Four Conventional Soybean [Glycine max (L.) Merrill] Seeds ExhibitDifferent Protein Profiles As Revealed by Proteomic AnalysisLuciana S. Gomes,† Raquel Senna,‡ Vanessa Sandim,# Mario A. C. Silva-Neto,‡,⊗ Jonas E. A. Perales,§,⊗

Russolina B. Zingali,#,⊗ Marcia R. Soares,⊥,⊗ and Eliane Fialho*,†

†Departamento de Nutricao Basica e Experimental, Instituto de Nutricao Josue de Castro, Universidade Federal do Rio de Janeiro,Av. Carlos Chagas Filho, Predio do CCS, Bloco J-2, Laboratorio 13, 393 Rio de Janeiro 21941-590, Brazil‡Laboratorio de Sinalizacao Celular, Instituto de Bioquımica Medica, Universidade Federal do Rio de Janeiro, Av. Carlos ChagasFilho, Predio do CCS, Bloco D, Subsolo, Sala 5, 373 Rio de Janeiro 21941-590, Brazil#Unidade de Espectrometria de Massas e Proteomica, Instituto de Bioquımica Medica, Universidade Federal do Rio de Janeiro,Av. Carlos Chagas Filho, Predio do CCS, Bloco D, 373 Rio de Janeiro 21941-590, Brazil§Instituto Oswaldo Cruz, Fundacao Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, RJ 21040-900, Brazil⊥Centro de Tecnologia, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos, 149, Rio de Janeiro 21941-909, Brazil⊗Rede proteomica do Rio de Janeiro, Rio de Janeiro, Brazil

*S Supporting Information

ABSTRACT: Soybeans have several functional properties due to their composition and may exert beneficial health effects thatare attributed to proteins and their derivative peptides. The present study aimed to analyze the protein profiles of four newconventional soybean seeds (BRS 257, BRS 258, BRS 267, and Embrapa 48) with the use of proteomic tools. Two-dimensional(2D) and one-dimensional (1D) gel electrophoreses were performed, followed by MALDI-TOF/TOF and ESI-Q-TOF massspectrometry analyses, respectively. These two different experimental approaches allowed the identification of 117 proteins from1D gels and 46 differentially expressed protein spots in 2D gels. BRS 267 showed the greatest diversity of identified spots inthe 2D gel analyses. In the 1D gels, the major groups were storage (25−40%) and lipid metabolism (11−25%) proteins. Thedifferences in protein composition between cultivars could indicate functional and nutritional differences and could direct thedevelopment of new cultivars.

KEYWORDS: Glycine max, seed proteome, protein composition, protein profile, proteomic analysis

■ INTRODUCTION

The soybean (Glycine max) is a legume of high nutritional valuewith outstanding nutritional characteristics. It is composed ofmono- and polyunsaturated fatty acids, is low in fat and carbo-hydrates, is a source of vitamins and minerals, and providesproteins with high biological value.1 The soybean has a proteindigestibility-corrected amino acid score of 1.00.2

Soybeans have several functional properties due to theircomposition and provide beneficial health effects and nourish-ment.3 Such properties are conferred by essential fatty acids;4

polyphenols such as isoflavones, anthocyanins, and procyani-dins;5,6 proteins; and low molecular weight peptides.7 Soybeanscontain from 37.0 to 44.5% protein,8,1 of which approximately70−83% are storage proteins. Storage proteins are divided intotwo main classes known as glycinins and β-conglycinins, whichare also classified as globulins 11S and 7S, respectively.9,10

Glycinins are hexameric proteins composed of differentsubunits that are linked together by disulfide bonds. The fivemajor subunits are classified into the following two groups:group I includes the G1 (A1aB2), G2 (A2B1a), and G3 (A1aB1b)subunits, and group II includes the G4 (A5A4B3) and G5 (A3B4)subunits.11 β-Conglycinins (which make up 17.8−23.0% of thetotal soybean protein content) have a trimeric structure made upof the subunits α, α′, and β, which have molecular weights of

approximately 76, 70, and 50 kDa, respectively. The threesubunits are linked by noncovalent bonds.12,13

Studies have shown that proteins and peptides derived fromsoybeans have beneficial health effects far beyond their basicfunctions. The trypsin and chymotrypsin inhibitor Bowman−Birk (BBI) has been associated with the prevention andtreatment of diseases such as colorectal cancer without toxicityto healthy cells.14 Furthermore, it has been shown that the Kunitztrypsin inhibitor (KTI) can protect mouse lung cells againstdamage caused by inflammatory processes.15

Evidence suggests that lectins, also known as hemagglutininsor agglutinins, inhibit the proliferation of liver and breast cancercells and inhibit the activity of the reverse transcriptase of thehuman immunodeficiency virus. These functions have also beendescribed for the Kunitz-type protease inhibitor.16

β-Conglycins and glycinins are bioactive compounds withprotective effects on cardiovascular health.17 Reductions inplasma total cholesterol, triglycerides, and LDL-c and increasesin the levels of HDL-c have been attributed to the oral

Received: June 25, 2013Revised: December 12, 2013Accepted: December 31, 2013Published: December 31, 2013

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administration of these proteins in hypercholesterolemic rats.18

Zhang et al.19 demonstrated that hydrolyzed soybean proteinsreduced the plasma level of very low-density lipoproteinscholesterol and triglycerides in mice. Additionally, the 11Sglobulin has hypotensive effects that are attributed to its ability toinhibit the angiotensin I-converting enzyme.20 Peptides derivedfrom the 7S globulin can act as blockers of fatty acid synthase,an enzyme present in large quantities in diseases such as cancer,obesity, and other metabolic disorders.21

