Detection of hydrolysis of lipid post-translational modifications during gel-electrophoresis-based proteomic protocol
Language English Country Germany Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Electrophoresis, Gel, Two-Dimensional * MeSH
- Time Factors MeSH
- Esters chemistry MeSH
- Hydrolysis MeSH
- Hordeum chemistry MeSH
- Hydrogen-Ion Concentration MeSH
- Lipids chemistry isolation & purification MeSH
- Lipoproteins chemistry MeSH
- Molecular Weight MeSH
- Protein Processing, Post-Translational * MeSH
- Proteomics methods MeSH
- Plant Proteins chemistry MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Esters MeSH
- Lipids MeSH
- Lipoproteins MeSH
- Plant Proteins MeSH
The influence of sample preparation on the identification of a lipid PTM was examined. Nonspecific lipid transfer protein 1 (LTP1) from barley is modified with a lipid-like molecule of mass of 294 Da. This modification was detected in the MS analysis of intact protein samples but no lipid-bound peptide was observed in the MS analysis of the in-gel digested LTP1 after an SDS-PAGE separation of the protein mixture. By using SEC instead of SDS-PAGE, the lipid-modified peptide was observed after in-solution enzymatic digestion of the SEC fraction containing LTP1. Conditions of individual steps of the gel-electrophoresis-based protocol were tested to find their effect on the removal of the lipid PTM from LTP1. The influences of particular solutions used in the gel-electrophoresis-based protocol on the hydrolysis of lipids were investigated. It was found that denaturing conditions, in combination with alkaline pH, have a major influence on the hydrolysis of the ester bond. Especially, the electrophoretic buffer has a strong influence on the hydrolysis of the lipid PTM (in the intact molecule) of LTP1.
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