基于不同提取方法的水稻叶片蛋白质组的二维液相色谱分离及高分辨质谱分析

建立了酚法提取-二维液相色谱分离-高分辨质谱分析水稻叶片蛋白质组的方法。水稻叶片蛋白质经过酚法提取，酶解肽段脱盐后用离线反相-反相二维液相色谱分离，然后用线性离子阱/静电场轨道阱组合式高分辨质谱分析，共鉴定到2712种蛋白质。比较了液相色谱分离系统（一维液相色谱与二维液相色谱）和水稻叶片蛋白质提取方法（酚法、十二烷基硫酸钠法（SDS法）和三氯乙酸/丙酮法（TCA/丙酮法））对鉴定蛋白质数量的影响，结果表明：在二维液相色谱条件下，酚法、SDS法和TCA/丙酮法鉴定到的蛋白质数目为2712、2415和1914，分别是一维液相色谱条件下鉴定到的蛋白质数目的2.7、2.5和1.9倍。二维液相色谱条件下，酚法鉴定到的蛋白质数目比SDS法和TCA/丙酮法分别多297和798。与SDS法和TCA/丙酮法相比，酚法不但鉴定到的蛋白质数量多，而且能够鉴定到一些极端蛋白质，如酸性、碱性及高等电点的蛋白质。此外，对二维液相色谱条件下3种蛋白质提取方法提取到的蛋白质进行生物学功能分类，发现3种方法鉴定到的蛋白质的功能存在互补性，但酚法鉴定到的蛋白质功能种类最多。该法为水稻蛋白质组学研究提供了技术支撑，同时也为其他作物的蛋白质组学研究技术提供重要的借鉴。

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

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.