Rapid and Cost-effective Purification of Endo-Lys-N from Maitake (Grifola Frondosa) Mushrooms for Proteomics Applications
Robert C. Pelot, Jon Reed, Gogce Crynen, James Evans, Ariel Hart, Thomas Plate, Prashanthi Vallabhaneni, Laila Abdullah, Mike Mullan and Fiona Crawford
Affiliation: Roskamp Institute Mass Spectrometry Laboratory, 2040 Whitfield Avenue, Sarasota, Florida, 34243, USA.
Bottom-up, or shotgun, proteomics typically relies heavily on trypsin for its ease-of-use and reproducibility.
Nonetheless, the need for greater sequencing depth and coverage has led researchers to develop complementary digestion
approaches using other proteases. One such protease, endo-Lys-N [EC 126.96.36.199] (Lys-N), has been explored in considerable
detail for its use in proteomics. Some of the advantages of Lys-N include the ability to perform digestions in
strongly-denaturing conditions and its ability to produce peptides that yield ETD spectra which are generally less complex
than their corresponding LysC or tryptic peptides. This attribute is particularly advantageous for de novo sequencing efforts
by MS. Despite these advantages, the high cost of commercially-available Lys-N poses a barrier to its wider use
within the research community. Here, we describe a novel protocol for a rapid purification of Lys-N from store-bought
mushrooms which exploits both its thermal stability and high affinity for carboxymethyl cellulose. The resulting preparation
exhibits comparable performance to a commercially available enzyme, as evidenced by LC-MS/MS analysis. The isolation
can be completed in a few hours, and yields active enzyme at a fraction of the commercial cost.
Keywords: Grifola frondosa, Lys-N, mass spectrometry, protein purification, proteomics, trypsin.
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