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 137

successful in the detailed structural elucidation of many different
types of carbohydrates ( 2 - 5 ) . The use of chromatographic meth-
ods, in combination with mass spectrometric techniques is very
advantageous for extremely complex samples and important for
the detection of minor oligosaccharide isomers in mixtures ( 5 ) .
Normal-phase HPLC (NP-HPLC) using amide-based columns is
a well-established, robust separation technique used by many lab-
oratories to obtain high-resolution separation of N -linked glycans
released from glycoproteins. We have combined this method with
MALDI-TOF/TOF-MS/MS for the detailed characterization of
carbohydrate mixtures. It is successfully employed in our labora-
tory for the detailed analysis of a wide range of carbohydrate-
based polymers including N -and O -linked glycans and plant
polysaccharides. The following protocol describes the preparation
and NP-HPLC-MALDI-TOF-MS of N -linked glycans released
from plant, insect and mammalian glycoproteins.

2. Materials

1. Ice-cold (-20 ◦ C) acetone.
2. 60% Trichloroacetic acid in H 2 O.

2.1.Protein
Precipitation

2.2.Release
ofN-Glycansfrom
PlantandInsect
Glycoproteins

1. Pepsin digestion buffer: 5% formic acid (vol:vol) pH
3 (adjust pH with 1 N NaOH).
2. Pepsin (Sigma; see Note 1 ) solution: 1 mg of enzyme in
1 mL of pepsin digestion buffer.
3. PNGase A digestion buffer: 50 mM ammonium acetate (pH
5; adjust pH with 5% acetic acid).
4. Peptide N -glycosidase A (PNGase A) (5 mU in 100 μ L;

Roche; see Note 2 ).
5. Pall NanoSep centrifugal devices with 3 K Omega Mem-
brane (Sigma).
6. Vacuum centrifuge (Speed Vac; Savant).

2.3.Release
ofN-Glycansfrom
Mammalian
Glycoproteins

1. 100 mM Ammonium bicarbonate (NH 4 HCO 3 ).
2. HPLC-grade acetonitrile (Rathburn Chemicals, Walker-
burn, Scotland).
3. 200 mM Dithiothreitol (DTT) in 100 mM NH 4 HCO 3 .
4. 1 M Iodoacetamide (IAA) in 100 mM NH 4 HCO 3

( see Note 3 ).
5. Bovine trypsin (protein sequencing grade; Roche) ( see
Note 4 )solutionin50mMNH 4 HCO 3 ( see Note 5 ).