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An improved protein extraction method applied to cotton leaves is compatible with 2-DE and LC-MS

文献类型： 外文期刊

作者： Jin, Xiang 1 ; Zhu, Liping 2 ; Tao, Chengcheng 2 ; Xie, Quanliang 2 ; Xu, Xinyang 1 ; Chang, Lili 3 ; Tan, Yanhua 3 ; Din 1 ;

作者机构： 1.Hainan Normal Univ, Coll Life Sci, Key Lab Ecol Trop Isl, Minist Educ, Haikou 571158, Hainan, Peoples R China

2.Shihezi Univ, Coll Life Sci, Key Lab Xinjiang Phytomed Resource & Utilizat, Minist Educ, Shihezi 832003, Peoples R China

3.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Haikou 571101, Hainan, Peoples R China

关键词： Cotton leaf; Protein extraction method; Proteomics; Polysaccharide and polyphenol; Two-dimensional electrophoresis; High-throughput LC-MS/MS

期刊名称：BMC GENOMICS （ 影响因子：3.969； 五年影响因子：4.478 ）

ISSN： 1471-2164

年卷期： 2019 年 20 卷

页码：

收录情况： SCI

摘要： Background; Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. Results: Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. Conclusion: Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.

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