Four Conventional Soybean [Glycine max (L.) Merrill] Seeds Exhibit

Article pubs.acs.org/JAFC

Four Conventional Soybean [Glycine max (L.) Merrill] Seeds Exhibit Different Protein Profiles As Revealed by Proteomic Analysis Luciana S. Gomes,† Raquel Senna,‡ Vanessa Sandim,# Mário A. C. Silva-Neto,‡,⊗ Jonas E. A. Perales,§,⊗ Russolina B. Zingali,#,⊗ Márcia R. Soares,⊥,⊗ and Eliane Fialho*,† †

Departamento de Nutriçaõ Básica e Experimental, Instituto de Nutriçaõ Josué de Castro, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, Prédio do CCS, Bloco J-2, Laboratório 13, 393 Rio de Janeiro 21941-590, Brazil ‡ Laboratório de Sinalizaçaõ Celular, Instituto de Bioquı ́mica Médica, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, Prédio do CCS, Bloco D, Subsolo, Sala 5, 373 Rio de Janeiro 21941-590, Brazil # Unidade de Espectrometria de Massas e Proteômica, Instituto de Bioquı ́mica Médica, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, Prédio do CCS, Bloco D, 373 Rio de Janeiro 21941-590, Brazil § Instituto Oswaldo Cruz, Fundaçaõ 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 proteômica 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 that are attributed to proteins and their derivative peptides. The present study aimed to analyze the protein profiles of four new conventional 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 mass spectrometry analyses, respectively. These two different experimental approaches allowed the identification of 117 proteins from 1D gels and 46 differentially expressed protein spots in 2D gels. BRS 267 showed the greatest diversity of identified spots in the 2D gel analyses. In the 1D gels, the major groups were storage (25−40%) and lipid metabolism (11−25%) proteins. The differences in protein composition between cultivars could indicate functional and nutritional differences and could direct the development of new cultivars. KEYWORDS: Glycine max, seed proteome, protein composition, protein profile, proteomic analysis

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INTRODUCTION The soybean (Glycine max) is a legume of high nutritional value with outstanding nutritional characteristics. It is composed of mono- and polyunsaturated fatty acids, is low in fat and carbohydrates, is a source of vitamins and minerals, and provides proteins with high biological value.1 The soybean has a protein digestibility-corrected amino acid score of 1.00.2 Soybeans have several functional properties due to their composition and provide beneficial health effects and nourishment.3 Such properties are conferred by essential fatty acids;4 polyphenols such as isoflavones, anthocyanins, and procyanidins;5,6 proteins; and low molecular weight peptides.7 Soybeans contain from 37.0 to 44.5% protein,8,1 of which approximately 70−83% are storage proteins. Storage proteins are divided into two main classes known as glycinins and β-conglycinins, which are also classified as globulins 11S and 7S, respectively.9,10 Glycinins are hexameric proteins composed of different subunits that are linked together by disulfide bonds. The five major 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 the total soybean protein content) have a trimeric structure made up of the subunits α, α′, and β, which have molecular weights of © 2013 American Chemical Society

approximately 76, 70, and 50 kDa, respectively. The three subunits are linked by noncovalent bonds.12,13 Studies have shown that proteins and peptides derived from soybeans have beneficial health effects far beyond their basic functions. The trypsin and chymotrypsin inhibitor Bowman− Birk (BBI) has been associated with the prevention and treatment of diseases such as colorectal cancer without toxicity to healthy cells.14 Furthermore, it has been shown that the Kunitz trypsin inhibitor (KTI) can protect mouse lung cells against damage caused by inflammatory processes.15 Evidence suggests that lectins, also known as hemagglutinins or agglutinins, inhibit the proliferation of liver and breast cancer cells and inhibit the activity of the reverse transcriptase of the human immunodeficiency virus. These functions have also been described for the Kunitz-type protease inhibitor.16 β-Conglycins and glycinins are bioactive compounds with protective effects on cardiovascular health.17 Reductions in plasma total cholesterol, triglycerides, and LDL-c and increases in the levels of HDL-c have been attributed to the oral Received: Revised: Accepted: Published: 1283

June 25, 2013 December 12, 2013 December 31, 2013 December 31, 2013 dx.doi.org/10.1021/jf404351g | J. Agric. Food Chem. 2014, 62, 1283−1293

Journal of Agricultural and Food Chemistry

Article

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 a thiourea/urea protocol from Glycine max cultivars (A) BRS 257, (B) BRS 258, (C) Embrapa 48, and (D) BRS 267. Identical numbers refer to the same spots, and distinct numbers correspond to differential spots present in each cultivar. The sizes of the protein markers in kilodaltons (kDa) are shown at the left of the images.

