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

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  • Wei Qing Wang, University of Southern Denmark, Institute of Botany, Chinese Academy of Sciences
  • ,
  • Ole Nørregaard Jensen, Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark
  • ,
  • Ian Max Møller
  • Kim H. Hebelstrup
  • Adelina Rogowska-Wrzesinska, University of Southern Denmark

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.

Original languageEnglish
Article number72
JournalPlant Methods
Number of pages13
Publication statusPublished - 25 Aug 2018

    Research areas

  • Barley, Hordeum vulgare, In solution digestion, Mass spectrometry, Sample preparation, Sodium deoxycholate

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