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 148

5. Other dominant ions include the cross-ring fragments (X n
and A n ). The X n ions provide only sequence information.
However, the A n ions, which incorporate the non-reducing
end, often give linkage information. The most abundant A n
ions observed in N -glycan high-energy CID are normally the
3,5 A n fragments that occur around the central

-mannose
residue and its neighbouring N -acetylglucosamine in the
chitobiose core ( see Fig. 8.3a and b ) .

β

4. Notes

1. Pepsin is stable at 60 ◦ C. The pH optimum for the enzyme
is 2-4 and the activity is irreversibly lost at pH>6. Pepsin
cleaves preferentially C-terminal to Phe, Leu and Glu; it
does not cleave Val, Ala and Gly. Other residues may also
be cleaved, with very variable rates.
2. PNGase A preparations should not contain glycerol, which
is present as a stabilizing reagent in commercially obtain-
able preparations, since this can interfere with subsequent
reductive amination reaction efficiencies. The enzyme is
therefore dialyzed into 50 mM ammonium acetate (pH
5.0), which can be added directly to the glycopeptide mix-
ture.
3. Solution is light sensitive, so wrap the container in alu-
minium foil and use immediately.
4. Trypsin is autolytic and is not very stable above 40 ◦ C. The
pH optimum for the enzyme is 7-9, and its activity is irre-
versibly lost at pH>11. Trypsin cleaves very specifically at
Arg-X and Lys-X bonds, but if X is Pro, then no cleavage
occurs.
5. Prepare freshly before use and keep it on ice; adjust trypsin
concentration in order to achieve a 1:100 to 1:20 trypsin
to protein ratio (between 200 and 500 ng/mL).
6. It is recommended that freeze-dried PNGase F be used and
not enzyme solutions in glycerol. If the enzyme contains
glycerol, make sure it is buffer exchanged into a volatile
buffer such as ammonium bicarbonate (pH 8) prior to use.
7. Classic Sep-Pak C 18 cartridges (Waters) should be kept at
room temperature, away from moisture and exposure to air
should be kept to a minimum.
8. Methanol is toxic by inhalation or if swallowed, thus it must
be handled in the fume hood.