Thylakoid Membrane Proteome: Separation by HPLC and Identification by Accurate Molecular Mass Determinations

Lello      Zolla; Anna   Maria   Timperio; Christian   G.   Huber

Abstract

This review covers the vast array of methods that appeared in the last few years for separation and identification of thylakoid membrane proteins present in chloroplast, a good model for setting up analytical methods suitable for membrane proteins. Although the major method for protein separation is gel electrophoresis (GE), we will summarise in this review the results of several studies performed to develop a rapid and straightforward method based on high-performance liquid chromatography (HPLC) to resolve most of the antenna proteins of the two photosystems, photosystem I (PSI) and II (PSII) and identify them by intact molecular mass determination using electrospray ionisation mass spectrometry (ESI-MS). The complex mixtures of these strongly hydrophobic membrane proteins are prefractionated by sucrose gradient ultracentrifugation in the first dimension followed by reversed-phase HPLC in the second dimension. The separation achieved is by far superior to that possible with GE. It is based on small differences in hydrophobicity of these very similar proteins. The correspondence between the intact molecular masses measured with those deduced from the DNA sequence enabled the identification of the different protein components of antenna complex. Isomeric forms of the proteins were readily revealed. On the contrary, peptide mass fingerprinting, commonly used for soluble proteins, does not seem to be very helpful for identification of these highly hydrophobic membrane proteins due to the limited number of peptides which can be obtained by proteolysis. Thus, separations and identifications of antenna proteins presented in this study may serve as reference for confident identification in future studies dealing with any membrane proteins.

Keywords: proteomics, thylakoid membrane, photosystems, antenna proteins, mass spectrometry, 2d-gel electrophoresis, liquid chromatography, intact molecular mass