administration of these proteins in hypercholesterolemic rats.18 Zhang et al.19 demonstrated that hydrolyzed soybean proteins reduced the plasma level of very low-density lipoproteins cholesterol and triglycerides in mice. Additionally, the 11S globulin has hypotensive effects that are attributed to its ability to inhibit the angiotensin I-converting enzyme.20 Peptides derived from 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 prevention and treatment of noncommunicable diseases, soybean proteins are the only proteins to offer benefits for cardiovascular disease that are certified by the American Dietetic Association (ADA).22 According to the ADA, soybean products that provide 6.25 g of soy protein per serving are allowed to state on the label that “25 g of soy protein a day as part of a diet low in saturated fat and cholesterol may reduce the risk of coronary heart disease”. The consumption of 3−4 tablespoons of soybeans (56−67 g of grains) each day meets the FDA requirements regarding the ingestion of 25 g of protein. Despite the nutritional and functional properties mentioned above, the consumption of soybeans in Brazil is limited due to the characteristic flavor caused by lipoxygenases, which oxidize lipids and cause a beany flavor. In addition, gastrointestinal distress may be caused by the presence of oligosaccharides, raffinose, stachyose, phytic acid, and protease inhibitors.23 Embrapa-Soja, Empresa Brasileira de Pesquisa Agropecuária, developed conventional cultivars in 2008 to stimulate consumption.24 These cultivars were obtained by crossing different varieties to mitigate flavor and improve nutritional value. This process resulted in lipoxygenase-free grains with higher levels of carbohydrates and improved protein content.

The use of proteomics in nutrition is diverse and offers different possibilities for nutritional interventions of great value. The proteome study represents an important tool when associated with nutrigenomics as it provides insights into promises of future dietary approaches.25 One proteomic strategy is the separation of proteins by polyacrylamide gel electrophoresis (in one or two dimensions), followed by the analysis and identification of proteins by mass spectrometry.26 Another approach is a combination of liquid chromatography and mass spectrometry, which has become a powerful approach for the identification of proteins and peptides occurring in complex mixtures.27 The production of conventional plants with improved sensory qualities and nutritional characteristics is an important strategy to increase soybean consumption. However, the impact of these changes and the soybean improvements should be investigated by methods such as comparative analysis of protein expression profiles. This analysis will allow for the determination of the legume proteins that interfere with the nutritional and functional properties. We analyzed the protein profiles of four conventional soybean seeds (BRS 257, BRS 258, Embrapa 48, and BRS 267) using proteomic tools.

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MATERIALS AND METHODS

Plant Materials. G. max seeds were kindly donated by the Empresa Brasileira de Pesquisa Agropecuária (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 cultivars were ground in a mechanical mill (Retsch ZM 1) using a metallic sieve with a pore size of 0.5 mm until a fine powder was obtained. The samples were stored at −20 °C until analysis. Extraction of Proteins from Seeds. The samples were delipidated with hexane until a colorless supernatant was obtained and then 1284

dx.doi.org/10.1021/jf404351g | J. Agric. Food Chem. 2014, 62, 1283−1293


Article

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 2

35 kDa seed maturation protein glycinin G1/A1aBx

gi|351726750 gi|225651

35.320/5.96 56.284/5.78

+d +

+ −

+ −

+ −

3

glycinin G1/A1aBx glycinin G2/A2B1 precursor glycinin G4/A5A4B3 subunit

gi|225651 gi|351725363 gi|126144648

56.284/5.78 54.927/5.46 64.196/5.17

+

−

−

−

4 5 6 7 8 9 10 11 12

lectin (soybean agglutinin) allergen Gly m Bd 28K allergen Gly m Bd 28K seed biotinylated protein 68 kDa isoform seed biotinylated protein 68 kDa isoform embryonic protein DC-8-like α-subunit of β-conglycinin glycinin G4/A5A4B3 subunit sucrose binding protein homologue S-64

gi|6729836 gi|12697782 gi|12697782 gi|240254706 gi|240254706 gi|356533407 gi|9967361 gi|121279 gi|6179947

27.555/5.15 52.780/5.73 52.780/5.73 67.963/6.18 67.963/6.18 48.766/6.12 65.160/5.23 64.005/5.29 56.142/6.32