Because of their health benefits, especially in the preventionand treatment of noncommunicable diseases, soybean proteinsare the only proteins to offer benefits for cardiovascular diseasethat are certified by the American Dietetic Association (ADA).22

According to the ADA, soybean products that provide 6.25 g ofsoy protein per serving are allowed to state on the label that “25 gof soy protein a day as part of a diet low in saturated fat andcholesterol may reduce the risk of coronary heart disease”. Theconsumption of 3−4 tablespoons of soybeans (56−67 g ofgrains) each day meets the FDA requirements regarding theingestion of 25 g of protein.Despite the nutritional and functional properties mentioned

above, the consumption of soybeans in Brazil is limited due to thecharacteristic flavor caused by lipoxygenases, which oxidize lipidsand cause a beany flavor. In addition, gastrointestinal distressmay be caused by the presence of oligosaccharides, raffinose,stachyose, phytic acid, and protease inhibitors.23 Embrapa-Soja,Empresa Brasileira de Pesquisa Agropecuaria, developed conven-tional cultivars in 2008 to stimulate consumption.24 Thesecultivars were obtained by crossing different varieties to mitigateflavor and improve nutritional value. This process resulted inlipoxygenase-free grains with higher levels of carbohydrates andimproved protein content.

The use of proteomics in nutrition is diverse and offersdifferent possibilities for nutritional interventions of greatvalue. The proteome study represents an important tool whenassociated with nutrigenomics as it provides insights intopromises of future dietary approaches.25 One proteomic strategyis the separation of proteins by polyacrylamide gel electro-phoresis (in one or two dimensions), followed by the analysisand identification of proteins by mass spectrometry.26 Anotherapproach is a combination of liquid chromatography andmass spectrometry, which has become a powerful approach forthe identification of proteins and peptides occurring in complexmixtures.27

The production of conventional plants with improved sensoryqualities and nutritional characteristics is an important strategy toincrease soybean consumption. However, the impact of thesechanges and the soybean improvements should be investigatedby methods such as comparative analysis of protein expressionprofiles. This analysis will allow for the determination of thelegume proteins that interfere with the nutritional and functionalproperties. We analyzed the protein profiles of four conventionalsoybean seeds (BRS 257, BRS 258, Embrapa 48, and BRS 267)using proteomic tools.

■ MATERIALS AND METHODSPlant Materials. G. max seeds were kindly donated by the Empresa

Brasileira de Pesquisa Agropecuaria (EMBRAPA - Soja, Londrina, PR,Brazil). The cultivars used in this study were the following: BRS 257,BRS 258, EMBRAPA 48, and BRS 267. The grains of these four cultivarswere ground in a mechanical mill (Retsch ZM 1) using a metallic sievewith a pore size of 0.5 mmuntil a fine powder was obtained. The sampleswere stored at −20 °C until analysis.

Extraction of Proteins from Seeds. The samples were delipidatedwith hexane until a colorless supernatant was obtained and then

Figure 1. Two-dimensional electrophoresis was carried out using a narrow-range 18 cm IPG strip of pH 4−7: analyses of the proteins extracted using athiourea/urea protocol from Glycine max cultivars (A) BRS 257, (B) BRS 258, (C) Embrapa 48, and (D) BRS 267. Identical numbers refer to the samespots, and distinct numbers correspond to differential spots present in each cultivar. The sizes of the protein markers in kilodaltons (kDa) are shown atthe left of the images.

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Table 1. Soybean Seed Proteins Identified by MALDI-TOF-TOF

SIDa proteinb NCBI accession no. MW/pIc BRS 257 BRS 258 EMBRAPA 48 BRS 267

1 35 kDa seed maturation protein gi|351726750 35.320/5.96 +d + + +2 glycinin G1/A1aBx gi|225651 56.284/5.78 + − − −

3 glycinin G1/A1aBx gi|225651 56.284/5.78 + − − −glycinin G2/A2B1 precursor gi|351725363 54.927/5.46glycinin G4/A5A4B3 subunit gi|126144648 64.196/5.17

4 lectin (soybean agglutinin) gi|6729836 27.555/5.15 + + − −5 allergen Gly m Bd 28K gi|12697782 52.780/5.73 + + − −6 allergen Gly m Bd 28K gi|12697782 52.780/5.73 + − − −7 seed biotinylated protein 68 kDa isoform gi|240254706 67.963/6.18 + − + −8 seed biotinylated protein 68 kDa isoform gi|240254706 67.963/6.18 + − − −9 embryonic protein DC-8-like gi|356533407 48.766/6.12 + − + −10 α-subunit of β-conglycinin gi|9967361 65.160/5.23 − + − −11 glycinin G4/A5A4B3 subunit gi|121279 64.005/5.29 − + − −12 sucrose binding protein homologue S-64 gi|6179947 56.142/6.32 − + − −

13 glycinin G1/A1aBx gi|225651 56.284/5.78 − + − −glycinin G2/A2B1 precursor gi|351725363 54.927/5.46

14 seed maturation protein PM31 gi|351722245 17.907/6.10 − + − −

15 seed maturation protein PM25 gi|6648966 25.827/4.99 − + − −seed maturation protein PM26 gi|351721132 26.201/4.83

