Institute of Experimental Botany AS CR
We are Discovering the Plant World

Hynek R., Maršálová L., Kučková Š., Koník P., Šantrůček J., Martinec J., Novotná Z., Kodíček M.
JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 34: 1004-1013, 2011

Keywords: expression levels, isoxaben, iTRAQ, mass spectrometry, membrane proteins, plasma membrane H+-ATPase, quantification, reversed-phase liquid chromatography
Abstract: 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 C4 resin and mass spectrometric techniques using iTRAQ (Isobaric tags for relative and absolute quantitation) labels. Reversed-phase chromatography on C4 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. IEB authors: Jan Martinec