Two-dimensional liquid chromatography separation and high resolution mass spectrometry analysis for proteome of rice leaves based on different extraction methods

Abstract

A two-dimensional liquid chromatography (2D LC) system was developed to separate proteins from rice leaves, which was extracted by phenol method, followed by the analysis with linear trap quadrupole orbitrap mass spectrometry (LTQ/Orbitrap MS). After proteins were extracted with phenol method, the enzymolytic peptides were separated by offline two-dimensional RP-RP system and detected by LTQ/Orbitrap MS, yielding 2712 proteins. Liquid chromatography separation system (1D LC and 2D LC) and protein extraction methods (phenol method, sodium dodecyl sulfate method (SDS method) and trichloroacetic acid/acetone method (TCA/acetone method)) were compared. Proteins identified by 2D LC were 2712, 2415 and 1914 with the above three extraction methods, respectively. The proteins were 2.7-fold, 2.5-fold and 1.9-fold the number of proteins identified by 1D LC respectively. And in terms of 2D LC, the proteins identified by phenol method were 297 and 798 more than SDS method and TCA/acetone method, respectively. Some proteins with extreme properties, such as very acidic or basic protein and high relative molecular mass proteins, were only identified in phenol method. Furthermore, proteins, which were extracted by different extraction methods and separated by 2D LC, were classified according to biological functions. It was found that protein functions by the three extraction methods were complementary. However, phenol method had the most variety of functions. The method provides technological support for rice proteomics and reference for research techniques of other crop proteomics.