16 alcohol dehydrogenase gi|4039115 37.042/6.13 − − + −17 glycinin G2/A2B1 precursor gi|351725363 54.927/5.46 − − + −18 lectin (soybean agglutinin) gi|6729836 27.555/5.15 − − + −19 lectin (soybean agglutinin) gi|6729836 27.555/5.15 − − + −20 glycinin G1/A1aBx gi|225651 56.284/5.78 − − + −

21 glycinin G1/A1aBx gi|225651 56.284/5.78 − − + −glycinin G2/A2B1 precursor gi|351725363 54.927/5.46

22 Kunitz trypsin inhibitor subtype B gi|125023 20.256/4.66 − − − +Kunitz-type trypsin inhibitor KTI1 gi|125722 22.817/4.97

23 Kunitz trypsin inhibitor subtype B gi|125023 20.256/4.66 − − − +24 2S albumin precursor gi|351727517 19.018/5.20 − − − +25 lectin (soybean agglutinin) gi|6729836 27.555/5.15 − − − +26 lectin (soybean agglutinin) gi|6729836 27.555/5.15 − − − +

27 glycinin G1/A1aBx gi|225651 56.284/5.78 − − − +lectin (soybean agglutinin) gi|6729836 27.555/5.15

28 lectin (soybean agglutinin) gi|6729836 27.555/5.15 − − − +29 allergen Gly m Bd 28K gi|12697782 52.780/5.73 − − − +30 allergen Gly m Bd 28K gi|12697782 52.780/5.73 − − − +31 allergen Ara h 1, clone P41B-like gi|356538162 82.003/5.32 − − − +32 allergen Gly m Bd 28K gi|12697782 52.780/5,73 − − − +33 uncharacterized protein LOC100305847 gi|351724719 27.847/5.72 − − − +

34 glycinin G1/A1aBx gi|225651 56.284/5.78 − − − +glycinin G2 precursor gi|351725363 54.927/5.46

35 isoflavone reductase homologue 2 gi|351726399 33.919/5.60 − − − +36 glucose and ribitol dehydrogenase homologue 1 gi|356505868 32.147/5.34 − − − +37 7S seed globulin precursor gi|1401240 47.006/8.68 − − − +38 glycinin G2 precursor gi|351725363 54.927/5.46 − − − +39 glyceraldehyde-3-dehydrogenase C subunit gi|351723699 36.815/6.72 − − − +40 elongation factor 1-α-like isoform 1 gi|356558807 49.717/9.14 − + − +

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lyophilized. A 50 mg lyophilized sample was added to 495 μL of buffercontaining 5 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.8%ampholytes at either pH 3−10 or pH 4−7, and 0.002% bromophenolblue. A 5 μL aliquot of a protease inhibitor cocktail (made up of 104 mMbenzenesulfonyl fluoride hydrochloride, 80 μM aprotinin, 4 mMbestatin hydrochloride, 1.4 mM E-64, 2 mM leupeptin, and 1.5 mMpepstatin A) was added. After sonication for 20 min, the samples werecentrifuged at 13000g for 15 min at room temperature, and supernatantswere stored at −80 °C according to the methodology described byHerman et al.28 withmodifications. The protein content was determinedusing the protocol described by Peterson.29

One-Dimensional Gel Electrophoresis (1D SDS-PAGE). For 1Danalysis, 90 μg of protein from each cultivar that had been previouslyextracted using a thiourea/urea method was applied to three lanes(30 μg in each lane) of a 12% SDS-PAGE gel (8.3× 7.3 cm dimensions).The gels were fixed and stained with CBB G-250. One-dimensionalseparation was performed following the method described byLaemmli.30 The experiments were performed with biological triplicates.After the image analysis, each lane was arbitrarily divided intoapproximately 2.5 mm slices and subjected to in-gel proteolysis withtrypsin.Two-Dimensional Gel Electrophoresis (2D SDS-PAGE). One

milligram of soybean protein that had been extracted using a thiourea/urea method was applied to 18 cm IPG strips. The first dimension(isoelectric focusing) was performed using linear IPG strips that covereda pH range of 4.0−7.0 in an Ettan IPGphor (Amersham Biosciences)with the following program: 12 V for 12 h (rehydration), 200 V for 1 h,500 V for 1 h, 1000 V for 1 h, 4000 V for 0.5 h, and 75067 V for 9.38 h.For the second dimension, the IPG strips were incubated with 50 mMTris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, 0.002%bromophenol blue, and 65 mM DTT for 15 min. The strips werealkylated with iodoacetamide (25 mg/mL) and subsequently placedonto 15% polyacrylamide gels. The gels were run in a DALTsix system(GE Healthcare) at 2.5 W/gel for 30 min followed by 100 W for six gelsuntil the end of the run. The reference gel was obtained after the analysisof biological triplicates. The experiments were performed in technicaltriplicates.Gel Staining, Scanning, and Image Analysis. After 2D gel

electrophoresis, gels were kept for 20 min in a fixation solution (2%orthophosphoric acid and 30% ethanol), washed with 2% orthophos-phoric acid, and stained with a solution containing 2% orthophosphoricacid, 15% ammonium sulfate, 18% ethanol, and 0.002% CBB G-250.Direct scanning and image analysis were performed using anImageScanner with ImageMaster 2D Platinum software (GE Health-care). For MALDI-TOF/TOFmass spectrometry analysis, the presenceor absence of spots in each cultivar was considered.In-Gel Digestion. Each 1D gel band or 2D gel spot was excised, cut

into smaller pieces, and destained overnight with a solution of 25 mMNH4HCO3 and 50% acetonitrile (ACN) at pH 8.0. For 1D gels, protein