+ + + + + + − − −

+ + − − − − + + +

− − − + − + − − −

− − − − − − − − −

13

glycinin G1/A1aBx glycinin G2/A2B1 precursor

gi|225651 gi|351725363

56.284/5.78 54.927/5.46

−

+

−

−

14

seed maturation protein PM31

gi|351722245

17.907/6.10

−

+

−

−

15

seed maturation protein PM25 seed maturation protein PM26

gi|6648966 gi|351721132

25.827/4.99 26.201/4.83

−

+

−

−

16 17 18 19 20

alcohol dehydrogenase glycinin G2/A2B1 precursor lectin (soybean agglutinin) lectin (soybean agglutinin) glycinin G1/A1aBx

gi|4039115 gi|351725363 gi|6729836 gi|6729836 gi|225651

37.042/6.13 54.927/5.46 27.555/5.15 27.555/5.15 56.284/5.78

− − − − −

− − − − −

+ + + + +

− − − − −

21

glycinin G1/A1aBx glycinin G2/A2B1 precursor

gi|225651 gi|351725363

56.284/5.78 54.927/5.46

−

−

+

−

22

Kunitz trypsin inhibitor subtype B Kunitz-type trypsin inhibitor KTI1

gi|125023 gi|125722

20.256/4.66 22.817/4.97

−

−

−

+

23 24 25 26

Kunitz trypsin inhibitor subtype B 2S albumin precursor lectin (soybean agglutinin) lectin (soybean agglutinin)

gi|125023 gi|351727517 gi|6729836 gi|6729836

20.256/4.66 19.018/5.20 27.555/5.15 27.555/5.15

− − − −

− − − −

− − − −

+ + + +

27

glycinin G1/A1aBx lectin (soybean agglutinin)

gi|225651 gi|6729836

56.284/5.78 27.555/5.15

−

−

−

+

28 29 30 31 32 33

lectin (soybean agglutinin) allergen Gly m Bd 28K allergen Gly m Bd 28K allergen Ara h 1, clone P41B-like allergen Gly m Bd 28K uncharacterized protein LOC100305847

gi|6729836 gi|12697782 gi|12697782 gi|356538162 gi|12697782 gi|351724719

27.555/5.15 52.780/5.73 52.780/5.73 82.003/5.32 52.780/5,73 27.847/5.72

− − − − − −

− − − − − −

− − − − − −

+ + + + + +

34

glycinin G1/A1aBx glycinin G2 precursor

gi|225651 gi|351725363

56.284/5.78 54.927/5.46

−

−

−

+

35 36 37 38 39 40

isoflavone reductase homologue 2 glucose and ribitol dehydrogenase homologue 1 7S seed globulin precursor glycinin G2 precursor glyceraldehyde-3-dehydrogenase C subunit elongation factor 1-α-like isoform 1

gi|351726399 gi|356505868 gi|1401240 gi|351725363 gi|351723699 gi|356558807

33.919/5.60 32.147/5.34 47.006/8.68 54.927/5.46 36.815/6.72 49.717/9.14

− − − − − −

− − − − − +

− − − − − −

+ + + + + +

1285



Article

Table 1. continued SIDa

proteinb

NCBI accession no.

MW/pIc

BRS 257

BRS 258

EMBRAPA 48

BRS 267

41

glycinin G1/A1aBx glycinin G2 precursor

gi|225651 gi|351725363

56.284/5.78 54.927/5.46

−

−

−

+

42 43

glycinin G1/A1aBx 18.2 kDa class I heat shock protein

gi|225651 gi|356501111

55.657/5.78 17.283/6.75

− −

− −

− −

+ +

44

glycinin G2 precursor glycinin G1/A1aBx

gi|351725363 gi|225651

54.927/5.46 56.284/5.78

−

−

−

+

45 46 47

glycinin G2 precursor glycinin G1/A1aBx glycinin

gi|351725363 gi|225651 gi|18641

54.927/5.46 56.284/5.78 64.35/5.21

− − +

− − +

− − +

+ + −

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

lyophilized. A 50 mg lyophilized sample was added to 495 μL of buffer containing 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% bromophenol blue. A 5 μL aliquot of a protease inhibitor cocktail (made up of 104 mM benzenesulfonyl fluoride hydrochloride, 80 μM aprotinin, 4 mM bestatin hydrochloride, 1.4 mM E-64, 2 mM leupeptin, and 1.5 mM pepstatin A) was added. After sonication for 20 min, the samples were centrifuged at 13000g for 15 min at room temperature, and supernatants were stored at −80 °C according to the methodology described by Herman et al.28 with modifications. The protein content was determined using the protocol described by Peterson.29 One-Dimensional Gel Electrophoresis (1D SDS-PAGE). For 1D analysis, 90 μg of protein from each cultivar that had been previously extracted 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-dimensional separation was performed following the method described by Laemmli.30 The experiments were performed with biological triplicates. After the image analysis, each lane was arbitrarily divided into approximately 2.5 mm slices and subjected to in-gel proteolysis with trypsin. 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 covered a 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 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, and 65 mM DTT for 15 min. The strips were alkylated with iodoacetamide (25 mg/mL) and subsequently placed onto 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 gels until the end of the run. The reference gel was obtained after the analysis of biological triplicates. The experiments were performed in technical triplicates. 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% orthophosphoric acid, and stained with a solution containing 2% orthophosphoric acid, 15% ammonium sulfate, 18% ethanol, and 0.002% CBB G-250. Direct scanning and image analysis were performed using an ImageScanner with ImageMaster 2D Platinum software (GE Healthcare). For MALDI-TOF/TOF mass spectrometry analysis, the presence or 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 mM NH4HCO3 and 50% acetonitrile (ACN) at pH 8.0. For 1D gels, protein

