Analysis of Carbohydrates on Proteins by Offline Normal-Phase Liquid Chromatography MALDI-TOF/TOF-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 138

6. PNGase F digestion buffer: 100 mM NH 4 HCO 3 (pH 8.4).
7. Lyophilized peptide N -glycosidase F (PNGase F; Roche) ( see
Note 6 ).
8. Vacuum centrifuge (Speed Vac; Savant).

1. Classic Sep-Pak C 18 1-cm 3 column (Waters) ( see Note 7 ).
2. HPLC-grade methanol (Fisher) ( see Note 8 ).
3. 5-mL Borosilicate glass Luer-Lip syringe (Sigma).
4. 5% (vol/vol) Acetic acid solution.
5. Dowex cation exchange resin (50WX8-100).
6. 4 M HCl.
7. HPLC-grade water ( see Note 9 ).
8. Disposable polystyrene columns (Pierce).
9. pH indicator strips.

2.4.N-Glycan
Purification

1. 2 M Acetic acid solution.

2.5.RemovalofSialic
Acidsfrom
Mammalian
N-Glycans

2.6.Reductive
AminationofGlycans

1. Vacuum centrifuge (Speed Vac; Savant).
2. Dry (anhydrous) DMSO (Romil; see Note 10 ).
3. Glacial acetic acid.
4. Anthranilic acid: 2-aminobenzoic acid (2-AA; Fluka) or
2-aminobenzamide acid (2-AB; Fluka).
5. Sodium cyanoborohydride (Aldrich) ( see Note 11 ).
6. GlykoClean S cartridge (Glyko) ( see Note 12 ).
7. HPLC-grade water.
8. HPLC-grade acetonitrile (Rathburn Chemicals, Walker-
burn, Scotland; see Note 13 ).

1. 80% Acetonitrile in water for sample loading.
2. Capillary HPLC system with UV detector set at 254 nm
(e.g., Ultimate HPLC system with capillary calibrator;
Dionex, UK).
3. MALDI plate spotter for automated fraction collection
(Probot; Dionex, UK).
4. Capillary amide-80 normal-phase column (300

2.7.Capillary
Normal-Phase
Chromatography

μ m ×

m particle size) (Dionex).
5. Solvent A: 50 mM ammonium formate made up with
HPLC-grade H 2 O and pH adjusted to 4.4 with formic acid
( see Note 14 ).

25 cm; 3 μ mor5 μ