- Home
- Protocols
-
In-Solution Digestion Of Purified Yeast Protein For LC-MS
Published: Apr 20, 2011 DOI: 10.21769/BioProtoc.62 Views: 14820
PDF 
Ask a question
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
LC-MS
Sample preparation
In-solution digestion
Yeast
Materials and Reagents
- Purified protein
- Sequencing grade modified trypsin (Promega Corporation, catalog number: V5113 )
- 6 M guanidine HCl
- Tris HCl (pH 8.0)
- 1 M DTT
- Triethylamine (TEA) (Sigma-Aldrich)
- HPLC solvent A (usually 10% acetonitrile in water)
- Acetic acid
Equipment
- Amicon Ultra centrifuge filters Ultracel 10 k MWCO (EMD Millipore)
- SpeedVac
- Heat block
- High Performance Liquid Chromatography (HPLC)
- Amicon filters
Procedure
Category
Systems Biology > Proteomics > Whole organism
Microbiology > Microbial proteomics > Whole organism
Biochemistry > Protein > Isolation and purification
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Post a Question
