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 139

6. Solvent B: 80% acetonitrile containing 20% of solvent A.
7. MALDI target.

1. 2,5-Dihydroxybenzoic acid (DHB; Fluka) solution: 2.8.Acquisition

ofMSSpectra

10 mg/mL DHB in 50% methanol ( see Note 15 ).
2. Desiccator with vacuum pump and solvent trap assembly.
3. 4700 Proteomics Analyser (Applied Biosystems, Foster City,
CA, USA).

3. Methods

This is carried out to concentrate and remove contaminants such
as salts and detergents from the glycoprotein samples. If the pro-
tein solution is very dilute (0.1-0.4mg/mL), the TCA method
gives the best protein recovery.

3.1.Protein
Precipitation

1. Suspend the glycoprotein sample into cold (-20 ◦ C) acetone
volume five times that of the protein samples to be precipi-
tated, mix well and incubate for 24 h at -20 ◦ C.
2. Centrifuge at 10,000

3.1.1.Acetone
Precipitation

g for 30 min at 4 ◦ C , decant and
properly dispose of the supernatant, being careful not to dis-
lodge the protein pellet.
3. Allow the acetone to evaporate from the uncapped tube at
room temperature for 30 min but make sure not to over-dry
the pellet as it may not dissolve properly.

×

1. To a dilute solution of glycoprotein (e.g., 200 μ Lat

3.1.2.TCAPrecipitation

L of 60% TCA.
2. Mix well and incubate mixture overnight on ice.
3. Centrifuge at 10,000

40-200 μ g/mL), add 40 μ

g for 30min at 4 ◦ C.
4. Carefully remove the supernatant and add 100

×

μ L of 90%

ice-cold acetone to wash the pellet.
5. Incubate on ice for 15min and centrifuge as above.
6. Carefully remove the acetone-containing supernatant and
allow acetone to evaporate from the uncapped tube at room
temperature for 30 min but make sure not to over-dry the
pellet as it may not dissolve properly.

3.2.N-Glycan
Release

1. Dissolve glycoprotein sample in 100 μ L of pepsin digestion

3.2.1.Plantand Insect
Glycoproteins

buffer and add 28 μ L of pepsin solution.