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Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves

文献类型：期刊论文

作者Wang, Wei-Qing; Jensen, Ole Norregaard; Moller, Ian Max; Hebelstrup, Kim H.; Rogowska-Wrzesinska, Adelina
刊名PLANT METHODS
出版日期2018
卷号14
关键词Barley Hordeum vulgare In solution digestion Mass spectrometry Sodium deoxycholate Sample preparation
ISSN号1746-4811
DOI10.1186/s13007-018-0341-4
文献子类Article
英文摘要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.
学科主题Biochemical Research Methods ; Plant Sciences
出版地LONDON
WOS关键词PLANT-TISSUES ; 2-DIMENSIONAL ELECTROPHORESIS ; EXTRACTION TECHNIQUES ; REDUCING AGENT ; PROTEINS ; DIGESTION ; REVEALS ; SURFACTANT ; MEMBRANE ; MIXTURES
语种英语
WOS记录号WOS:000442569000002
出版者BMC
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)
源URL[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.[Moller, Ian Max
2.Univ Southern Denmark, VILLUM Ctr Bioanalyt Sci, Campusvej 55, DK-5230 Odense M, Denmark
3.[Wang, Wei-Qing
4.Jensen, Ole Norregaard
5.Univ Southern Denmark, Dept Biochem & Mol Biol, Campusvej 55, DK-5230 Odense M, Denmark
6.Chinese Acad Sci, Key Lab Plant Mol Physiol, Inst Bot, Beijing 100093, Peoples R China
7.Hebelstrup, Kim H.] Aarhus Univ, Dept Mol Biol & Genet, DK-4200 Slagelse, Denmark
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).

入库方式： OAI收割

来源：植物研究所

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