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 140

2. Incubate at 37 ◦ C for 24 h.
3. Inactivate pepsin by incubation at 100 ◦ Cfor5minand
immediately cool the digest mixture on ice ( see Note 16 ).
4. Freeze and lyophilize sample using a freeze dryer.
5. Redissolve 2.5 mU of commercially available peptide-
N -glycosidase A ( see Note 2 ) in 250 L of HPLC-grade

water and apply it to dialysis by threefold centrifugation
in Nanosep centrifugal devices (3K) at 5,000

μ

× g for about

30 min at 4 ◦ C.
6. For a complete exchange of buffer, dilute the concentrated
enzyme in the upper layer (volume L) each time

with ammonium acetate buffer to final volume of 500

∼ 50 μ

μ

L.
After three cycles of centrifugation, dilute the concentrated
enzyme with ammonium acetate buffer (pH 5) to an enzyme
concentration of 5 mU in 100 L.

7. Dissolve the lyophilized samples in 100

μ

μ L of PNGase A

digestion buffer and add 5 μ L of the buffer-exchanged

PNGase A enzyme.
8. Incubate samples at 37 ◦ C for 16-18 h or overnight.
9. Stop digestion by drying the samples.

Mammalian glycans often contain sialic acid residues, which are
extremely susceptible to cleavage at elevated temperatures under
acidic conditions. Therefore, pepsin digestion is not recom-
mended and the following, more lengthy procedure should be
followed to efficiently release N -glycans:
1. Redissolve the protein pellet

3.2.2.Mammalian
Glycoproteins

in 90 μ Lof50mM

NH 4 HCO 3 under agitation ( see Note 17 ).
2. Supplement with 10% acetonitrile (add 10

μ L).

L of DTT solution and heat it up to 95 ◦ Cfor 3. Add 5 μ

5min.
4. Alkylate the protein by adding 4

μ

L of IAA solution. The
reaction will take place at 25 ◦ C for 45 min in the darkness.
5. Stop the alkylation by adding 20 L of DTT solution and

by incubating the solution at 25 ◦ C for 45 min.
6. TCA precipitate according to Section 3.1.2 .
7. Redissolve in 90

μ

μ Lof50mMNH 4 HCO 3 and add 10 μ L

of acetonitrile ( see Note 17 ).
8. Prepare a trypsin solution in NH 4 HCO 3 and add it to the
protein solution in a trypsin to protein ratio of 1:50 to
1:20.
9. Incubate at 37 ◦ C for 16-18 h or overnight.
10. Inactivate the trypsin by incubation at 95 ◦ Cinawaterbath
and cool the digest for 30 min on ice.