Rapid and Cost-effective Purification of Endo-Lys-N from Maitake (Grifola Frondosa) Mushrooms for Proteomics Applications

ISSN: 1875-6247 (Online)
ISSN: 1570-1646 (Print)


Volume 11, 4 Issues, 2014


Download PDF Flyer




Current Proteomics

Aims & ScopeAbstracted/Indexed in


Submit Abstracts Online Submit Manuscripts Online

Editor-in-Chief:
Bernd Rehm
Institute of Molecular BioSciences Massey University
Private Bag 11222
Palmerston North
New Zealand


View Full Editorial Board

Subscribe Purchase Articles Order Reprints

Current: 0.44

Rapid and Cost-effective Purification of Endo-Lys-N from Maitake (Grifola Frondosa) Mushrooms for Proteomics Applications

Author(s): 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.

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.

Purchase Online Rights and Permissions

  
  



Article Details

Volume: 10
Issue Number: 4
First Page: 292
Last Page: 297
Page Count: 6
DOI: 10.2174/15701646113106660003
Advertisement

Related Journals



Webmaster Contact: urooj@benthamscience.org Copyright © 2014 Bentham Science