disulfide bonds were reduced with 10 mM DTT in 25 mM NH4HCO3at 56 °C for 1 h. The supernatant was then removed, and the gelpieces were incubated with 55 mM iodoacetamide for 30 min at roomtemperature in the dark. The pieces were washed with 25 mMNH4HCO3 to remove excess reagent. Finally, the 1D and 2D gel pieceswere dehydrated with 100% ACN for 5 min and dried completely usinga Speed-Vac system. The excised spots and bands were then rehydratedin 15 μL of digestion buffer containing 10 ng/μL of trypsin (Promega,modified sequencing grade) in 25 mM NH4HCO3 in an ice-cold bathfor 10 min. The digestion was performed at 37 °C for 20 h. Followingthe incubation, the microcentrifuge tubes containing the samples werecentrifuged, and 4 μL of 1% formic acid was added. The liquid from eachgel piece was transferred into a clean microcentrifuge tube. The peptideswere extracted from the gel pieces twice by incubation with 50% ACNin 5% trifluoroacetic acid (TFA) for 30 min. The resulting solutionsfrom the two extractions were pooled together and concentrated to neardryness using a Speed-Vac system. Each sample was then solubilized in20 μL of deionized water. Prior to mass spectrometry, peptides were

Table 1. continued

SIDa proteinb NCBI accession no. MW/pIc BRS 257 BRS 258 EMBRAPA 48 BRS 267

41 glycinin G1/A1aBx gi|225651 56.284/5.78 − − − +glycinin G2 precursor gi|351725363 54.927/5.46

42 glycinin G1/A1aBx gi|225651 55.657/5.78 − − − +43 18.2 kDa class I heat shock protein gi|356501111 17.283/6.75 − − − +

44 glycinin G2 precursor gi|351725363 54.927/5.46 − − − +glycinin G1/A1aBx gi|225651 56.284/5.78

45 glycinin G2 precursor gi|351725363 54.927/5.46 − − − +46 glycinin G1/A1aBx gi|225651 56.284/5.78 − − − +47 glycinin gi|18641 64.35/5.21 + + + −

aNumber of spot identified. bProteins identified by MALDI-TOF-TOF. cMolecular weight (MW) and isoelectric point (pI). d“+” indicates thepresence of a spot and “−” the absence of a spot in the soybean cultivar. Protein spot data for this analysis were recorded (Supporting Information).

Figure 2. 1D SDS-PAGE analysis of protein extracts suitable for 2DSDS-PAGE from BRS 257, BRS 258, Embrapa 48, and BRS 267. Excisedbands are identified at the right of the gel (numbered 1−12). The sizesof the protein markers in kilodaltons (kDa) are shown at the left of thefigure.

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Table 2. Soybean Seed Proteins Identified by ESI-Q-TOF Mass Spectrometry

banda proteinbNCBI accession

no. MM/pIcBRS257

BRS258

EMBRAPA48

BRS267

1 α-subunit of β-conglycinin gi|9967357 63.184/4.92 +d + + +uncharacterized protein LOC100780139 gi|356515096 117.977/8.55 + + − +uncharacterized protein LOC100801440 gi|356561627 96.970/5.94 + + − +β-subunit of β-conglycinin gi|356575855 50.468/5.88 + + − +glycinin G1/A1aBx gi|225651 56.284/5.78 + − − +glycinin G2 precursor gi|351725363 54.927/5.46 + + − −P24 oleosin isoform B gi|351722277 23.378/8.89 + − − +sucrose-binding protein-like gi|356536206 58.353/6.08 + + - +P24 oleosin isoform A gi|356571311 23.575/8.89 + - - +seed maturation protein PM39 gi|5802248 46.665/5.69 + + − +lipoxygenase 2 gi|126404 97.370/6.27 − − − +lipoxygenase 3 gi|126406 97.970/5.94 − − − +glycinin G5/A3B4 subunit gi|126144646 58.120/5.78 − − − +lipoxygenase 1 gi|351727907 94.580/5.91 − − − +

2 lipoxygenase 3 gi|161318157 97.107/6.12 − + + +lipoxygenase 2 gi|295388395 97.472/6.15 − + + +chain a lipoxygenase-1 (soybean) i553l mutant gi|171849009 94.580/5.91 − + + +seed linoleate 9S-lipoxygenase-2 gi|356525977 97.082/6.70 − + − −α-subunit of β-conglycinin gi|9967357 63.184/4.92 − + − −α-subunit of β-conglycinin gi|9967357 63.184/4.92 − − − +lipoxygenase 5 gi|161318161 91.337/6.08 − + − −glycinin G1/A1aBx gi|225651 56.284/5.78 − + − −lipoxygenase 9 gi|351724717 96.637/6.54 − + + +β-subunit of β-conglycinin gi|356575855 50.468/5.88 − − + −lipoxygenase 10 gi|351725145 97.419/5.99 − + − −

