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Unravelling hidden components of the chloroplast envelope proteome
Chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling exchanges of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides…) of the plant cell. However, chloroplast envelope membranes remain the hidden part of the chloroplast proteome and many envelope proteins remain to be characterized (known but uncharacterized envelope proteins) or identified (orphan known envelope-associated functions carried by unidentified proteins) Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.25% of the whole cell proteome). When comparing the composition crude leaf extract and purified envelope vesicles, a high theoretical enrichment factor (EF) of specific envelope proteins is thus expected. This is especially true for minor envelope proteins (e.g. representing 1/100 or 1/1000 of the envelope protein content). Sensitivity of MS technique has been greatly improved during the last decade. Today, thanks to the continuous improvement of MS techniques, we are able to detect more components in a complex sample, to generate quantitative data and to statistically validate above-cited enrichment factor. Here, we have taken advantage of present-days better MS sensitivity towards a better definition (differentiate genuine envelope proteins from contaminants) of the chloroplast envelope proteome. This MS- and statistical-based analysis relied on an enrichment factor calculated for each protein identified in purified envelope fractions when compared to the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1376 proteins was detected in purified envelope fractions, of which, more than 500 could be assigned an envelope localization combining MS-based statistical analyses and manual annotation using data from the literature or prediction tools. Interestingly, many of such proteins being unknown or unexpected envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.
Sample Processing Protocol
Arabidopsis thaliana plants, Wassilewskija background (Ws), were grown in culture chambers at 23°C (12-h light cycle) with a light intensity of 150 µmol/m2/s in standard conditions for four weeks. Crude cell extracts and envelope fractions were prepared in independent triplicates. Intact chloroplasts were obtained from 400–500 g of rosette leaves and purified by isopyknic centrifugation using Percoll gradient. Purified intact chloroplasts were lysed in hypotonic medium containing protease inhibitors (10 mM MOPS-NaOH, pH 7.8, 4 mM MgCl2, 1 mM PMSF, 1 mM benzamidine, and 0.5 mM amino caproic acid), and envelope was purified from the lysate by centrifugation on discountinous sucrose gradients. To recover the envelope proteins, the yellow band of the sucrose gradient containing the envelope proteins was then carefully aspirated with a pipette. Recovered envelope proteins were then diluted in 10 mM MOPS–NaOH pH 7.8 buffer (containing protease inhibitors), and concentrated as a pellet by centrifugation at 110,000 g for 1 h (Beckman SW 41 Ti rotor). Envelope proteins were diluted in 100 µL of the same medium containing protease inhibitors (an average of ~100 μg of envelope proteins was obtained from each preparation). Crude cell extracts were obtained from 2-3 leaves of Arabidopsis (Arabidopsis thaliana Ws). Leaf material was homogenized in 200 µL of extraction buffer (30 mM tetrasodium pyrophosphate, 50 mM Tris pH 6.8, SDS 1% [v/v]) and then centrifuged at 16,000 g for 5 minutes. Crude leaf extracts were then recovered by carefully aspirating the supernatant. Each protein sample (10 µg) was stacked in the top of a SDS-PAGE gel (NuPAGE 4–12%, Invitrogen) before Coomassie blue staining (R250, Bio-Rad). Gel bands of concentrated proteins were manually excised and cut in pieces before being washed by 6 successive incubations of 15 min in 25 mM NH4HCO3 containing 50% (v/v) acetonitrile. Gel pieces were then dehydrated in 100% acetonitrile and incubated at 53°C with 10 mM DTT in 25 mM NH4HCO3 for 45 min and in the dark with 55 mM iodoacetamide in 25 mM NH4HCO3 for 35 min. Alkylation was stopped by adding 10 mM DTT in 25 mM NH4HCO3 and mixing for 10 min. Gel pieces were then washed again by incubation in 25 mM NH4HCO3 before dehydration with 100% acetonitrile. Modified trypsin (Promega, sequencing grade) in 25 mM NH4HCO3 was added to the dehydrated gel pieces for an overnight incubation at 37°C. Peptides were then extracted from gel pieces in three 15-min sequential extraction steps in 30 μL of 50% acetonitrile, 30 μL of 5% formic acid and finally 30 μL of 100% acetonitrile. The pooled supernatants were then vacuum-dried. The dried extracted peptides were resuspended in 5% acetonitrile and 0.1% trifluoroacetic acid and analyzed by online nanoLC–MS/MS (NCS, and Q-Ex_HF, Thermo Fischer Scientific). Peptides were sampled on a 300 μm x 5 mm PepMap C18 precolumn and separated on a reprosyl 25 cm 1.9 µm (Cluzeau). The nanoLC method consisted in a 140-min gradient ranging from 4% to 40% acetronitrile in 0.1% formic acid in 123 min and wash to 90% and equilibration at 4% at a flow rate of 300 nL.min-1. MS and MS/MS data were acquired using Xcalibur (Thermo Fischer Scientific). Spray voltage and heated capillary were set at 2 kV and 270°C, respectively. Survey full-scan MS spectra (m/z = 400–1600) were acquired in the Orbitrap with a resolution of 60,000 after accumulation of 1e6 ions (maximum filling time: 200 ms). The twenty most intense ions from the preview survey scan delivered by the Orbitrap were fragmented by collision induced dissociation (collision energy 30%) in the LTQ after accumulation of 1e5 ions (maximum filling time: 50 ms).
Data Processing Protocol
Peaklists files were generated using Mascot Daemon. MS/MS spectra were searched using Mascot 2.6.0 (Matrix Science) against the target-decoy version of a compilation of the A. thaliana protein database (nuclear, mitochondrial and plastid genome; TAIR v10.0; December 14, 2010; 35,386 entries) and a home-made list of contaminants, frequently observed in proteomics analyses (249 entries). Trypsin/P was chosen as the enzyme and maximum of 2 missed cleavage allowed. Precursor and fragment mass error tolerances were set at 10 ppm and 0.025 Da, respectively. Peptide modifications allowed during the search were: carbamidomethyl (C, fixed) acetyl (Protein N-term, variable) and oxidation (M, variable). The Proline software (http://proline.profiproteomics.fr/) was used to filter the results (filters at replicate level were conservation of only rank 1 peptides, peptide identification FDR < 1% as calculated on peptide scores by employing the reverse database strategy, minimum peptide score of 25, peptide length ≥ 7, and minimum of 1 specific peptide per identified protein group). Next identifications were merged over the whole experiment and the minimum of 1 specific peptide per protein group filter was applied again. Spectral count were computed within each replicate.
Bouchnak I, Brugière S, Moyet L, Le Gall S, Salvi D, Kuntz M, Tardif M, Rolland N. Unravelling hidden components of the chloroplast envelope proteome: opportunities and limits of better MS sensitivity. Mol Cell Proteomics. 2019 PubMed: 30962257
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