Figure 2. 1D SDS-PAGE analysis of protein extracts suitable for 2D SDS-PAGE from BRS 257, BRS 258, Embrapa 48, and BRS 267. Excised bands are identified at the right of the gel (numbered 1−12). The sizes of the protein markers in kilodaltons (kDa) are shown at the left of the figure. disulfide bonds were reduced with 10 mM DTT in 25 mM NH4HCO3 at 56 °C for 1 h. The supernatant was then removed, and the gel pieces were incubated with 55 mM iodoacetamide for 30 min at room temperature in the dark. The pieces were washed with 25 mM NH4HCO3 to remove excess reagent. Finally, the 1D and 2D gel pieces were dehydrated with 100% ACN for 5 min and dried completely using a Speed-Vac system. The excised spots and bands were then rehydrated in 15 μL of digestion buffer containing 10 ng/μL of trypsin (Promega, modified sequencing grade) in 25 mM NH4HCO3 in an ice-cold bath for 10 min. The digestion was performed at 37 °C for 20 h. Following the incubation, the microcentrifuge tubes containing the samples were centrifuged, and 4 μL of 1% formic acid was added. The liquid from each gel piece was transferred into a clean microcentrifuge tube. The peptides were extracted from the gel pieces twice by incubation with 50% ACN in 5% trifluoroacetic acid (TFA) for 30 min. The resulting solutions from the two extractions were pooled together and concentrated to near dryness using a Speed-Vac system. Each sample was then solubilized in 20 μL of deionized water. Prior to mass spectrometry, peptides were 1286



Article

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

proteinb

NCBI accession no.

MM/pIc

BRS 257

BRS 258

EMBRAPA 48

BRS 267

1

α-subunit of β-conglycinin uncharacterized protein LOC100780139 uncharacterized protein LOC100801440 β-subunit of β-conglycinin glycinin G1/A1aBx glycinin G2 precursor P24 oleosin isoform B sucrose-binding protein-like P24 oleosin isoform A seed maturation protein PM39 lipoxygenase 2 lipoxygenase 3 glycinin G5/A3B4 subunit lipoxygenase 1

gi|9967357 gi|356515096 gi|356561627 gi|356575855 gi|225651 gi|351725363 gi|351722277 gi|356536206 gi|356571311 gi|5802248 gi|126404 gi|126406 gi|126144646 gi|351727907

63.184/4.92 117.977/8.55 96.970/5.94 50.468/5.88 56.284/5.78 54.927/5.46 23.378/8.89 58.353/6.08 23.575/8.89 46.665/5.69 97.370/6.27 97.970/5.94 58.120/5.78 94.580/5.91

+d + + + + + + + + + − − − −

+ + + + − + − + + − − − −

+ − − − − − − − − − − −

+ + + + + − + + + + + + + +

2

lipoxygenase 3 lipoxygenase 2 chain a lipoxygenase-1 (soybean) i553l mutant seed linoleate 9S-lipoxygenase-2 α-subunit of β-conglycinin α-subunit of β-conglycinin lipoxygenase 5 glycinin G1/A1aBx lipoxygenase 9 β-subunit of β-conglycinin lipoxygenase 10

gi|161318157 gi|295388395 gi|171849009 gi|356525977 gi|9967357 gi|9967357 gi|161318161 gi|225651 gi|351724717 gi|356575855 gi|351725145

97.107/6.12 97.472/6.15 94.580/5.91 97.082/6.70 63.184/4.92 63.184/4.92 91.337/6.08 56.284/5.78 96.637/6.54 50.468/5.88 97.419/5.99