3 α-subunit of β-conglycinin gi|9967361 65.161/5.23 + + + +β-subunit of β-conglycinin gi|356575855 50.468/5.88 + + + +heat shock protein 90-1 gi|351726363 80.700/4.94 + + + +methionine synthase gi|351724907 84.401/5.93 + − + +glycinin G1/A1aBx gi|225651 56.284/5.78 + + + −5-methyltetrahydropteroyltriglutamate-homocysteinemethyltransferase-like

gi|356508448 89.065/6.41 − − + +

heat shock protein 83-like gi|356530818 80.624/4.96 + − + −24 kDa oleosin isoform gi|18720 15.801/8.23 + − − −sucrose-binding protein-like gi|356536206 58.353/6.08 + + + +glycinin G4/A5A4B3 subunit gi|255224 64.097/5.38 + − − −glycinin G5/A3B4 subunit gi|126144646 58.120/5.78 − + + +uncharacterized protein LOC100527853 gi|351720923 16.490/9.78 − + − −lipoxygenase 3 gi|161318157 97.107/6.12 − − − +lipoxygenase 2 gi|126404 97.370/6.27 − − − +lipoxygenase 1 gi|351727907 94.538/5.95 − − − +

4 α-subunit of β-conglycinin gi|9967357 63.184/4.92 + + + +β-subunit of β-conglycinin gi|356575855 50.468/5.88 + + + +seed biotinylated protein 68 kDa isoform gi|240254706 67.963/6.18 − + + +heat shock 70 kDa protein-like gi|356500683 71.981/5.20 + − + +endoplasmic reticulum HSC70-cognate binding protein precursor gi|2642238 73.822/5.15 + + + +sucrose-binding protein-like gi|356536206 58.353/6.08 + + + +glycinin G2 precursor gi|351725363 54.927/5.46 + − + +glycinin G1/A1aBx gi|225651 56.284/5.78 + + + +glycinin G4/A5A4B3 subunit gi|255224 64.097/5.38 + + + −glycinin G3/A1ab1B subunit gi|121278 54.835/5.73 − − + −glycinin G5/A3B4 subunit gi|126144646 58.120/5.78 − − − +glycyl-tRNA synthetase 1 mitochondrial-like gi|356527475 81.691/7.03 − − + +

5 sucrose-binding protein-like gi|356536206 58.353/6.08 + + + +α-subunit of β-conglycinin gi|9967357 63.184/4.92 + + + +β-subunit of β-conglycinin gi|341603993 50.044/6.14 − + − −

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Table 2. continued

banda proteinbNCBI accession

no. MM/pIcBRS257

BRS258

EMBRAPA48

BRS267

protein disulfide isomerase gi|171854980 58.953/5.13 − − + +glycinin G1/A1aBx gi|225651 56.284/5.78 − − − +lipoxygenase 3 gi|161318157 97.107/6.12 − − − +

6 β-subunit of β-conglycinin gi|341603993 50.044/6.14 + + + +α-subunit of β-conglycinin gi|9967361 65.160/5.23 + + + +unknown gi|255636348 50.590/5.81 − + − −elongation factor 1-α gi|1352345 49.689/9.14 − + + −uncharacterized protein LOC100794313 gi|359807071 43.082/6.28 − − + +glycinin gi|18641 64.351/5.21 − − + −pea protein precursor gi|351727923 49.484/7.08 − − + −uncharacterized protein LOC100797606 gi|359807483 42.140/6.90 − − − +

7 glyceraldehyde-3-dehydrogenase C subunit gi|351723699 36.815/6.72 + + + +glycinin G5/A3B4 subunit gi|126144646 58.120/5.78 − + + +glycinin G1/A1aBx gi|225651 56.284/5.78 − + + +fructose-bisphosphate aldolase. cytoplasmic isozyme-like gi|356500825 38.469/7.12 − − + +α-subunit of β-conglycinin gi|9967357 63.184/4.92 − − − +glycinin G2 precursor gi|351725363 54.927/5.46 − − − +hydroxysteroid 11-β-dehydrogenase 1-like protein-like gi|356539128 40.972/5.89 − − − +β-subunit of β-conglycinin gi|356575855 50.468/5.88 − − − +P24 oleosin isoform A gi|356571311 23.575/8.89 − − − +seed maturation protein PM34 gi|351722943 32.032/6.60 − − − +cytosolic malate dehydrogenase gi|351727793 35.846/6.32 − − − +P24 oleosin isoform B gi|351722277 23.378/8.89 − − − +Kunitz trypsin inhibitor subtype B gi|125023 20.256/4.66 − − − +

8 glycinin G1/a1ab1b gi|356505023 56.299/5.89 + + + +glycinin G2 precursor gi|351725363 54.927/5.46 + + + +glycinin G4/A5A4B3 subunit gi|255224 64.351/5.21 + − + −seed maturation protein PM34 gi|351722943 32.032/6.60 − + − −

9 lectin (soybean agglutinin) gi|6729836 27.555/5.15 + + + +glycinin G2 precursor gi|351725363 54.927/5.46 − + − +uncharacterized protein LOC100806472 gi|363807732 36.000/7.72 − + + −glycinin G1/A1aBx gi|225651 56.284/5.78 − + + +uncharacterized protein LOC100809384 gi|359807588 32.097/6.38 − + − −P24 oleosin isoform A gi|1709459 23.487/8.01 − + − −seed maturation protein PM34 gi|351722943 32.032/6.60 − + − −P24 oleosin isoform B gi|351722277 23.378/8.89 − + − −dehydrin gi|119709430 25.370/6.10 − + − −seed maturation protein PM34 gi|351722943 32.032/6.60 − − + −

10 lectin (soybean agglutinin) gi|6729836 27.555/5.15 + − + +glycinin G1/A1aBx gi|225651 56.284/5.78 + − − −uncharacterized protein LOC100809384 gi|359807588 32.097/6.38 − + − +glycinin G2 precursor gi|351725363 54.927/5.46 − + − −seed maturation protein PM26 gi|351721132 26.201/4.83 − − + −ribosomal protein L2 gi|351723983 28.224/10.45 − − − +7S seed globulin precursor gi|1401240 47.006/8.68 − − − +

