A NOVEL APPROACH FOR THE DETERMINATION OF MEMBRANE PROTEIN EXPRESSION LEVELS BASED ON LC-MS/MS

Membrane proteins play crucial roles in a number of cellular processes. Thus, the determination of their expression levels under various physiological conditions is crucial in many biological and biochemical studies. However, the extremely hydrophobic nature of proteins possessing transmembrane doma...

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Bibliographic Details
Published in:Journal of liquid chromatography & related technologies 2011-07, Vol.34 (12), p.1004-1013
Main Authors: Hynek, Radovan, Marsalova, Lucie, Kuckova, Stepanka, Konik, Peter, Santrucek, Jiri, Martinec, Jan, Novotna, Zuzana, Kodicek, Milan
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
ATPase
Biological and medical sciences
Chemical control
Control
expression levels
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods
isoxaben
iTRAQ
mass spectrometry
membrane proteins
Membranes
Phytopathology. Animal pests. Plant and forest protection
Plasma
plasma membrane H
Proteins
Protozoa. Invertebrates
quantification
reversed-phase liquid chromatography
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Summary:Membrane proteins play crucial roles in a number of cellular processes. Thus, the determination of their expression levels under various physiological conditions is crucial in many biological and biochemical studies. However, the extremely hydrophobic nature of proteins possessing transmembrane domains complicates their analysis. Therefore, we developed a new approach toward quantification of membrane proteins in complex mixtures combining reversed-phase chromatography on C 4 resin and mass spectrometric techniques using iTRAQ (Isobaric tags for relative and absolute quantitation) labels. Reversed-phase chromatography on C 4 resin with stepwise elution with 2-propanol was used to reduce contamination with hydrophilic proteins and to fractionate membrane proteins according to their hydrophobicity. The method was tested on an artificially prepared defined ratio of plasma membrane proteins obtained from cell cultures of Arabidopsis thaliana by 2-phase partitioning of microsomal fractions. Then, the technique was applied for determination of the influence of the pesticide isoxaben on plasma membrane protein expression levels, particularly plasma membrane H + -ATPase. It was found that isoxaben increases expression levels of plasma membrane, H + -ATPase 1, H + -ATPase 2, and H + -ATPase 3, which was also supported by the results of Western blotting with antibodies against H + -ATPase. The developed technique seems to be promising for the determination of expression levels of membrane proteins in general.
ISSN:1082-6076
1520-572X
DOI:10.1080/10826076.2011.569808