Ian Max Møller

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

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Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves. / Wang, Wei Qing; Jensen, Ole Nørregaard; Møller, Ian Max; Hebelstrup, Kim H.; Rogowska-Wrzesinska, Adelina.

I: Plant Methods, Bind 14, Nr. 1, 72, 25.08.2018.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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Wang, Wei Qing ; Jensen, Ole Nørregaard ; Møller, Ian Max ; Hebelstrup, Kim H. ; Rogowska-Wrzesinska, Adelina. / Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves. I: Plant Methods. 2018 ; Bind 14, Nr. 1.

Bibtex

@article{d7afd6da8d974e898220d31baebfc4c3,
title = "Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves",
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",
author = "Wang, {Wei Qing} and Jensen, {Ole N{\o}rregaard} and M{\o}ller, {Ian Max} and Hebelstrup, {Kim H.} and Adelina Rogowska-Wrzesinska",
year = "2018",
month = "8",
day = "25",
doi = "10.1186/s13007-018-0341-4",
language = "English",
volume = "14",
journal = "Plant Methods",
issn = "1746-4811",
publisher = "BioMed Central",
number = "1",

}

RIS

TY - JOUR

T1 - Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves

AU - Wang, Wei Qing

AU - Jensen, Ole Nørregaard

AU - Møller, Ian Max

AU - Hebelstrup, Kim H.

AU - Rogowska-Wrzesinska, Adelina

PY - 2018/8/25

Y1 - 2018/8/25

N2 - 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.

AB - 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.

KW - Barley

KW - Hordeum vulgare

KW - In solution digestion

KW - Mass spectrometry

KW - Sample preparation

KW - Sodium deoxycholate

UR - http://www.scopus.com/inward/record.url?scp=85052305732&partnerID=8YFLogxK

U2 - 10.1186/s13007-018-0341-4

DO - 10.1186/s13007-018-0341-4

M3 - Journal article

C2 - 30159003

AN - SCOPUS:85052305732

VL - 14

JO - Plant Methods

JF - Plant Methods

SN - 1746-4811

IS - 1

M1 - 72

ER -