11 7S seed globulin precursor gi|1401240 47.006/8.68 − + − −dehydrin gi|37495451 23.774/5.97 − + + +7S seed globulin precursor gi|1401240 47.006/8.68 − − + −glycinin G2 precursor gi|351725363 54.927/5.46 − − − +glycinin G1/A1aBx gi|225651 56.284/5.78 − − − +

12 glycinin G1/A1aBx gi|225651 56.284/5.78 + − + +glycinin G4 subunit gi|255224 64.097/5.38 + − + +glycinin G5/A3B4 subunit gi|126144646 58.120/5.78 + − + +

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desalted using a mini-reverse phase column PerfectPure C-18 Tip(Eppendorf). The tips were conditioned in 100% acetonitrile andwashed three times with water. The peptide solution was aspirated andexpelled from the tips 10 times. After three washings of the tip withwater, the peptides were eluted with 50% ACN. The samples were driedusing a Speed-Vac system and resuspended in solution of 0.1% formicacid and 3% ACN.MALDI-TOF/TOF Mass Spectrometry Analysis. The mass

spectrometry (MS) and tandem mass spectrometry (MS/MS) experi-ments were performed using a 5800 Proteomics Analyzer (AppliedBiosystems, Foster City, CA). Approximately 0.6 μL of the extractedpeptide solution was mixed with an equal volume of α-cyano-4-hydroxycinnamic acid matrix solution (Aldrich, Milwaukee,WI, USA) at10 mg/mL in 50% ACN/0.1% TFA. Samples were placed on the targetplate and allowed to dry at room temperature. Both MS and MS/MSdata were acquired with a neodymium-doped yttrium aluminum garnet(Nd:YAG) laser with a 200 Hz repetition rate. Typically, 1600 shotswere accumulated for spectra in the MS mode and 2400 shots wereaccumulated for spectra in the MS/MS mode. MS and MS/MS spectrawere acquired in reflector mode. Up to eight of the most intense ionsignals with signal-to-noise ratios above 30 were selected as precursorsfor MS/MS acquisition. The external calibration in MS mode wasperformed using a mixture of the following four peptides: des-Arg1-bradykinin (m/z 904.468), angiotensin I (m/z 1296.685), Glu1-fibrinopeptide B (m/z 1570.677), and adrenocorticotropic hormone(18−39) (m/z 2465.199). MS/MS spectra were externally calibratedusing known fragment ion masses observed in the MS/MS spectrum ofangiotensin I.ESI-Q-TOF Mass Spectrometry Analysis. The peptides extracted

from the 1D gel slices were desalted and fractionated by liquidchromatography (LC) coupled online with an electrospray ionizationquadrupole time-of-flight mass spectrometer (ESI-Q-TOF). A 7.5 μLaliquot of the sample was loaded on a Waters nanoACQUITY UPLCSystem (Waters, Milford, MA, USA) with a Waters symmetry C18 trapcolumn coupled to a Q-Tof MicroMass spectrometer. Afterward,the peptides were fractioned by a nanoEase BEH 130 C18 100 mm ×100 μm column (Waters) at a flow rate of 0.5 μL/min and eluted witha linear acetonitrile gradient (from 10 to 40%) of 0.1% formic acid.The ESI voltage was set at 3.5 kV using a metal needle. The sourcetemperature was 80 °C, and the cone voltage was 40 V. Instrumentcontrol and data acquisition were conducted using a MassLynx datasystem (version 4.1, Waters). The experiments were performed byscanning a mass-to-charge ratio (m/z) of 300−2000 using a scan timeof 1 s during the entire chromatographic process. A maximum of threeions with charge states of 2, 3, or 4 were selected for MS/MS from asingleMS survey. Collision-induced dissociation (CID)MS/MS spectrawere obtained using argon as the collision gas at a pressure of 1 bar.The collision voltage varied between 22 and 60 eV depending on themass and charge of the precursor. The reference ion used was themonocharged ion m/z 588.8692 of phosphoric acid. Mass spectracorresponding to each signal from the total ion current (TIC) chro-matogram were averaged, allowing for an accurate molecular mass

determination. Exact mass MS/MS was automatically determined usingthe LockSpray source (Waters).

The acquired peak lists were analyzed by searching the NationalCenter for Biotechnology Information (NCBI) database using theMASCOT search engine (www.matrixscience.com) for identificationbased on the MS/MS ions. Under standard thresholds (mass errortolerance, ±50 ppm for MALDI-TOF-MS and 0.2 Da for ESI-Q-TOFmass spectrometry analysis; MS/MS tolerance, ±0.2 Da for MALDI-TOF-MS and ESI-Q-TOF mass spectrometry analysis; fixed mod-ifications, Cys-carbamidomethylation; variable modifications, methio-nine oxidation; one tolerated missed cleavage), proteins with aMOWSEscore of at least 54 were considered as significantly identified. Thefunctional classification of the identified proteins was performed bysearching the http://www.ncbi.nlm.nih.gov, http://www.uniprot.org,and www.genome.jp/kegg databases.

Statistical Analysis.The statistical analysis was performed using theprogram GraphPad Prism 5.0 for Windows. A one-way ANOVA wasapplied followed by Tukey’s test (p ≤ 0.05).

