Evaluation of the Effect of Trypsin Digestion Buffers on Artificial Deamidation

Nonenzymatic
deamidation occurs readily under the condition of
trypsin digestion, resulting in the identification of many artificial
deamidation sites. To evaluate the effect of trypsin digestion buffers
on artificial deamidation, we compared the three commonly used buffers
Tris-HCl (pH 8), ammonium bicarbonate (ABC), and triethylammonium
bicarbonate (TEAB), and ammonium acetate (pH 6), which was reported
to reduce Asn deamidation. iTRAQ quantification on rat kidney tissue
digested in these four buffers indicates that artificial Asn deamidation
is produced in the order of ammonium acetate < Tris-HCl < ABC
< TEAB, and Gln deamidation has no significant differences in all
tested buffers. Label-free experiments show the same trend, while
protein and unique peptide identification are comparable using these
four buffers. To explain the differences of these four buffers in
producing artificial Asn deamidation, we determined the half-life
of Asn deamidation in these buffers using synthetic peptides containing
-Asn-Gly- sequences. It is 51.4 ± 6.0 days in 50 mM of ammonium
acetate (pH 6) at 37 °C, which is about 23, 104, and 137 times
that in Tris-HCl, ABC, and TEAB buffers, respectively. In conclusion,
ammonium acetate (pH 6) is more suitable than other tested buffers
for characterizing endogenous deamidation and N-glycosylation.