− − − − − − − − − − −

+ + + + + − + + + − +

+ + + − − − − − + + −

+ + + − − + − − + − −

3

α-subunit of β-conglycinin β-subunit of β-conglycinin heat shock protein 90-1 methionine synthase glycinin G1/A1aBx 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase-like heat shock protein 83-like 24 kDa oleosin isoform sucrose-binding protein-like glycinin G4/A5A4B3 subunit glycinin G5/A3B4 subunit uncharacterized protein LOC100527853 lipoxygenase 3 lipoxygenase 2 lipoxygenase 1

gi|9967361 gi|356575855 gi|351726363 gi|351724907 gi|225651 gi|356508448

65.161/5.23 50.468/5.88 80.700/4.94 84.401/5.93 56.284/5.78 89.065/6.41

+ + + + + −

+ + + − + −

+ + + + + +

+ + + + − +


80.624/4.96 15.801/8.23 58.353/6.08 64.097/5.38 58.120/5.78 16.490/9.78 97.107/6.12 97.370/6.27 94.538/5.95

+ + + + − − − − −

− − + − + + − − −

+ − + − + − − − −

− − + − + − + + +

4

α-subunit of β-conglycinin β-subunit of β-conglycinin seed biotinylated protein 68 kDa isoform heat shock 70 kDa protein-like endoplasmic reticulum HSC70-cognate binding protein precursor sucrose-binding protein-like glycinin G2 precursor glycinin G1/A1aBx glycinin G4/A5A4B3 subunit glycinin G3/A1ab1B subunit glycinin G5/A3B4 subunit glycyl-tRNA synthetase 1 mitochondrial-like

gi|9967357 gi|356575855 gi|240254706 gi|356500683 gi|2642238 gi|356536206 gi|351725363 gi|225651 gi|255224 gi|121278 gi|126144646 gi|356527475

63.184/4.92 50.468/5.88 67.963/6.18 71.981/5.20 73.822/5.15 58.353/6.08 54.927/5.46 56.284/5.78 64.097/5.38 54.835/5.73 58.120/5.78 81.691/7.03

+ + − + + + + + + − − −

+ + + − + + − + + − − −

+ + + + + + + + + + − +

+ + + + + + + + − − + +

5

sucrose-binding protein-like α-subunit of β-conglycinin β-subunit of β-conglycinin

gi|356536206 gi|9967357 gi|341603993

58.353/6.08 63.184/4.92 50.044/6.14

+ + −

+ + +

+ + −

+ + −

1287



Article

Table 2. continued NCBI accession no.

MM/pIc

BRS 257

BRS 258

EMBRAPA 48

BRS 267

protein disulfide isomerase glycinin G1/A1aBx lipoxygenase 3

gi|171854980 gi|225651 gi|161318157

58.953/5.13 56.284/5.78 97.107/6.12

− − −

− − −

+ − −

+ + +

6

β-subunit of β-conglycinin α-subunit of β-conglycinin unknown elongation factor 1-α uncharacterized protein LOC100794313 glycinin pea protein precursor uncharacterized protein LOC100797606

gi|341603993 gi|9967361 gi|255636348 gi|1352345 gi|359807071 gi|18641 gi|351727923 gi|359807483

50.044/6.14 65.160/5.23 50.590/5.81 49.689/9.14 43.082/6.28 64.351/5.21 49.484/7.08 42.140/6.90

+ + − − − − − −

+ + + + − − − −

+ + − + + + + −

+ + − − + − − +

7

glyceraldehyde-3-dehydrogenase C subunit glycinin G5/A3B4 subunit glycinin G1/A1aBx fructose-bisphosphate aldolase. cytoplasmic isozyme-like α-subunit of β-conglycinin glycinin G2 precursor hydroxysteroid 11-β-dehydrogenase 1-like protein-like β-subunit of β-conglycinin P24 oleosin isoform A seed maturation protein PM34 cytosolic malate dehydrogenase P24 oleosin isoform B Kunitz trypsin inhibitor subtype B

gi|351723699 gi|126144646 gi|225651 gi|356500825 gi|9967357 gi|351725363 gi|356539128 gi|356575855 gi|356571311 gi|351722943 gi|351727793 gi|351722277 gi|125023

36.815/6.72 58.120/5.78 56.284/5.78 38.469/7.12 63.184/4.92 54.927/5.46 40.972/5.89 50.468/5.88 23.575/8.89 32.032/6.60 35.846/6.32 23.378/8.89 20.256/4.66

