Evaluation of Recombinant, Chemically Treated Trypsin in Proteomics and Protein Characterization Assays

Judy Boland, Nicolas Caffarelli, Gordon Nicol, Amber Henry, Jeffrey L. Turner, Kevin Ray

Overview

  • We compared recombinant trypsin to native-source in various MS workflows.
  • Native source trypsin is dimethylated to prevent autolysis and TPCK treated to inhibit chymotrypsin.
  • Native and recombinant trypsins gave similar amounts of proteins and peptides identified in a complex mixture.
  • TIC of a single-protein digest revealed more complete peptide processing by recombinant trypsins relative to native trypsin.
  • Trypsin dimethylation leads to slower processing but with fewer autolytic peptides present.

Introduction

Porcine Pancreatic Trypsin

Figure 1.Porcine Pancreatic Trypsin

Proteomics grade trypsin is:

  1. Dimethylated (prevent autolysis)
  2. TPCK treated (inhibit chymotrypsin)
Dimethylated

Figure 2.Dimethylated

Are there better options?

Materials and Methods

Figure 3.Materials and Methods

Results

SOLu-Trypsin Stability

Figure 4.SOLu-Trypsin Stability

Table 1.Human Protein Extract Digest

Human protein extract was digested using native and recombinant trypsins in a traditional overnight 37 °C digest and a rapid 2 hr
45 °C protocol. In all cases, similar numbers of proteins were identified.

Apolipoprotein A-1 Digest

Figure 5.Apolipoprotein A-1 Digest

To Dimethylate or Not Dimethylate

Figure 6.To Dimethylate or Not Dimethylate

Conclusions

  • Recombinant trypsin digests yielded comparable amounts of peptides and proteins identified across all conditions tested versus standard sequencing-grade trypsin.
  • Dimethylation was shown to reduce the presence of autolytic fragments, whereas unmodified enzyme yielded faster processing of certain cut sites.
  • SOLu-Trypsin is solution-stable for >49 days at 37 °C and >800 days at 4 °C based on accelerated stability studies.
  • Recombinant solution-stable trypsin can be used in lieu of native trypsin with no changes to work flow.
Materials
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