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Evaluation of the Effect of Trypsin Digestion Buffers on Artificial Deamidation

ABSTRACT: This work comprehensively compared 50 mM of ammonium acetate (pH 6), Tris-HCl (pH 8), ABC and TEAB as in-solution trypsin digestion buffers. Both iTRAQ quantification and label-free results indicate that ammonium acetate (pH 6) is more suitable than other buffers for studying endogenous deamidation and N-glycosylation due to the significant decrease of artificial Asn deamidation in comparison to other buffers without affecting protein and peptide identification and Gln deamidation. Determination of the half-life of Asn deamidation in the four buffers further validates the conclusion. Our results also indicate that among the commonly used tryspin digestion buffers, ABC and TEAB are not suitable for studying Asn deamidation and N-glycosylation, but Tris-HCl may be used if trypsin digestion has to be done at around pH 8.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Kidney

SUBMITTER: Piliang Hao

LAB HEAD: Siu Kwan Sze

PROVIDER: PXD001650 | Pride | 2022-02-28

REPOSITORIES: Pride

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Evaluation of the effect of trypsin digestion buffers on artificial deamidation.

Hao Piliang P Ren Yan Y Datta Arnab A Tam James P JP Sze Siu Kwan SK

Journal of proteome research 20141222 2

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 ...[more]

PMID: 25495137

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