+ − − − − − − − − − − − −

+ + + − − − − − − − − − −

+ + + + − − − − − − − − −

+ + + + + + + + + + + + +

8

glycinin G1/a1ab1b glycinin G2 precursor glycinin G4/A5A4B3 subunit seed maturation protein PM34

gi|356505023 gi|351725363 gi|255224 gi|351722943

56.299/5.89 54.927/5.46 64.351/5.21 32.032/6.60

+ + + −

+ + − +

+ + + −

+ + − −

9

lectin (soybean agglutinin) glycinin G2 precursor uncharacterized protein LOC100806472 glycinin G1/A1aBx uncharacterized protein LOC100809384 P24 oleosin isoform A seed maturation protein PM34 P24 oleosin isoform B dehydrin seed maturation protein PM34

gi|6729836 gi|351725363 gi|363807732 gi|225651 gi|359807588 gi|1709459 gi|351722943 gi|351722277 gi|119709430 gi|351722943

27.555/5.15 54.927/5.46 36.000/7.72 56.284/5.78 32.097/6.38 23.487/8.01 32.032/6.60 23.378/8.89 25.370/6.10 32.032/6.60

+ − − − − − − − − −

+ + + + + + + + + −

+ − + + − − − − − +

+ + − + − − − − − −

10

lectin (soybean agglutinin) glycinin G1/A1aBx uncharacterized protein LOC100809384 glycinin G2 precursor seed maturation protein PM26 ribosomal protein L2 7S seed globulin precursor

gi|6729836 gi|225651 gi|359807588 gi|351725363 gi|351721132 gi|351723983 gi|1401240

27.555/5.15 56.284/5.78 32.097/6.38 54.927/5.46 26.201/4.83 28.224/10.45 47.006/8.68

+ + − − − − −

− − + + − − −

+ − − − + − −

+ − + − − + +

11

7S seed globulin precursor dehydrin 7S seed globulin precursor glycinin G2 precursor glycinin G1/A1aBx

gi|1401240 gi|37495451 gi|1401240 gi|351725363 gi|225651

47.006/8.68 23.774/5.97 47.006/8.68 54.927/5.46 56.284/5.78

− − − − −

+ + − − −

− + + − −

− + − + +

12

glycinin G1/A1aBx glycinin G4 subunit glycinin G5/A3B4 subunit

gi|225651 gi|255224 gi|126144646

56.284/5.78 64.097/5.38 58.120/5.78

+ + +

− − −

+ + +

+ + +

banda

proteinb

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Table 2. continued banda

proteinb

NCBI accession no.

MM/pIc

BRS 257

BRS 258

EMBRAPA 48

BRS 267


54.927/5.46 20.256/4.66 24.275/4.99 22.817/4.97 23.071/6.14 47.006/8.68 54.953/5.28 20.310/4.61 19.018/5.20

+ − + − − + + − −

− − − − − − − − −

+ − − + − + + + −

+ + − + + + − − +

glycinin G2 precursor Kunitz trypsin inhibitor subtype B Kunitz trypsin inhibitor subtype A precursor Kunitz-type trypsin inhibitor KTI1 Kunitz-type trypsin inhibitor KTI2 7S seed globulin precursor glycinin G3 precursor soybean trypsin inhibitor 2S albumin precursor

Number 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). a

desalted using a mini-reverse phase column PerfectPure C-18 Tip (Eppendorf). The tips were conditioned in 100% acetonitrile and washed three times with water. The peptide solution was aspirated and expelled from the tips 10 times. After three washings of the tip with water, the peptides were eluted with 50% ACN. The samples were dried using a Speed-Vac system and resuspended in solution of 0.1% formic acid and 3% ACN. MALDI-TOF/TOF Mass Spectrometry Analysis. The mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments were performed using a 5800 Proteomics Analyzer (Applied Biosystems, Foster City, CA). Approximately 0.6 μL of the extracted peptide solution was mixed with an equal volume of α-cyano-4hydroxycinnamic acid matrix solution (Aldrich, Milwaukee, WI, USA) at 10 mg/mL in 50% ACN/0.1% TFA. Samples were placed on the target plate and allowed to dry at room temperature. Both MS and MS/MS data were acquired with a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser with a 200 Hz repetition rate. Typically, 1600 shots were accumulated for spectra in the MS mode and 2400 shots were accumulated for spectra in the MS/MS mode. MS and MS/MS spectra were acquired in reflector mode. Up to eight of the most intense ion signals with signal-to-noise ratios above 30 were selected as precursors for MS/MS acquisition. The external calibration in MS mode was performed using a mixture of the following four peptides: des-Arg1bradykinin (m/z 904.468), angiotensin I (m/z 1296.685), Glu1fibrinopeptide B (m/z 1570.677), and adrenocorticotropic hormone (18−39) (m/z 2465.199). MS/MS spectra were externally calibrated using known fragment ion masses observed in the MS/MS spectrum of angiotensin I. ESI-Q-TOF Mass Spectrometry Analysis. The peptides extracted from the 1D gel slices were desalted and fractionated by liquid chromatography (LC) coupled online with an electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF). A 7.5 μL aliquot of the sample was loaded on a Waters nanoACQUITY UPLC System (Waters, Milford, MA, USA) with a Waters symmetry C18 trap column 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 with a 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 source temperature was 80 °C, and the cone voltage was 40 V. Instrument control and data acquisition were conducted using a MassLynx data system (version 4.1, Waters). The experiments were performed by scanning a mass-to-charge ratio (m/z) of 300−2000 using a scan time of 1 s during the entire chromatographic process. A maximum of three ions with charge states of 2, 3, or 4 were selected for MS/MS from a single MS survey. Collision-induced dissociation (CID) MS/MS spectra were obtained using argon as the collision gas at a pressure of 1 bar. The collision voltage varied between 22 and 60 eV depending on the mass and charge of the precursor. The reference ion used was the monocharged ion m/z 588.8692 of phosphoric acid. Mass spectra corresponding to each signal from the total ion current (TIC) chromatogram were averaged, allowing for an accurate molecular mass

