Detection of hydrolysis of lipid post-translational modifications during gel-electrophoresis-based proteomic protocol.
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.
Zídková
Jitka
J
Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
Rehulka
Pavel
P
Chmelík
Josef
J
D015180:Electrophoresis, Gel, Two-Dimensional; D004952:Esters / Q000737:chemistry; D001467:Hordeum / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry* / Q000302:isolation & purification; D008074:Lipoproteins / Q000737:chemistry; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013696:Temperature; D013997:Time Factors
D004952:Esters; D008055:Lipids; D008074:Lipoproteins; D010940:Plant Proteins