Dataset Information

Barley leaf sample preparation

ABSTRACT: Barley is an important cereal crop all over the world. Detailed molecular characterization of barley provides the basis for development of improved cultivars, stress and drought-resistant 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 (SDS) or sodium deoxycholate (SDC), and three in-solution digestion (ISD) protocols using SDC or trichloroacetic acid/acetone precipitation. The proteomics workflow identified up to 1800 barley proteins based on sequencing of up to 7700 peptides per sample. The two spin filter-based protocols provided a 17-38% 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 labeling 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. We provide detailed protocols for efficient barley protein sample preparation for LC-MS/MS-based proteomics studies. Spin filter-based protocols are the most efficient for the preparation of barley leaf samples for MS-based proteomics, however, a simple protocol provides comparable results although with different peptide digestion profile.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Hordeum Vulgare (barley)

TISSUE(S): Leaf

SUBMITTER: Wei-Qing Wang

LAB HEAD: Wei-Qing Wang

PROVIDER: PXD008938 | Pride | 2018-09-03

REPOSITORIES: Pride

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

Wang Wei-Qing WQ Jensen Ole Nørregaard ON Møller Ian Max IM Hebelstrup Kim H KH Rogowska-Wrzesinska Adelina A

Plant methods 20180825

<h4>Background</h4>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 bas ...[more]

PMID: 30159003

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			Action	DRS
	FASP-R1.msf	Msf
	FASP-R1.raw	Raw
	FASP-R2.msf	Msf
	FASP-R2.raw	Raw
	FASP-R3.msf	Msf