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

Wei-Qing Wang; Ole Nørregaard Jensen; Ian Max Møller; Kim H Hebelstrup; Adelina Rogowska-Wrzesinska

doi:10.1186/s13007-018-0341-4

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

Plant Methods. 2018 Aug 25:14:72. doi: 10.1186/s13007-018-0341-4. eCollection 2018.

Authors

Wei-Qing Wang^{1

2}, Ole Nørregaard Jensen¹, Ian Max Møller³, Kim H Hebelstrup³, Adelina Rogowska-Wrzesinska¹

Affiliations

¹ 1Department of Biochemistry and Molecular Biology and VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
² 3Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093 China.
³ 2Department of Molecular Biology and Genetics, Aarhus University, Flakkebjerg, 4200 Slagelse, Denmark.

Abstract

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.

Keywords: Barley; Hordeum vulgare; In solution digestion; Mass spectrometry; Sample preparation; Sodium deoxycholate.