Project PXD009670

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Biological Dataset
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Summary

Title

impact of Cd-exposure on medicago sativa cell wall

Description

The heavy metal cadmium (Cd) accumulates in the environment due to anthropogenic influences. It is unessential and harmful to all life forms. The plant cell wall forms a physical barrier against environmental stress and changes in the cell wall structure have been observed upon Cd exposure. In the current study, changes in the cell wall composition and structure of Medicago sativa stems were investigated after long-term exposure to Cd. Liquid chromatography coupled to mass spectrometry (LC-MS) for quantitative protein analysis was complemented with targeted gene expression analysis and combined with analyses of the cell wall composition. Compared to most studies the plants were exposed to the heavy metal for an entire season, including the repeated cutting of the above-ground biomass as is done in agriculture.

Sample Processing Protocol

Cell wall and soluble protein extraction of five replicates from M. sativa stems were done as described before (Printz et al Frontiers Plant Science https://doi.org/10.3389/fpls.2015.00237). Digestion of proteins was performed using an Amicon Ultra-4 10K Centrifugal filter device (Millipore). Cell wall and soluble proteins, 20 µg of each sample, were reduced with 10 mM DTT in 100 mM ammonium bicarbonate (AmBic) for 20 min and subsequently washed with 100 mM AmBic (30 min, 4700 g, 4°C). Reduced samples remained on top of the filter and were alkylated with 50 mM iodoacetamide dissolved in 100 mM AmBic for 30 mins in the dark. After two washing steps, samples were digested with 40 µL trypsin Gold (Promega), 5 ng/ml trypsin in 20 mM AmBic, at 40°C overnight. Afterwards, 100 µL H2O was added on the filter, devices were centrifuged (40 min, 4700 g, 4°C) and peptides collected from the bottom of the tube. The peptides were dried under vacuum and solubilized in 40 µL of 5 % v/v acetonitrile (ACN) and 0.01 % v/v trifluoroacetic acid (TFA). LC-MS/MS peptide separation and analysis Peptides were analysed with a NanoLC-2D System (Eksigent) coupled to a TripleTOF 5600+ MS (Sciex). After desalting and enrichment on a C18 pre-column (PepMap™ 100, 5 μm, 5 mm x 300 μm I.D.,Thermo Scientific), the peptides were separated with a C18 reverse phase column (PepMap™ 100, 3 μm, 100 Å, 75 μm I.D. × 15 cm, Thermo Scientific) using a linear binary gradient (solvent A: 0.1 % formic acid; solvent B: 80 % ACN, 0.1 % formic acid) at a flow rate of 300 nL/min. Peptides were eluted from 5 % to 55 % solvent B over 40 min, afterwards eluent B increased to 100 % to wash the column and the column was re-equilibrated. The LC was coupled to the mass spectrometer with a NanoSpray III source. The CID fragmentations for MS/MS spectra acquisitions used the automatically adjusted system of rolling collision energy voltage. A full MS scan was performed (scan range: 300 – 1250 m/z, accumulation time: 250 ms) and the 20 most intense precursors were selected for fragmentation.

Data Processing Protocol

The CID spectra were analysed with Mascot-Daemon (version 2.4.2, Matrix Science) by searching against the alfalfa EST database downloaded from the Samuel Roberts Noble website (675750 sequences; 304231702 residues, released on 3rd of November 2015) using the following parameters: 2 missed cleavages, mass accuracy precursor: 20 ppm, mass accuracy fragments: ± 0.5 Da, fixed modifications: carbamidomethyl (C), dynamic modifications: Oxidation (M and P), Acetyl (protein N-term), Didehydro (F) and tryptophan to kynurenine. Proteins were considered as identified when at least two peptides passed the MASCOT-calculated score of ≥ 25 and the same peptides were identified in at least 80 % of the replicates. Mascot data were imported in PROGENESIS QI software for proteomics (NonLinear Dynamics) for quantitative analysis. Quantitative results were statistically evaluated by means of a one-way ANOVA p-value (p≤0.05) as well as a fold-change of 1.5 to reveal proteins with a significantly different abundance. In the quantitative analysis only unique peptides were considered. Proteins, for which a significant change was observed, were manually validated to avoid false positive identifications. The subcellular location was determined by the TargetP online tool using standard parameters. A protein was considered as cell-wall targeted when TargetP predicted a secretion signal peptide or the subcellular localization was found based on literature. Identified cell wall proteins were categorised into functional classes following DOI:10.1002/pmic.201600449.

Contact

kjell sergeant, Luxembourg Institute of Science and Technology (LIST) Environmental Research and Innovation (ERIN)
Jean-François Hausman, Environmental Research and innovation Environmental and Industrial Biotechnologies Plant Biotechnologies ( lab head )

Submission Date

04/05/2018

Publication Date

11/06/2019

Cell Type

plant cell

Instrument

TripleTOF 5600

Software

Not available

Experiment Type

Shotgun proteomics

Assay count

4

Publication

Publication pending

Assay

Showing 1 - 4 of 4 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 91732 F146448 sol prot for pride submission.mzid 1187 55608 4667 34719 0
2 91730 F146092 Li for PRIDE submission.mzid 406 15735 2395 41154 0
3 91731 F146450 EGTA for pride submission.mzid 564 19744 1493 17832 0
4 91729 F146449 CaCl for pride submission.mzid 729 40159 2402 29635 0