Efficient proteolysis is a major requirement for protein mass spectrometry analysis. Incomplete digestion has multiple ramifications including decreased number of identified proteins, compromised analytical reproducibility and protein quantitation, etc. Trypsin is one of the most robust proteases and is characterized by efficient proteolysis. Typical trypsin reactions do not digest proteins to completion, missing 15–30% of cleavage sites. Incomplete digestion affects protein identification, reproducibility of mass spectrometry analysis and accuracy of protein quantitation. Supplementing Trypsin with Lys-C compensates for the majority of missed cleavages.
The number of missed cleavage sites may be even higher if the protein is poorly purified or contains protease-inhibiting contaminants. For example, guanidine hydrochloride, a common agent for solubilizing and denaturing proteins, inhibits trypsin even at low concentrations. While both trypsin and the Trypsin/Lys-C Mix are inhibited by guanidine to some extent, the Trypsin/Lys-C Mix is more tolerant of guanidine chloride than trypsin alone. For example, in the presence of 0.5M guanidine chloride, the level of missed cleavages in yeast protein extract digest with trypsin is more than 44%, whereas this level drops to 21.5% with Trypsin/Lys-C (1). Due to this inhibition, use guanidine chloride as a protein solubilizer to prepare proteins for mass spectrometry only when absolutely necessary.
Tightly folded proteins represent another challenge for trypsin. Theoretically, proteolytically resistant proteins can be digested under strong denaturing conditions; however, denaturing conditions inhibit trypsin. Using the Trypsin/Lys-C Mix instead of trypsin alone may help overcome these limitations. The proteolysis is performed in two steps (Figure 2). The first step uses a strong protein denaturing agent such as urea, which allows Lys-C to cleave previously inaccessible sites. Lys-C digests proteins into relatively large fragments, while trypsin activity is inhibited. In the second step, the digestion mixture is diluted to reduce urea concentration. This reactivates trypsin and allows complete proteolysis.
- Saveliev, S. et al. (2013) Trypsin/Lys-C protease mix for enhanced protein mass spectrometry analysis. Nature Methods 10, i–ii.
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