As the protease most commonly used to digest proteins into peptides for mass spec analysis, trypsin is a key part of most proteomic workflows, with the performance of the enzyme potentially affecting a variety experimental outcomes including sequence coverage and reproducibility.
A number of vendors sell trypsin, with their offerings ranging significantly in price. For instance, porcine trypsin from Promega – generally considered the gold standard within the field – costs $0.41 per μg. Bovine trypsin from Sigma-Aldrich, meanwhile, runs less than $0.01 per μg.
Typically, trypsin represents a small fraction of the overall cost of a proteomics experiment. In some cases, however, relatively large amounts of the enzyme are required – which raises the question of just how much the top-dollar brands are actually worth.
A group of researchers led by Henrik Molina, director of the Proteomics Resource Center at Rockefeller University, recently set out to answer this question, comparing the performance of six varieties of trypsin in digesting a standard protein mixture. They published their findings last month in a paper in the Journal of Proteome Research.
Molina's interest in this question stemmed from his work in Rockefeller's proteomics core, where, he said, "we have a lot of people coming in with all kinds of samples," and all of them, he joked, are "notorious cheapskates."
And while for many experiments trypsin costs are not significant, in the case of experiments where researchers start off with large amounts of proteins – such as post-translational modification studies requiring target enrichment – "you actually need to use a lot of trypsin, and then [the cost] makes a difference," he said.
For example, an experiment analyzing 5 μg of protein will typically use around 0.1 μg of trypsin, Molina noted. "But on the other hand, if you want to work with proteins or peptides where you really need to enrich, then you might be starting out with, say, 50 milligrams of protein and then you need to use 1 milligram of trypsin, and that suddenly becomes a concern."
Beyond the shotgun-style proteomics experiments Molina described, trypsin costs have also arisen as a potential issue for targeted clinical proteomics work where large amounts of trypsin are required to ensure complete digestion of samples, which is key for assay reproducibility.
That has led some researchers focused on clinical proteomics to try out cheaper grades of trypsin. For instance, in a 2010 interview, Andy Hoofnagle, director of clinical mass spectrometry at the University of Washington's Department of Laboratory Medicine, told ProteoMonitor, that his lab had been working with less expensive varieties of trypsin from Sigma and that these had "worked just fine (PM 10/22/2010)."
Leigh Anderson, CEO of SISCAPA Assay Technologies, likewise told ProteoMonitor that while "in proteomics we've all just submitted to marketing that says, 'Oh, we should use Promega Gold' [trypsin]," he was "convinced it will turn out that we really can use vastly cheaper grades of trypsin."
Going into the analysis, Molina said his expectation was, indeed, that the more expensive trypsin would offer the best performance. "I think that is why most of us have been using Promega trypsin," he said. "Because most of us don't want to take any chances, and we knew that it was working."
To test this assumption, he and his colleagues compared six commercial trypsins: porcine trypsin from Promega; porcine trypsin, bovine trypsin, and bovine trypsin expressed in maize from Sigma-Aldrich; bovine trypsin from bovine pancreas from Worthington Biochemical; and porcine trypsin expressed in Pichia pastoris from Roche Applied Sciences.
The most expensive of the six was the Roche product, which cost $0.80 per μg. Least expensive was the Sigma-Aldrich bovine trypsin, which cost $0.0007 per μg, making for a 1,000-fold difference in price across the six varieties.
The researchers used the enzymes to digest a mix of eight standard proteins, which they then analyzed in triplicate by both tandem mass tag and label-free LC-MS/MS on either a Thermo Fisher Scientific Orbitrap Velos or Q Exactive instrument.
In general, they found little difference in performance between the bovine and porcine trypsins, and all six generated tryptic peptides in roughly equal amounts. The inexpensive Sigma-Aldrich bovine trypsin, however, had significantly higher chymotryptic activity than the other five varieties due to higher levels of contamination with the protease chymotrypsin.
This non-tryptic activity isn't necessarily a bad thing, though, Molina suggested.
"Where I really need a lot of trypsin, it is very often for experiments where, [for instance], I am looking for phosphorylated peptides, or peptides that are acetylated," he said. "And there I would see it as a feature having an enzyme that is a little less specific – though not too unspecific – because this way I might be able to get some peptides that I would not be getting otherwise."
This lack of specificity, however, hurts the identification of low-abundance peptides, Molina noted, as the addition of the non-tryptic peptides from high-abundance proteins can mask the presence of low-abundance tryptic peptides.
Broadly speaking, though, Molina said, the study left him comfortable with the idea of using lower grade trypsin like the inexpensive Sigma product.
"If I had to go out and do an experiment where I needed a lot of trypsin, I would now probably use the cheap Sigma [reagent.]," he said. "I would not be hesitating."