Protein Protocols Protein Protocols

HPLC of Fluorescently Labeled Glycans 867
Fig. 1. The steps involved in complete analysis are shown diagrammatically. The Roman
numerals relate to the stages given in the Introduction.
2. Materials
2.1. Equipment
1. HPLC system: High-pressure mixing two-solvent system capable of delivering
0.1–1.0 mL/min with shallow gradient (see Note 1).
2. Solvent degasser: Additional solvent degasser aids reproducibility.
3. Detector fluorescent detector capable of excitation at λ 330nm and emission at λ 420nm (see
Note 2).
4. PC computer system for data analysis.
5. Data acquisition software, for example, Waters Millenium.
6. Curve-fitting software, Microsoft Excel or Peak time (see Note 3).
2.2. Reagents
1. Anhydrous hydrazine was prepared by distillation from reagent grade hydrazine (Pierce
Aldrich 21515-1) by mixture with calcium oxide and toluene as previously described (34)
(see Note 4). This is available commercially from Glyko.
2. Acetic anhydride (ACS reagent, Sigma).
3. Anion-exchange resin: Bio-Rad AG50 X12.
4. Acetonitrile: HPLC grade with low background fluorescence such as E Chromosolv ®
(Reiedel-de-Haën, from Sigma).
5. Formic acid aristar grade (BDH).
6. 26% Extra pure ammonia solution (Reiedel-de-Haëen, from Sigma).
7. Water MilliQ or equivalent, subboiling point double distilled water (for mass spectrometric
analysis).