determination. Exact mass MS/MS was automatically determined using the LockSpray source (Waters). The acquired peak lists were analyzed by searching the National Center for Biotechnology Information (NCBI) database using the MASCOT search engine (www.matrixscience.com) for identification based on the MS/MS ions. Under standard thresholds (mass error tolerance, ±50 ppm for MALDI-TOF-MS and 0.2 Da for ESI-Q-TOF mass spectrometry analysis; MS/MS tolerance, ±0.2 Da for MALDITOF-MS and ESI-Q-TOF mass spectrometry analysis; fixed modifications, Cys-carbamidomethylation; variable modifications, methionine oxidation; one tolerated missed cleavage), proteins with a MOWSE score of at least 54 were considered as significantly identified. The functional classification of the identified proteins was performed by searching 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 the program GraphPad Prism 5.0 for Windows. A one-way ANOVA was applied followed by Tukey’s test (p ≤ 0.05).

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RESULTS AND DISCUSSION Plant breeding is one of the strategies used to enhance the sensory characteristics and increase the nutritional properties of foods. However, the impact of this technique on both environmental and human health should be monitored. In this study, we investigated the proteome profiles of four new soybean varieties (BRS 257, BRS 258, Embrapa 48, and BRS 267) and the relationships between the protein expression profiles and the nutritional 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 is in accordance with our results. The thiourea/urea method of protein extraction provided the following amounts of protein (μg/g dry matter ± SD): BRS 257 had a protein content of 144000 ± 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 by 2D SDS-PAGE. Proteins from four soybean cultivars were extracted using a urea/thiourea protocol. These proteins were then separated by 2D SDS-PAGE (Figure 1), followed by protein identification 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 were 102 ± 22, 124 ± 5, 113 ± 32, and 99 ± 23, respectively. After this analysis, the differentially expressed protein spots were identified using MALDI-TOF-TOF mass spectrometry (Figure 1). A total of 47 protein spots were identified (Table 1). 1289



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

modifications of glycinin that occur during seed development, such as dissociation and reassembly.12 Conversely, some authors attribute the occurrence of different spots for the same protein to the natural variation among different cultivars.34 Comparative Analysis of the Soybean Seed Proteomes by 1D SDS-PAGE. A 1D SDS-PAGE (Figure 2) analysis was performed using a protein extract suitable for 2D separation. The gels were stained with CBB, and proteins were identified by ESI-Q-TOF mass spectrometry. After image analysis, each intense Coomassie staining lane of the gel was divided into approximately 2.5 mm slices; the slices were then subjected to in-gel proteolysis with trypsin. A total of 117 proteins were identified from 12 bands in the 1D SDS-PAGE gel of soybean seed proteins.

It has been reported that various soybean seeds may have different protein profiles due to genetic variability.32,33 Among the analyzed spots, we found storage, allergenic, maturation, and agglutinin proteins as well as trypsin inhibitors. The comparative study of the 2D gels showed that the cultivar BRS 267 had the highest diversity for the identified spots (approximately 53%); this cultivar also had a greater number of spots corresponding to allergenic proteins and antinutrients, such as lectin and Kunitz inhibitor. Eighteen glycinin and β-conglycinin spots were detected in the four soybean seeds. As the data show, these protein spots are distributed over a wide pI range in the same MW region. This pattern suggests the presence of isoforms or post-translational 1290



