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; Fiona      Crawford

doi:10.2174/15701646113106660003

Abstract

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 3.4.24.20] (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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