Dataset Information

Tef (Eragrostis tef) iTRAQ-labelled protein fractions

ABSTRACT: The orphan crop, Eragrostis tef, was subjected to controlled drought conditions to observe the physiological parameters and proteins changing in response to dehydration stress. Physiological measurements involving electrolyte leakage, chlorophyll fluorescence and ultra-structural analysis showed tef plants to tolerate water loss to 50% RWC before adverse effects in leaf tissues were observed. Proteomic analysis using iTRAQ mass spectrometry and appropriate database searching enabled the detection of 5727 proteins, of which 211 proteins, including a number of spliced variants, were found to be differentially regulated with imposed stress conditions. Validation of the stress-related proteins, fructose-bisphosphate aldolase (FBA), glutamine synthetase (GLN) and the protective antioxidant proteins, monodehydroascorbate reductase (MDHAR) and peroxidase (POX), using immunodetection and enzymatic assay confirmed protein presence according to iTRAQ findings and showed increased protein abundance levels and enzymatic activity in response to water-deficit. GO-term enrichment and analysis revealed terms involved in biotic and abiotic stress response, signaling, transport, cellular homeostasis and pentose metabolic processes, to be enriched in tef up-regulated proteins, while terms linked to ROS-producing processes under water-deficit, such as photosynthesis and associated light harvesting reactions, manganese transport and homeostasis, the synthesis of sugars and cell wall catabolism and modification, to be enriched in tef down-regulated proteins.

INSTRUMENT(S): 6520 Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Hordeum Vulgare (barley) Oryza Sativa (rice) Zea Mays (maize)

TISSUE(S): Leaf

SUBMITTER: Rizqah Kamies

LAB HEAD: Dr Suhail Rafudeen

PROVIDER: PXD007907 | Pride | 2017-11-21

REPOSITORIES: Pride

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Publications

A Proteomic Approach to Investigate the Drought Response in the Orphan Crop Eragrostis tef.

Kamies Rizqah R Farrant Jill M JM Tadele Zerihun Z Cannarozzi Gina G Rafudeen Mohammed Suhail MS

Proteomes 20171115 4

The orphan crop, <i>Eragrostis tef</i>, was subjected to controlled drought conditions to observe the physiological parameters and proteins changing in response to dehydration stress. Physiological measurements involving electrolyte leakage, chlorophyll fluorescence and ultra-structural analysis showed tef plants tolerated water loss to 50% relative water content (RWC) before adverse effects in leaf tissues were observed. Proteomic analysis using isobaric tag for relative and absolute quantifica ...[more]

PMID: 29140297

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Project description:In order to identify molecular targets of USP2-45 in the small intestine, we carried out a quantitative proteomics iTraq (Isobaric tag for relative and absolute quantitation) screen to compare proteins from WT and knockout mice. With this analysis we identified, among others, the scaffolding protein Na(+)/H(+) Exchange Regulatory cofactor NHERF4 to be up-regulated (4.81-fold) in the duodenum of Usp2-KO mice. Methods : Usp2-KO and WT littermates males were entrained to 12h L:12h D for at least two weeks in light-light housing boxes. Mice were sacrificed by cervical disolcation at ZT13 under red dim light and duodenal mucosa was dissected by scraping. The tissue samples were quickly frozen in liquid nitrogen before total protein extraction. Proteins were extracted in a lysis buffer containing 8M Urea, 20mM Tris-HCl, pH 7.5, 14 µg/ml Aprotinin, 0.7 µg /ml PepstatinA 0.7 µg /ml Leupeptin, 1 mM NaVO4, 10 mM Na-Pyrophosphate, 50 µM MG132 and 1mM PMSF. Protein concentration was measured by Bradford quantitation. Relative quantitation of proteins in 4 pools of 3 KO and 4 pools of 3 WT tissue samples was carried out by iTRAQ (Isobaric Tags for Relative and Absolute Quantitation). After reduction and alkylation of cysteines, proteins were precipitated, redissolved in 8M Urea buffer and digested with trypsin (1:50 w:w) overnight. 80ug of digested material for each sample was labeled by reaction with one vial of iTRAQ 8-plex reagent (ABSciex). Samples were pooled , desalted and fractionated by off-gel electrofocusing. The 24 fractions obtained were analysed by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) on a hybrid linear trap LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) on a two-hour gradient. Full MS survey scans were performed at 60’000 resolution and the ten most intense multiply charged precursor ions detected in the full MS survey scan were selected for HCD (Higher energy Collision Dissociation) fragmentation and Orbitrap analysis (7500 resolution). Each spectrum was acquired at two relative collision energies (35% and 45%) and the spectra were summed. Data files were analysed with MaxQuant 1.3.0.5 incorporating the Andromeda search engine. Further data analysis was performed using the R statistical programming language version 2.15.2 (R core team 2012). Intensities for the reporter ions 113 to 121 were normalized using the Variance Stabilizing method (R package version 3.26.0). Proteins with non-zero intensities in all channels were evaluated for differential expression using the Local-Pooled-Error method (R package version 1.32.0) followed by correction for multiple testing according to Benjamini and Hochberg.

Dataset Information

Tef (Eragrostis tef) iTRAQ-labelled protein fractions

Publications

A Proteomic Approach to Investigate the Drought Response in the Orphan Crop Eragrostis tef.

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