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In-Solution Digestion Of Purified Yeast Protein For LC-MS

Xiyan Li Xiyan Li

Published: Apr 20, 2011 DOI: 10.21769/BioProtoc.62 Views: 14802

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Abstract

This method describes the preparation of total yeast protein extract for mass spectrometry analysis. The protein extract is digested by trypsin in a solution with strong denaturants. The digested sample is dried and re-constituted in a mixture compatible with HPLC separation. Samples of isobaric labels should be processed in parallel experiments starting from trypsin digestion.

Keywords: Proteomics search LC-MS search Sample preparation search In-solution digestion search Yeast search

Materials and Reagents

  1. Purified protein
  2. Sequencing grade modified trypsin (Promega Corporation, catalog number: V5113 )
  3. 6 M guanidine HCl
  4. Tris HCl (pH 8.0)
  5. 1 M DTT
  6. Triethylamine (TEA) (Sigma-Aldrich)
  7. HPLC solvent A (usually 10% acetonitrile in water)
  8. Acetic acid

Equipment

  1. Amicon Ultra centrifuge filters Ultracel 10 k MWCO (EMD Millipore)
  2. SpeedVac
  3. Heat block
  4. High Performance Liquid Chromatography (HPLC)
  5. Amicon filters

Procedure

Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.

Category

Systems Biology > Proteomics > Whole organism

Microbiology > Microbial proteomics > Whole organism

Biochemistry > Protein > Isolation and purification

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