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| Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves | |
| Wang, Wei-Qing; Jensen, Ole Norregaard; Moller, Ian Max; Hebelstrup, Kim H.; Rogowska-Wrzesinska, Adelina | |
| 2018 | |
| 发表期刊 | PLANT METHODS
 |
| ISSN | 1746-4811 |
| 卷号 | 14 |
| 摘要 | Background: Sample preparation is a critical process for proteomic studies. Many efficient and reproducible sample preparation methods have been developed for mass spectrometry-based proteomic analysis of human and animal tissues or cells, but no attempt has been made to evaluate these protocols for plants. We here present an LC-MS/MS-based proteomics study of barley leaf aimed at optimization of methods to achieve efficient and unbiased trypsin digestion of proteins prior to LC-MS/MS based sequencing and quantification of peptides. We evaluated two spin filter-aided sample preparation protocols using either sodium dodecyl-sulphate or sodium deoxycholate (SDC), and three in-solution digestion (ISD) protocols using SDC or trichloroacetic acid/acetone precipitation. Results: The proteomics workflow identified and quantified up to 1800 barley proteins based on sequencing of up to 6900 peptides per sample. The two spin filter-based protocols provided a 12-38% higher efficiency than the ISD protocols, including more proteins of low abundance. Among the ISD protocols, a simple one-step reduction and S-alkylation method (OP-ISD) was the most efficient for barley leaf sample preparation; it identified and quantified 1500 proteins and displayed higher peptide-to-protein inference ratio and higher average amino acid sequence coverage of proteins. The two spin filter-aided sample preparation protocols are compatible with TMT labelling for quantitative proteomics studies. They exhibited complementary performance as about 30% of the proteins were identified by either one or the other protocol, but also demonstrated a positive bias for membrane proteins when using SDC as detergent. Conclusions: We provide detailed protocols for efficient plant protein sample preparation for LC-MS/MS-based proteomics studies. Spin filter-based protocols are the most efficient for the preparation of leaf samples for MS-based proteomics. However, a simple protocol provides comparable results although with different peptide digestion profile. |
| 关键词 | Barley Hordeum vulgare In solution digestion Mass spectrometry Sodium deoxycholate Sample preparation |
| 学科领域 | Biochemical Research Methods ; Plant Sciences |
| DOI | 10.1186/s13007-018-0341-4 |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS关键词 | PLANT-TISSUES ; 2-DIMENSIONAL ELECTROPHORESIS ; EXTRACTION TECHNIQUES ; REDUCING AGENT ; PROTEINS ; DIGESTION ; REVEALS ; SURFACTANT ; MEMBRANE ; MIXTURES |
| WOS记录号 | WOS:000442569000002 |
| 出版者 | BMC |
| 文献子类 | Article |
| 出版地 | LONDON |
| 资助机构 | China Scholarship CouncilChina Scholarship Council ; National Natural Sciences Foundation of ChinaNational Natural Science Foundation of China (NSFC) [31400292] ; Danish Council for Independent Research - Technology and Production (FTP) |
| 作者邮箱 | wwq0814@ibcas.ac.cn ; adelinar@bmb.sdu.dk |
| 作品OA属性 | Green Published, gold |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.ibcas.ac.cn/handle/2S10CLM1/20394 |
| 专题 | 中科院植物分子生理学重点实验室 |
| 作者单位 | 1.[Wang, Wei-Qing 2.Jensen, Ole Norregaard 3.Univ Southern Denmark, Dept Biochem & Mol Biol, Campusvej 55, DK-5230 Odense M, Denmark 4.Univ Southern Denmark, VILLUM Ctr Bioanalyt Sci, Campusvej 55, DK-5230 Odense M, Denmark 5.[Moller, Ian Max 6.Hebelstrup, Kim H.] Aarhus Univ, Dept Mol Biol & Genet, DK-4200 Slagelse, Denmark 7.Chinese Acad Sci, Key Lab Plant Mol Physiol, Inst Bot, Beijing 100093, Peoples R China |
| 推荐引用方式 GB/T 7714 | Wang, Wei-Qing,Jensen, Ole Norregaard,Moller, Ian Max,et al. Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves[J]. PLANT METHODS,2018,14. |
| APA | Wang, Wei-Qing,Jensen, Ole Norregaard,Moller, Ian Max,Hebelstrup, Kim H.,&Rogowska-Wrzesinska, Adelina.(2018).Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves.PLANT METHODS,14. |
| MLA | Wang, Wei-Qing,et al."Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves".PLANT METHODS 14(2018). |
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| Wang-2018-Evaluation（3280KB） | 期刊论文 | 出版稿 | 开放获取 | CC BY-NC-SA | 浏览 请求全文 | |
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