While many proteases are used in bottom-up mass spectrometric (MS) analysis (1–3), trypsin (4,5) is the de facto protease for most applications. There are several reasons for this: Trypsin is highly efficient and has good digestive activity and specificity for arginine and lysine; Additionally, tryptic peptides (generated by digestion with trypsin) produced after proteolysis are ideally suited in terms of both size (350–1,600 Daltons) and charge (+2 to +4) to MS analysis.
One significant drawback to trypsin digestion is the long sample preparation time, which typically ranges from 4 hours to overnight for most protocols. Achieving efficient digestion usually requires that protein substrates first be unfolded either with surfactants or denaturants such as urea or guanidine. These chemical additives can have negative effects, including protein modification, inhibition of trypsin or incompatibility with downstream LC-MS/MS. Accordingly, additional steps are typically required to remove chemical additives prior to analysis.
Additional steps might include disulfide reduction using a reducing agent such as tris(2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT). The free sulfhydryl groups on cysteine residues are then alkylated with reagents such as iodoacetamide or iodoacetic acid, which prevents free sulfhydryls from reforming disulfide bonds. These two steps typically take about 2 hours.
A New, Rapid Trypsin
In an effort to shorten the time required to prepare samples for LC-MS/MS analysis, we have developed a specialized trypsin preparation that supports rapid and efficient digestion at temperatures as high as 70°C. (Figure 1). There are several benefits to this approach. First, proteolytic reaction times are dramatically shortened. Second, because no chemical denaturants have been added, off-line sample cleanup is not necessary, leading to shorter preparation times and diminished sample loss.
Figure 1. Comparison of the Rapid Digestion-Trypsin Kit workflow to standard trypsin digestions.
The Rapid Digestion Kits are highly flexible. They can accommodate a variety of additives including reducing and alkylating agents. There are no restrictions on sample volume or substrate concentrations with these kits. Furthermore, the protocol is simple to follow and requires no laboratory equipment beyond a heat block. Complete digestion is achieved using an in-solution approach (Figure 2). Since the enzyme is not immobilized on beads, the protocol does not require rapid shaking and/or off-line filtering to remove beads.
Figure 2. Comparison of peptide spectral matches for Rapid Digestion–Trypsin and standard overnight digestion. Overnight digestions were conducted at 37°C using Trypsin Gold in 50mM ammonium bicarbonate buffer for 16 hours. Rapid digestions were conducted at 70°C using Rapid Digestion-Trypsin in 1X Rapid Digestion Buffer for 30 minutes.
While reduction and alkylation are often needed for applications like peptide mapping, where the entire protein is characterized, other applications such as quantitative analysis of complex mixtures or biomarker studies don’t require reduction or alkylation. Because the samples are heated (and therefore denatured), the protocols do not require (or recommend) denaturants. However, the procedure is amenable to the use of reducing/alkylating agents if so desired.
The Rapid Digestion-Trypsin protocol typically results in protein digests within 30–60 minutes. However, samples such as complex mixtures or substrates that are more difficult to digest (i.e., disulfide-rich proteins or membrane proteins) may have slightly longer digestion times (up to 3 hours). We recommend that you optimize the Rapid Digestion protocol by performing a time course to identify the best conditions for your samples. Titration with several enzyme/substrate (E/S) ratios is also recommended.
In addition to the benefits and flexibility of Rapid Digestion-Trypsin, we have developed a Rapid Digestion-Trypsin/Lys-C mixture, a updated version of Trypsin/Lys-C Mix, previously developed for maximally efficiently proteolytic digests, particularly for complex mixtures. The benefits of Trypsin/Lys-C compared to standard Trypsin are shown in Figure 3. Rapid Digestion-Trypsin/Lys C Kit is ideally suited for studies that require improved reproducibility across samples, and that would benefit from a shortened digestion time.
Figure 3. Improved digestion of a complex protein mixture with the Trypsin/Lys-C Mix. A typical one-step digestion using yeast or mouse protein extracts with Trypsin (left panel) resulted in 18.6% (yeast extract) or 6.6% (mouse extract) missed cleavages at lysine (K). When using Trypsin/Lys-C Mix (right panel) to digest the same extracts the missed cleavages at lysine were reduced to 2.6% (yeast extract) or 2.9% (mouse extract). When using either Trypsin or Trypsin/Lys-C Mix, missed cleavages at arginine (R) were identical (~4% for yeast extract and ~1.5% for mouse extract).
The Rapid Digestion-Trypsin and Rapid Digestion-Trypsin/Lys-C Kits are designed to shorten protein digestion times to 60 minutes versus the typical 4–20 hours required for protein digestion. The protocol is flexible, can accommodate a large range of sample volumes and protein concentrations and requires no special laboratory equipment, no additional denaturants and no off-line desalting.
- Giansanti, P. et al. (2016) Six alternative proteases for mass spectrometry based proteomics beyond trypsin. Nat. Protocols 5(11), 993–1006.
- Tsiatsiani, L. and Heck, A.J.R. (2015) Proteomics beyond trypsin. FEBS J. 282, 2612–26.
- Swaney, D.L. et al. (2010) Value of using multiple proteases for large-scale mass spectrometry-based proteomics. J. Prot. Res. 9, 1323–9.
- Aebersold, R. and Mann, M. (2003) Mass spectrometry-based proteomics. Nature 422, 198–207.
- Shevchenko, A. et al. (1996) Linking genome and proteome by mass spectrometry: Large scale identification of yeast proteins from two dimensional gels. Proc. Natl. Acad. Sci. USA 93, 14440–5.