IPG-strips versus off-gel fractionation: advantages and limits of two-dimensional PAGE in separation of microsomal fractions of frequently used plant species and tissues.

The crucial cellular role of membrane proteins is generally known for all life forms. Depending on the species, tissue, compartment, function and physiological condition, membranes differ in their protein and lipid profiles. Additionally, occurrence of microdomains hampers quantitative protein solubilisation and therefore membrane proteomics remain a major challenge. In the present study sample preparation (TCA/acetone and methanol/chloroform precipitation with and without SDS pre-solubilisation) for two-dimensional PAGE were compared for microsomal fractions of leaves (Arabidopsis thaliana, Nicotiana tabaccum, Pisum sativum) and roots (P. sativum, Zea mays). Generally, pre-solubilisation with SDS impaired the resolution of the gels. All samples showed higher spot yields with TCA/acetone precipitation. Finally, we compared the results of conventional 2D-PAGE (IPG/SDS-PAGE) and the combination of off-gel fractionation in the first-dimension, 10% urea-SDS-PAGE in the second-dimension. Results showed that more spots are present in the alkaline pH range after off-gel fractionation then on conventional 2D-PAGE. For the first time, off-gel fractionation was combined with SDS/SDS-PAGE and BAC/SDS-PAGE to improve the resolution after off-gel fractionation. Transmembrane domains and GRAVY were calculated for all significantly identified spots resulting from the MALDI-TOF-TOF mass spectrometry showing that in the second dimension after off-gel fractionation 10.3% more transmembrane proteins were identified compared to IPG/SDS-PAGE.