适合杨树叶片蛋白质组学样品制备方法的比较

【目的】为了提高植物叶片中低丰度蛋白质的检测效率,建立有效的杨树叶片蛋白质组学分析的蛋白质提取方法。【方法】利用三氯乙酸-丙酮结合PEG分级提取方法和SDS-酚结合PEG分级提取方法对杨树叶片蛋白质进行提取,并采用SDS-PAGE和双向凝胶电泳(2-DE)对提取结果进行评价。【结果】SDS-PAGE实验结果显示,三氯乙酸-丙酮结合PEG方法优于SDS-酚结合PEG分级方法。三氯乙酸-丙酮结合PEG分级和非结合PEG分级提取蛋白质的双向电泳结果表明,PEG分级能有效地去除高丰度蛋白质Rubisco,提高低丰度蛋白质的检测。三氯乙酸-丙酮结合PEG分级法检测的蛋白质比非结合PEG分级法多90个蛋白质点。【结论】以LTQ-Orbitrap XL分析三氯乙酸-丙酮结合PEG分级分离的一些蛋白质显示,三氯乙酸-丙酮结合PEG分级对于2-DE和MS鉴定低丰度蛋白质是一种有效的分离方法。

Comparison of sample preparation methods suitable for poplar leaf proteomics

【Objective】In order to improve the detection of low abundance proteins in plant leaves, the effective extraction methods were established in poplar leaves for proteomics analysis.【Method】 Trichloroacetic acid(TCA)-acetone coupled with polyethylene glycol(PEG)fractionation and sodium dodecyl sulfate(SDS)-phenol coupled with PEG fractionation were evaluated, combined with SDS-polyacrylamide gel electrophoresis(SDS-PAGE)and two-dimensional electrophoresis(2-DE).【Result】The SDS-PAGE results showed that TCA-acetone coupled with PEG fractionation is superior to SDS-phenol coupled with the PEG fractionation method. Further, proteins with PEG fractionation and no PEG fractionation coupled with TCA-acetone were compared by using 2-DE, and the gel maps displayed that PEG fractionation is a good way to deplete the Rubiscos and improve the detection of low abundant proteins. The PEG fractionation method can detect nearly 90 more spots than nonfractionation can. 【Conclusion】LTQ-Orbitrap XL was used to identify several proteins from the PEG fractionation coupled with TCA-acetone. The TCA-acetone coupled with PEG fractionation is an effective approach for the detection of 2-DE analysis and mass spectroscopy(MS)identification of lower abundance proteins.