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Revisiting the effect of trypsin digestion buffers on artificial deamidation

ABSTRACT: Deamidation of asparagine and glutamine residues occurs spontaneously, accelerated by adjacent amino acid residues and influenced by pH, temperature, and incubation time. Standard proteolytic digestion induces significant deamidation, which is especially problematic in bottom-up proteomic studies. Trypsin digestion protocol modifications, including shorter incubation times and specific lysis buffers are shown to minimize deamidation. Herein, HEPES is proven to be most optimal due to reduced deamidation coupled with its compatibility for both proteins and trypsin digestion, highlighting its suitability for maintaining sample integrity in biological research contexts.

INSTRUMENT(S): Orbitrap Ascend

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Emmajay Sutherland

LAB HEAD: Nicholas M. Riley

PROVIDER: PXD054603 | Pride | 2025-03-11

REPOSITORIES: Pride

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Revisiting the Effect of Trypsin Digestion Buffers on Artificial Deamidation.

Sutherland Emmajay E Veth Tim S TS Riley Nicholas M NM

Journal of the American Society for Mass Spectrometry 20250131 3

Deamidation of asparagine and glutamine residues occurs spontaneously, is influenced by pH, temperature, and incubation time, and can be accelerated by adjacent amino acid residues. Incubation conditions used for proteolytic digestion in bottom-up proteomic studies can induce significant deamidation that affects results, either knowingly or unknowingly. This has prompted studies into modifications to common trypsin digestion protocols to minimize chemical deamidation, including shorter incubatio ...[more]

PMID: 39887243

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			Action	DRS
	3T3_AmBic_r1.raw	Raw
	3T3_AmBic_r1_psm.tsv	Tabular
	3T3_AmBic_r2.raw	Raw
	3T3_AmBic_r2_psm.tsv	Tabular
	3T3_AmBic_r3.raw	Raw