■ RESULTS AND DISCUSSION

Plant breeding is one of the strategies used to enhance thesensory characteristics and increase the nutritional properties offoods. However, the impact of this technique on both environ-mental and human health should be monitored. In this study,we investigated the proteome profiles of four new soybeanvarieties (BRS 257, BRS 258, Embrapa 48, and BRS 267) and therelationships between the protein expression profiles and thenutritional properties of these cultivars.Alves et al.31 showed that BRS 258 has higher protein content

than BRS 257, Embrapa 48, and BRS 267. Such a finding isin accordance with our results. The thiourea/urea method ofprotein extraction provided the following amounts of protein(μg/g dry matter ± SD): BRS 257 had a protein content of144000 ± 10324, BRS 258 had a protein content of 168700 ±12587, Embrapa 48 had a protein content of 153400 ± 46528,and BRS 267 had a protein content of 138450 ± 46315.

Comparative Analysis of Soybean Seed Proteomes by2D SDS-PAGE. Proteins from four soybean cultivars wereextracted using a urea/thiourea protocol. These proteins werethen separated by 2D SDS-PAGE (Figure 1), followed by proteinidentification using mass spectrometry (Table 1).The image data from the triplicate 2D gel experiment were

analyzed for each cultivar. The numbers of proteins spots (±SD)detected for BRS 257, BRS 258, Embrapa 48, and BRS 267 were102± 22, 124± 5, 113± 32, and 99± 23, respectively. After thisanalysis, the differentially expressed protein spots were identifiedusing MALDI-TOF-TOF mass spectrometry (Figure 1). A totalof 47 protein spots were identified (Table 1).

Table 2. continued

banda proteinbNCBI accession

no. MM/pIcBRS257

BRS258

EMBRAPA48

BRS267

glycinin G2 precursor gi|351725363 54.927/5.46 + − + +Kunitz trypsin inhibitor subtype B gi|125023 20.256/4.66 − − − +Kunitz trypsin inhibitor subtype A precursor gi|351724949 24.275/4.99 + − − −Kunitz-type trypsin inhibitor KTI1 gi|125722 22.817/4.97 − − + +Kunitz-type trypsin inhibitor KTI2 gi|125723 23.071/6.14 − − − +7S seed globulin precursor gi|1401240 47.006/8.68 + − + +glycinin G3 precursor gi|99909 54.953/5.28 + − + −soybean trypsin inhibitor gi|3318877 20.310/4.61 − − + −2S albumin precursor gi|351727517 19.018/5.20 − − − +

aNumber of band identified. bProteins identified by ESI-Q-TOF mass spectrometry. cMolecular weight (MW) and isoelectric point (pI). d“+”indicates the presence of a protein and “−” the absence of a protein in the soybean cultivar band. Protein band data for this analysis were recorded(Supporting Information).

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It has been reported that various soybean seeds may havedifferent protein profiles due to genetic variability.32,33 Amongthe analyzed spots, we found storage, allergenic, maturation, andagglutinin proteins as well as trypsin inhibitors. The comparativestudy of the 2D gels showed that the cultivar BRS 267 had thehighest diversity for the identified spots (approximately 53%);this cultivar also had a greater number of spots corresponding toallergenic proteins and antinutrients, such as lectin and Kunitzinhibitor.Eighteen glycinin and β-conglycinin spots were detected in

the four soybean seeds. As the data show, these protein spotsare distributed over a wide pI range in the sameMW region. Thispattern suggests the presence of isoforms or post-translational

modifications of glycinin that occur during seed development,such as dissociation and reassembly.12 Conversely, some authorsattribute the occurrence of different spots for the same protein tothe natural variation among different cultivars.34

Comparative Analysis of the Soybean Seed Proteomesby 1D SDS-PAGE. A 1D SDS-PAGE (Figure 2) analysis wasperformed using a protein extract suitable for 2D separation.The gels were stained with CBB, and proteins were identified byESI-Q-TOFmass spectrometry. After image analysis, each intenseCoomassie staining lane of the gel was divided into approximately2.5 mm slices; the slices were then subjected to in-gel proteolysiswith trypsin. A total of 117 proteins were identified from 12bands in the 1D SDS-PAGE gel of soybean seed proteins.

Figure 3. Functional classification of the soybean proteins isolated from BRS 257, BRS 258, Embrapa 48, and BRS 267 identified by 1D gelelectrophoresis followed by ESI-Q-TOF mass spectrometry.

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Distinct differences between the cultivars were found. Table 2shows a comparative scheme indicating the presence or absence ofthe proteins identified in each cultivar.Band 1 is the result of proteolysis. Lipoxygenase was detected

in this band only in the BRS 267 cultivar. BRS 257 contains thelipid storage P24 oleosin isoforms A and B in this band, whereasonly the α-subunit of β-conglycinin was identified in Embrapa 48.These data confirm that BRS 257 is a cultivar without

lipoxygenase, which is in accordance with information fromEmbrapa-soja.24 Scientists have recently verified that a soybeanseed lacking lipoxygenase had higher contents of calcium,magnesium, glucosides, and the aglycone forms of isoflavonesand phytate compared to cultivars containing lipoxygenase.35

Other proteins were found only in the BRS 267 cultivar,including the following: 2S albumin precursor (band 12 andspot 24), cytosolic malate dehydrogenase (band 7), and hydroxy-steroid 11-β-dehydrogenase 1-like protein-like (band 7). A24 kDa oleosin isoform was found in BRS 257. The antinutrientslectin and Kunitz trypsin inhibitor were present in all soybeanseeds.The analyses of band 11 of BRS 257 and band 12 of BRS 258

were not reliable in any replicate. It is possible that some form ofcontamination occurred.Functional Classification of the Identified Soybean

