity at least t en-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 chemicall y modified heparin derivatives. Advances in sequencing techniques for hepar an sulfate saccharides will permit detailed structure-function studies and will in the future underpin novel proteomics-based approaches aimed at stud ying their diverse functional roles as protein regulators.