Optimization of Alternative Proteases for Bottom-Up Proteomics

Alternate Proteases CoverBottom-up proteomics focuses on the analysis of protein mixtures after enzymatic digestion of the proteins into peptides. The resulting complex mixture of peptides is analyzed by reverse-phase liquid chromatography (RP-LC) coupled to tandem mass spectrometry (MS/MS). Identification of peptides and subsequently proteins is completed by matching peptide fragment ion spectra to theoretical spectra generated from protein databases.

Trypsin has become the gold standard for protein digestion to peptides for shotgun proteomics. Trypsin is a serine protease. It cleaves proteins into peptides with an average size of 700-1500 daltons, which is in the ideal range for MS (1). It is highly specific, cutting at the carboxyl side of arginine and lysine residues. The C-terminal arginine and lysine peptides are charged, making them detectable by MS. Trypsin is highly active and tolerant of many additives.

Even with these technical features, the use of trypsin in bottom-up proteomics may impose certain limits in the ability to grasp the full proteome, Tightly-folded proteins can resist trypsin digestion. Post-translational modifications (PTMs) present a different challenge for trypsin because glycans often limit trypsin access to cleavage sites, and acetylation makes lysine and arginine residues resistant to trypsin digestion.

To overcome these problems, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated when using these alternative proteases have not been systematically documented.

In a recent reference (2), optimized protocols for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, Lys-C, Lys-N, Asp-N, Glu-C and Arg-C have been created.

Data describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in approximately in 2 days.


  1. Laskay, U. et al. (2013) Proteome Digestion Specificity Analysis for the Rational Design of Extended Bottom-up and middle-down proteomics experiments. J of Proteome Res. 12, 5558–69.
  2. Giansanti, P. et. al. (2016) Six alternative protease for mass spectrometry based proteomics beyond trypsin. Nat. Protocols 11, 993–6

Free Webinar: Protein Reference Materials for Mass Spectrometry

MSextractcroppedOnce the domain of analytical chemistry labs, mass spectrometry instruments are now used in basic life science research, drug discovery research, environmental and industrial laboratories, and in many other biological laboratories. These instruments, which are used to perform critical analyses, need to be monitored for performance and quality.

How do you determine the system suitability and quality for your experiments? How do you evaluate a sample preparation protocol for mass spec analysis to ensure you get the best results possible without introducing artifacts? And, if you are trying to optimize the instrument method, what standard do you use ?

Currently, there is no commercially available reference reagent to help you monitor and test all LC (liquid chromatography) and MS (mass spec) parameters, particularly sensitivity and dynamic range, in a single run.

But what if there were an optimized peptide mixture that could report all key instrument performance parameters in a single run? Even better, what if that mixture came with free software to make analysis of all of the data you can generate even easier?

The upcoming free webinar: The 6 × 5 LC-MS/MS Peptide Reference Mixture and Software Analysis Tool describes such a standard reagent and analysis software.
If you are interested in learning about standardized reagent and software to help you monitor your instrument or optimize methods or protocols, register for this free webinar today!