Proteins. Classification according to the biological function ofthe proteins identified for each cultivar is illustrated in Figure 3.The proteins identified by ESI-Q-TOF-MS from the 1D SDS-PAGE analysis were grouped into nine categories according totheir possible biological functions, based on molecular function,using the Universal Protein Knowledgebase (UniprotKB) andNCBI databases.Comparison of the four soybean cultivars revealed similar

protein function profiles. The distribution of proteins from thecultivars BRS 257 and BRS 258 had greater differentiationbetween categories than was observed in BRS 267 and Embrapa48. Cultivars BRS 267 and Embrapa 48 had similar distributionsof proteins between the categories; among the four cultivars, BRS267 and Embrapa 48 had the greatest amounts of total sugar.These cultivars are described in the literature as having similarcharacteristics and as containing more sucrose.36

The highest percentage of storage proteins was found inBRS 257 (40%), followed by Embrapa 48 (31%), BRS 258(25%), and BRS 267 (25%). The second largest protein groupwas unclassified proteins, which ranged from 16 to 28% of thetotal protein content.The third major group of proteins observed was related to lipid

metabolism (11−25%). Although BRS 257 does not possesslipoxygenases, lipid metabolism proteins represented 12% of thetotal protein content. Other observed protein categories wereprotein biosynthesis (6−14%) and stress response proteins (6−8%). Protein disulfide isomerase, a redox homeostasis enzyme,was observed only in the cultivars Embrapa 48 and BRS 267.Amino acid metabolism proteins were not observed in BRS 258.According to the literature, BRS 258 is a soybean cultivar with

a milder flavor and high protein content,31 which is in agreementwith our finding of a higher extraction yield of protein, althoughthe values found were similar to those of other cultivars. Anotherdistinctive feature of this cultivar is the significant percentage ofproteins related to lipid metabolism.An investigation of the proteomics of seed filling in soybean

was reported by Hajduch et al.37 The authors demonstratedthat the largest functional class was unclassified proteins (28%),followed by metabolic proteins (22%; mainly lipid and sterol

metabolism) and protein destination and storage proteins(10%). We noted that the maximal percentages in our studywere similar for unclassified proteins and those associated withlipid metabolism. It must be noted that the protein profile ischanged during seed filling; there is a decrease in lipid and sterolmetabolism proteins and an increase in storage proteins.Asakura et al.38 characterized the gene expression patterns

of developing soybeans. This group demonstrated that geneexpression changes according to the stages of seed development.These changes could contribute to the differences in proteincontent. Kottapalli et al.39 identified variations between fourpeanut cultivars. Proteins such as storage proteins, antinutritiveproteins, and allergens were present in some peanut cultivars andabsent in others. According to their study, the expression of someidentified protein spots was cultivar specific.According to Embrapa-soja,24 BRS 257, BRS 258, Embrapa 48,

and BRS 267, are promising options for human nutrition dueto their elevated levels of carbohydrates and the high quality ofthe proteins. The diversity of proteins among the four soybeanseeds in this study suggests that there are significant differencesin the functional and nutritional properties of these seeds.Nevertheless, little is known about the protein composition ofnew conventional soybean varieties. The results obtained in thisstudy indicate which cultivars are most suitable for humanconsumption and can direct the development of new cultivarswith consideration of the protein composition.

■ ASSOCIATED CONTENT*S Supporting InformationSpreadsheets containing molecular weight, isoelectric point,matched peptides, percent sequence coverage, and MOWSE scoresof protein for identified proteins in the spots and bands from 2DSDS-PAGE and 1D SDS-PAGE, respectively, of soybean cultivarsBRS 257, BRS 258, Embrapa 48, and BRS 267. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*(E.F.) Mailing address: Departamento de Nutricao Basica eExperimental, Instituto de Nutricao Josue de Castro, Centro deCiencias da Saude, Universidade Federal do Rio de Janeiro,UFRJ, Caixa Postal 68041, Cidade Universitaria, Ilha do Fundao,Rio de Janeiro, CEP 21941-590, Brazil. E-mail: fialho@nutricao.ufrj.br. Fax: + 55 21 2280 8343. Phone: + 55 21 2562 6599.FundingThis work was supported by Coordenacao de Aperfeicoamentode Pessoal de Nıvel Superior (CAPES) and Fundacao deAmparo a Pesquisa Carlos Chagas Filho do Estado do Rio deJaneiro (FAPERJ).NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Ana Lucia O. Carvalho and Augusto Vieira Magalhaes(IBqM-UFRJ) for their helpful assistance. We also thankEmbrapa-soja for the kind donation of soybean seeds.

■ ABBREVIATIONS USEDACN, acetonitrile; CBB, Coomassie brilliant blue; DTT,dithiothreitol; EMBRAPA, Empresa Brasileira de PesquisaAgropecuaria; ESI-Q-TOF, electrospray ionization-quadrupole-time of flight; FDA, U.S. Food and Drug Administration; HDL-c,

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high-density lipoprotein cholesterol; IPG, immobilized pHgradient; kDa, kilodalton; KTI, Kunitz trypsin inhibitor; LDL-c, low-density lipoprotein cholesterol; MALDI-TOF, matrix-assisted laser desorption ionization−time of flight; MOWSE,molecular weight search; MS, mass spectrometry; NCBI,National Center for Biotechnology Information; PAGE,polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate;TFA, trifluoroacetic acid

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