Which Trypsin Do You Use?

Although this topic is not directly related to bioinformatics, I think it is generally useful information to many proteomic researchers. Trypsin is a universal proteinase for mass spec experiment. Trypsin generates nice peptides that can be ionized well and is not so expensive for routine in-gel digestion or in-solution digestion.  Protocol has been established very well and many people assume what they are doing is optimal condition. Is it true?

Today I came across a paper published recently online on Journal of Proteome Research.

Benchmark_of_6_trypsin

In this article, the authors compared 6 commonly used trypsins in terms of specificity, efficiency, contaminants and so on. They used 8 standard proteins bought from Sigma. These proteins are solublized in 6M urea/TEAB, reduced and alkylated. Then the protein solution is diluted with TEAB and digested with trypsin overnight. They compared these trypsins

Promega (#5113): Sequencing grade modified trypsin$41/100ug
Sigma (T6567-5X20UG): Trypsin (porcin) $49.7/100ug
Sigma (T1426-50MG): Trypsin (bovine) $36.1/50mg
Sigma (T3568): TrypZean (bovine expressed in maize) $165/10mg
Worthington-Biochem (LS02120): Tryspin (bovine) $56/100ug
Roche (03708985001): Trypsin (porcin expressed in yeast) $80/100ug

As authors mentioned the price varies dramatically, Roche is most expensive of all (1.25ug/$) and bovine trypsin from sigma is the cheapest (1385ug/$).

The mass spec analysis they used is both label free and TBT-labeling. Peptides are analyzed with nLC/MS/MS Orbi Velos or Q-Exactive.

Here is the conclusion

Contaminants:
Sigma (bovine): Chymotrypsin
Roche: 5 yeast proteins
Sigma (TrypZean): 5 maize proteins

Specificity & Efficiency:
Fully tryptic: more is better
Promega=TrypZean>Sigma Porcine=Roche>Sigma=Worth.Bio

Missed cleavage: fewer is better
Promega=Sigma Bobine=TrypZean<Sigma Porcine=Roche<Worth.Bio

Non-tryptic, tryptic only N- or C-term: fewer is better
Promega=Sigma Procine=TrypZean=Worth.Bio=Roche<<Sigma Bovine

Based on these results, Promega trypsin is a good choice to start as this product has been around for a long time. I personally use this for most of application. However, if you need to use a large amount of trypsin, TrypZean may be worth considering. Especially if you want to study post-translational modification, you probably need to digest a large amount of protein first, then purify specifically modified peptides. Bovine trypsin from Sigma is cheap but it contains chymotrypsin which increase non-specific cleavages.

They also tested different protein:trypsin ratio 1:20 to 1:100. They found that the number of identified peptides did change so much, but at higher ratio (1:20), they observed more non-tryptic peptides due to increased activity of contaminated chymotrypsin or other proteases. So no need to use more trypsin than necessary.

About bioinfomagician

Bioinformatic Scientist @ UCLA

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