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Thermal denaturation produced degenerative proteins and interfered with MS for proteins dissolved in lysis buffer in proteomic analysis

文献类型： 外文期刊

作者： Wang, Xuchu 1 ; Wang, Haiyan 1 ; Wang, Dan 1 ; Wang, Dongyang 1 ; Han, Bing 1 ; Tian, Weimin; Guo, Anping 1 ;

作者机构： 1.Chinese Acad Trop Agr Sci, Minist Agr, Inst Trop Biosci & Biotechnol, Key Lab Trop Crop Biotechnol, Haikou Hainan 571101, Peoples R China

2.Chinese Acad Trop Agr Sci, Minist Agr, Rubber Res Inst, Key Lab Rubber Biol, Danzhou Hainan, Peoples R China

3.Chinese Acad Trop Agr Sci, Minist Agr, Rubber Res Inst, Key Lab Rubber Biol, Dan

关键词： Lysis buffer;MS;Plant proteomics;Protein marker;Thermal denaturation

期刊名称：ELECTROPHORESIS （ 影响因子：3.535； 五年影响因子：2.731 ）

ISSN： 0173-0835

年卷期： 2011 年 32 卷 3-4 期

页码：

收录情况： SCI

摘要： In 1-DE, proteins were traditionally mixed with the standard Laemmli buffer and boiled for several minutes. Recently, proteins dissolved in lysis buffer were used to produce better-resolved 2-DE gels, but thermal denaturation procedure still remained in some proteomic analysis. To determine the detailed effects of thermal denaturation on SDS-PAGE and MS, both 1-DE and 2-DE were performed using proteins heated at 100 degrees C for different periods of time, and 17 protein bands/spots were positively identified by MALDI TOF/TOF MS/MS. Protein profiles on both 1-DE and 2-DE gels changed obviously and more polydisperse bands/spots were observed with increased heating time for over-heated samples. Based on these observations, an alternative protein marker-producing method was designed by directly dissolving protein standards without BSA into lysis buffer. This new kind of protein marker could be stored at room temperature for a long time, thus was more convenient for using and shipping. The identification of 17 proteins via MS and comparison of their identities revealed MASCOT-searched scores, number of both matched peptides, total searched peptides and sequence coverage became progressively lower with increasing denaturation intensity, probably due to the interference of thermal denaturation on trypsin cleavage efficiency and produced redundant modified peptides. Therefore, it was concluded that thermal denaturation not only changed the protein profiles and produced more polydisperse protein bands/spots, but also heavily interfered with the subsequent MS analysis, hence not recommended in future proteomic analysis for proteins dissolved in lysis buffer.

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