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

Figure 1. Porcine Pancreatic Trypsin

Proteomics grade trypsin is:

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

Figure 2. Dimethylated

Are there better options?

Figure 3. Materials and Methods

Results

Figure 4. SOLu-Trypsin Stability

Table 1. 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.

Figure 5. Apolipoprotein A-1 Digest

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.