FAQ: Can Endoproteinase LysC used in a double digest with Trypsin-ultra?

Trypsin-LysC co-digests have been found to improve protein coverage in proteomics studies especially among low abundance proteins. Many different conditions have been used successfully. Please refer to the references below for more detailed discussions. A good standard Trypsin-LysC digest is to set up a LysC digest in 8M urea buffer for 4hrs at 37 °C, then dilute to 1.5 M urea for a trypsin digest for 4 hrs at 37 °C. Both digests should be 1:20-50 protease to susbtrate protein.

  • Giansanti, P. et al 2016 “Six alternative proteases for mass-spectrometry based proteomics beyond trypsin” Natur Protoc. 11, 993-1006.
  • Swaney, D. L. et al 2010 “The value of using multiple proteases for large-scale mass spectrometry-based proteomics” J. Proteome Res. 9, 1323-1329.
  • Wang, Y. et al 2016 “Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid-chromatography-mass spectrometry” Anal. Biochem. 495, 21-28.