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

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

Proteomics grade trypsin is:

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

Are there better options?

Results

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.

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.