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

By: Judy Boland, Nicolas Caffarelli, Gordon Nicol, Amber Henry, Jeffrey L. Turner, and Kevin Ray, MilliporeSigma, A business of Merck KGaA, Darmstadt, Germany, St. Louis MO 63103

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

Porcine Pancreatic Trypsin


Proteomics grade trypsin is:

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

Dimethylated

Are there better options?

 

Materials and Methods

Materials and Methods

 

Results

SOLu-Trypsin Stability

SOLu-Trypsin Stability

Human Protein Extract Digest
 

Trypsin Average number of proteins identified
37°C for 18 hr 45°C for 2 hr
Proteomics
Grade Trypsin
2,021 2,167
SOLu-Trypsin 2,055 2,128
SOLu-Trypsin
Dimethylated
2,025 2,162

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

Apolipoprotein A-1 Digest

 

To Dimethylate or Not Dimethylate

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

     
Related Links