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Proteins
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Tools
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Proteins
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Contacts
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2D Electrophoresis Proteins were first separated by electrophoresis according to charge. Isoelectrofocusing (IEF) was carried out with protein samples with an equivalent to an extract of 8 400 seeds, corresponding to about 150µg protein for all samples. Proteins from the various extracts were separated using gel strips forming an immobilized non-linear pH gradient from 3 to 10 (ImmobilineTM DryStrip pH 3-10 NL, 18 cm; Amersham Pharmacia Biotech). Strips were rehydrated for 14 h at 22°C with the thiourea/urea lysis buffer containing 2% (v/v) Triton X-100, 20 mM DTT and the protein extracts. IEF was performed at 22°C in the Multiphor II system (Amersham Pharmacia Biotech) for 1 h at 300 V and 7 h at 3500 V. Proteins were then separated according to size. Prior to the second dimension, the gel strips were equilibrated for 2 x 20 min in 2 x 100 ml equilibration solution containing 6 M urea, 30% (v/v) glycerol, 2.5% (w/v) SDS, 0.15 M bis-Tris, and 0.1 M HCl (Görg et al., 1987; Harder et al., 1999). DTT (50 mM) was added to the first equilibration solution, and iodoacetamide [4% (w/v)] to the second (Harder et al., 1999). Equilibrated gel strips were placed on top of vertical polyacrylamide gels [10% (v/v) acrylamide, 0.33% (w/v) piperarine diacrylamide, 0.18 M Trizma base, 0.166 M HCl, 0.07% (w/v) ammonium persulfate, 0.035% (v/v) Temed]. A denaturing solution [1% (w/v) low-melting agarose (Gibco BRL), 0.4% (w/v) SDS, 0.15 M bis-Tris, and 0.1 M HCl] was loaded on gel strips. After agarose solidification, electrophoresis was performed at 10°C in a buffer (pH 8.3) containing 25 mM Trizma base, 200 mM taurine, and 0.1% (w/v) SDS, for 1 h at 35 V and 14 h at 110 V. Ten gels (200 x 250 x 1.0 mm) were run in parallel (Isodalt system from Amersham Pharmacia Biotech). For each condition analyzed, 2D gels were made in triplicate and from two independent protein extractions. |
Protein Staining and Analysis of 2D Gels Gels were stained with either silver nitrate according to a modified procedure of Blum et al. (1987) or the GelCode blue stain reagent from Pierce (Rockford, IL, USA), using the Hoefer® Automated Gel Stainer apparatus from Amersham Pharmacia Biotech. Silver stained gels were scanned with the Sharp JX-330 scanner equipped with the Labscan version 3.00 from Amersham Pharmacia Biotech. Image analysis was carried out with the ImageMaster 2-D Elite version 3.01 software (Amersham Pharmacia Biotech), according to the instruction booklet “ImageMaster® 2D Elite” from Amersham Pharmacia Biotech. After spot detection and background substraction (mode: average on boundary), 2D gels were aligned, matched, and the quantitative determination of the spot volumes was performed (mode: total spot volume normalization). For each analysis, statistical data showed a high level of reproducibility between normalized spot volumes of gels produced in triplicate from the two independent protein extractions. |
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Protein Identification by Mass Spectrometry Spots
of interest were excised from GelCode-stained 2D gels and digested
by sequence grade trypsin (Promega Biotec, Madisson, WI, USA). After
digestion, the supernatant containing peptides was concentrated by
batch adsorption on POROS 50 R2 beads (Roche Molecular Biochemicals,
Basel, Switzerland), and used for MALDI-MS analysis on a Bruker Reflex
II MALDI-TOF spectrometer after on-target desorption with matrix solution
(Gevaert et al., 1998). Before each analysis, the instrument was externally
calibrated using two synthetic peptides spotted as near as possible
to the biological sample. Proteins were identified
by searching the protein databases using MASCOT (http://www.matrixscience.com).
Theoretical masses and isoelectric points of identified proteins were
predicted by entering the sequence at http://www.expasy.org/tools/peptide-mass.html.
To denote a protein as unambiguously identified, the following criteria
were used: coverage of the protein by the matching peptides must reach
a minimum of 10% and at least four independent peptides should match
within a stringent 10-ppm maximum deviation of mass accuracy. In some
cases, protein identities were further confirmed from Post Source
Decay (PSD) spectra, generated from selected peptides. |
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