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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Publications

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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Project description:Here we exploit the essential process of X-chromosome dosage compensation to elucidate basic mechanisms that control the assembly, genome-wide binding, and function of gene regulatory complexes that act over large chromosomal territories. We demonstrate that a subunit of C. elegans MLL/COMPASS, a gene-activation complex, acts within the dosage compensation complex (DCC), a condensin complex, to target the DCC to both X chromosomes of hermaphrodites and thereby reduce chromosome-wide gene expression. The DCC binds to two categories of sites on X: rex sites that recruit the DCC in an autonomous, sequence- dependent manner, and dox sites that reside primarily in promoters of expressed genes and bind the DCC robustly only when attached to X. We find that DCC mutants that abolish rex-site binding do not eliminate dox-site binding, but instead reduce it to the level observed at autosomal binding sites in wild-type animals. Changes in DCC binding to these non-rex sites occur throughout development and correlate with transcriptional activity of adjacent genes. Moreover, autosomal DCC binding is enhanced by rex-site binding in cis in X-autosome fusion chromosomes. Thus, dox and autosomal sites exhibit similar binding properties. Our data support a model for DCC binding in which low-level DCC binding at dox and autosomal sites is dictated by intrinsic properties correlated with high transcriptional activity. Sex-specific DCC recruitment to rex sites then greatly elevates DCC binding to dox sites in cis, which lack intrinsically high DCC affinity on their own. We also show here that the C. elegans DCC achieves dosage compensation through its effects on transcription. ChIP-chip experiments using antibodies against DPY-27, SDC-3, DPY-30, DPY-26, MIX-1, SMC-4, ASH-2 in wild-type embryos. ChIP-chip experiments using antibodies against SDC-3, DPY-27, DPY-30, ASH-2, and IgG in different DCC mutants. ChIP-chip experiments using antibodies against RNA Pol II (hypophosphorylated and S2 and S5) in wild type and sdc-2 partial loss of function mutants.

Dataset Information

Barley leaf sample preparation

Publications

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

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