Methods

Fraction processing

After centrifugation at 10,000× g for 10 min, each clarified fraction was combined 1:1 with Tris-saturated phenol (pH 8.8), vortexed vigorously at room temperature for 5 min, and centrifuged at 10,000× g for 10 min. The upper aqueous fraction was back-extracted once with an equal volume of Tris-saturated phenol, and the two phenol phases were combined and re-extracted with 100mM Tris-HCl (pH 8.8) with 10mM DTT (Dithiothreitol). Next, 4 volumes of 100% methanol containing 100mM ammonium acetateand 10mM DTT (chilled to -80oC) was added to the pooled phenolic fractions and these were allowed to precipitate for 2 h at -80oC, then were centrifuged at 10,000× g for 10 min. The proteins were washed three times with 100% methanol containing 10mM DTT, followed by centrifugation at 10,000× g for 10 min. The final pellet was then washed with acetone and dried under vacuum.

SDS-PAGE

The pellet was dissolved in sample buffer solution containing 10% 1M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.002% bromophenol blue and vortexed at medium speed for 1 h at room temperature. Sonication, consisting of two or three pulses, 15 s each, at 22 W was also performed to improve dissolution. Protein solutions were clarified by ultracentrifugation at 14,000× g for 10 min at 18oC. Protein concentrations were determined using the Bradford method. Protein samples were separated by SDS-PAGE in 12% polyacrylamide minigels (Bio-Rad).

Silver staining and image analysis

After gel separation, the proteins were visualized by silver staining as described by (Méchin et al. 2003).The gel profile of the protein bands was scanned using the ImageScanner (Amersham Bioscience), following the image analysis in the Gel-ProAnalyzer 4.5 software (Media Cybernetics). The abundance of each individual protein band was determined and calculated for definition of % volume. Three replicates were made for each gel.

Protein identification based on peptidemass fingerprint/mass spectrum(PMF/MS)

Protein bands were excised from the gels and destained with 100mM NH4HCO3 in 30% ACN. After removing the destaining buffer, the gel pieces were lyophilized and rehydrated in 30 μL of 50mM NH4HCO3 containing 50ng trypsin (sequencing grade; Promega). After overnight digestion at 37℃, the peptides were extracted three times with 0.1% TFA in 60% ACN. Extracts were pooled together and lyophilized. The resulting lyophilized tryptic peptides were kept at -80℃ until mass spectrometric analysis.A protein-free gel piece was treated as above and used for a control to identify autoproteolysis products derived from trypsin.

All mass spectra were acquired on an AutoFlex Matrix-assisted laser desorption/ionization tandem time-of-flight(MALDI-TOF/TOF) mass spectrometer (Bruker Daltonics). Tryptic digests were prepared on an AnchorChip sample plate (Bruker Daltonics) according to the manufacturer’s directions. MS data were acquired with a N2 laser at a 25-Hz sampling rate.

PMF data was submitted to MASCOT for protein identification. The National Center for Biotechnology non-redundant (NCBInr) database (updated on 20080422), with Arabidopsis thaliana as taxonomy, was searched. The other parameters for searching were trypsin enzyme; one missed cleavage; fixed modifications of carbamidomethyl (C); variable modifications of oxidation (Met). A peptide tolerance of 100 ppm; a fragment mass tolerance of ± 0.5 Da; and a peptide charge of 1+ were selected. Only significant hits, as defined by the MASCOT probability analysis (p<0.05), were accepted.

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