Twelve peanut genotypes were used in this study. There were six runner-type peanut genotypes: Georgia Green, A100, A104, GK7, A13 and Tifrunner, and six Spanish-bunch type peanut genotypes: ICGV 95435 (International Crops Research Institute for the Semi-Arid Tropics, Patancheru, India), MXHY and ZQ48 (Chinese landraces), and GT-YY20, GT-YY7 and GT-YY79 (Spanish derivatives with runner type peanut in their pedigrees, obtained from Crops Research Institute, Guangdong Academy of Agricultural Sciences, China). To avoid the effects of different locations, all genotypes were grown in Tifton, GA in 2003. Seeds were harvested at full maturity per normal production practices. After harvest, seeds were air-dried at 40°C and stored at 4°C before use.
Total protein extraction
The total protein extraction was modified from TCA/Acetone protein extraction protocol  with the first step of de-fatting using hexane. Dry peanut kernels (20 g) of each genotype were frozen in liquid nitrogen and ground to powder in a mill and defatted with hexane (10 ml/g dry weight) at -20°C overnight. The defatted samples were collected by centrifugation (15,000 × g for 10 min at 4°C), air-dried, and ground to a fine powder in a pre-chilled mortar and pestle in liquid nitrogen. Protein extraction and precipitation were performed in 10% (w/v) trichloroacetic acid in cold acetone with 0.07% (v/v) β-mercaptoethanol at -20°C for 2 h, followed by centrifugation at 10,000 × g for 10 min at 4°C. The pellets were washed twice with cold acetone containing 0.07% β-mercaptoethanol, followed by washing twice with cold 80% acetone and then centrifuged at 10,000 × g for 10 min at 4°C. The pellets were air dried and stored at 4°C overnight. The total proteins were dissolved in lysis buffer (10 μl/mg) containing 9.5 M urea, 4% Igepal CA-360 (Sigma, St. Louis, MO), 2.5% ampholytes (0.5% pH 3.0–10, 0.5% pH 4–6, and 1.5% pH 6–8) (Sigma), 5% β-mercaptoethanol, and kept at 35°C for 30 min. After centrifugation (15,000 × g, 20 min, 25°C), the supernatant was collected for loading in first-dimension gel electrophoresis, or alternatively, for storing at -20°C until use. The supernatant protein concentration was determined using the Bradford  assay. The experiment was conducted twice, and each genotype was run at least three times.
SDS-PAGE and two-dimensional PAGE electrophoresis
Total protein samples from these twelve peanut genotypes were first profiled using SDS-PAGE (15% separating gel with 4% stacking gel) according to the method of Laemmli  with the Mini-PROTEIN ®II Dual Slab Cell System (BIO-RAD, Hercules, CA) . Total proteins (100 μg) from each sample were loaded onto SDS-PAGE gels. Low-range protein markers (Sigma) were used as molecular mass standard. The gels were electrophoresed (120 V, 1.5 h), stained with 0.125% Coomassie blue R-250 in 40% methanol and 10% acetic acid. For 2-D PAGE, total seed proteins (1 mg) were loaded into tube gels (8 M urea, 4% acrylamide, 2% Igepal CA-630, 0.5% ampholyte pH 3.0–10, 0.5% ampholyte pH 4–6, 1.5% ampholyte pH 6–8, 0.01% ammonium persulfate, and 0.1% TEMED), and overlaid with 20 μl sample overlay buffer (4 M urea, 0.25% ampholyte pH 3.0–10, 0.25% ampholyte pH 4–6, 0.75% ampholyte pH 6–8, 2.5% β-mercaptoethanol, 1% Igepal CA-360, and 0.05% Bromophenol blue). Isoelectric focusing (IEF) was conducted by using Mini-Protean® 2-D Electrophoresis Cell (BIO-RAD). The upper and lower chamber buffers were 100 mM NaOH and 10 mM H3PO4 respectively. IEF conditions were 200 V for 15 min, 300 V for 15 min, 400 V for 30 min, and 750 V for 6 h. The focused tube gels were equilibrated immediately for 30 min in 10 ml SDS equilibration buffer (60 mM Tris-HCl, pH6.8, 2% SDS, 10% glycerol, and 0.05% Bromophenol blue), or kept at -20°C until use. After equilibration, the tube gels were embedded in a 1% agarose solution at the top of the 2-D gel. The second dimension was run on 15% polyacrylamide-SDS gels in a Mini-Protean® 3 Cell (BIO-RAD), 120 V for 90 min. The gels were stained with Coomassie Brilliant Blue R250 and all gels were scanned and the spot intensities were analyzed using the software Image Master-2D (BIO-RAD). The interesting spots of seed protein among the genotypes were identified by gel-to-gel comparison. For molecular weight determination, low molecular weight standard (Sigma) was used.
Protein peptides were excised from the 2-D gels and PVDF membranes for peptide sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS) to obtain internal peptide sequences and using the conventional Edman degradation method to obtain N-terminal sequences. Protein spots from the gels were excised with combined total protein amount up to 10 pg, and were subjected to in-gel digestion and analysis by ESI-MS/MS to obtain peptide sequence information at the Protein Chemistry Core Facility, Baylor College of Medicine (Houston, TX). When peptide sequences could not be obtained unambiguously by using ESI-MS/MS, Edman degradation was performed using an Applied Biosystems Procise cLC sequencer to obtain sequence information for protein identification.
Electrobloting and N-terminal sequence
To prevent N-terminal blockage during second-dimension gel electrophoresis, gels were poured at least 24 hr prior to running and 0.1 mM thiodiglycolate was added as a scavenger in the upper running buffer. 2-D gels were equilibrated for 30 min in 25 mM Tris, 192 mM glycine, 10% MeOH (pH 8.3), and then electroblotted to Immobilon-p PVDF-membrane (Millipore, Bedford, MA, USA) at 300 mA for 4 hr in a Mini Trans-Blot® Electrophoretic Transfer Cell (BIO-RAD). The membrane was subsequently equilibrated for 5 min in deionized water and proteins stained with 0.05% Coomassie Blue in 1% acetic acid and 50% methanol for a few min, destained in 50% methanol until background was pale blue. The membrane was rinsed for 5–10 min in deionized water and air-dried. Spots were excised and used for N-terminal amino acid microsequencing at Baylor Medical School (Houston, TX).
Database sequence homology analysis
Internal and N-terminal peptide sequence homology identification was performed using basic local alignment search tool (BLAST)  against known or translated open reading frames of expressed sequence tags (ESTs) in the databases at the National Center for Biotechnology Information (NCBI) and SWISS-Prot.