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metabolism) and protein destination and storage proteins (10%). We noted that the maximal percentages in our study were similar for unclassified proteins and those associated with lipid metabolism. It must be noted that the protein profile is changed during seed filling; there is a decrease in lipid and sterol metabolism proteins and an increase in storage proteins. Asakura et al.38 characterized the gene expression patterns of developing soybeans. This group demonstrated that gene expression changes according to the stages of seed development. These changes could contribute to the differences in protein content. Kottapalli et al.39 identified variations between four peanut cultivars. Proteins such as storage proteins, antinutritive proteins, and allergens were present in some peanut cultivars and absent in others. According to their study, the expression of some identified 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 due to their elevated levels of carbohydrates and the high quality of the proteins. The diversity of proteins among the four soybean seeds in this study suggests that there are significant differences in the functional and nutritional properties of these seeds. Nevertheless, little is known about the protein composition of new conventional soybean varieties. The results obtained in this study indicate which cultivars are most suitable for human consumption and can direct the development of new cultivars with consideration of the protein composition.

Distinct differences between the cultivars were found. Table 2 shows a comparative scheme indicating the presence or absence of the 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 the lipid storage P24 oleosin isoforms A and B in this band, whereas only 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 from Embrapa-soja.24 Scientists have recently verified that a soybean seed lacking lipoxygenase had higher contents of calcium, magnesium, glucosides, and the aglycone forms of isoflavones and 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 and spot 24), cytosolic malate dehydrogenase (band 7), and hydroxysteroid 11-β-dehydrogenase 1-like protein-like (band 7). A 24 kDa oleosin isoform was found in BRS 257. The antinutrients lectin and Kunitz trypsin inhibitor were present in all soybean seeds. 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 of contamination occurred. Functional Classification of the Identified Soybean Proteins. Classification according to the biological function of the proteins identified for each cultivar is illustrated in Figure 3. The proteins identified by ESI-Q-TOF-MS from the 1D SDSPAGE analysis were grouped into nine categories according to their possible biological functions, based on molecular function, using the Universal Protein Knowledgebase (UniprotKB) and NCBI databases. Comparison of the four soybean cultivars revealed similar protein function profiles. The distribution of proteins from the cultivars BRS 257 and BRS 258 had greater differentiation between categories than was observed in BRS 267 and Embrapa 48. Cultivars BRS 267 and Embrapa 48 had similar distributions of proteins between the categories; among the four cultivars, BRS 267 and Embrapa 48 had the greatest amounts of total sugar. These cultivars are described in the literature as having similar characteristics and as containing more sucrose.36 The highest percentage of storage proteins was found in BRS 257 (40%), followed by Embrapa 48 (31%), BRS 258 (25%), and BRS 267 (25%). The second largest protein group was unclassified proteins, which ranged from 16 to 28% of the total protein content. The third major group of proteins observed was related to lipid metabolism (11−25%). Although BRS 257 does not possess lipoxygenases, lipid metabolism proteins represented 12% of the total protein content. Other observed protein categories were protein 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 agreement with our finding of a higher extraction yield of protein, although the values found were similar to those of other cultivars. Another distinctive feature of this cultivar is the significant percentage of proteins related to lipid metabolism. An investigation of the proteomics of seed filling in soybean was reported by Hajduch et al.37 The authors demonstrated that the largest functional class was unclassified proteins (28%), followed by metabolic proteins (22%; mainly lipid and sterol

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ASSOCIATED CONTENT

S Supporting Information *

Spreadsheets containing molecular weight, isoelectric point, matched peptides, percent sequence coverage, and MOWSE scores of protein for identified proteins in the spots and bands from 2D SDS-PAGE and 1D SDS-PAGE, respectively, of soybean cultivars BRS 257, BRS 258, Embrapa 48, and BRS 267. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(E.F.) Mailing address: Departamento de Nutriçaõ Básica e Experimental, Instituto de Nutriçaõ Josué de Castro, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, UFRJ, Caixa Postal 68041, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, CEP 21941-590, Brazil. E-mail: fialho@nutricao. ufrj.br. Fax: + 55 21 2280 8343. Phone: + 55 21 2562 6599. Funding

This work was supported by Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı ́vel Superior (CAPES) and Fundaçaõ de Amparo à Pesquisa Carlos Chagas Filho do Estado do Rio de Janeiro (FAPERJ). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Ana Lúcia O. Carvalho and Augusto Vieira Magalhães (IBqM-UFRJ) for their helpful assistance. We also thank Embrapa-soja for the kind donation of soybean seeds.

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ABBREVIATIONS USED ACN, acetonitrile; CBB, Coomassie brilliant blue; DTT, dithiothreitol; EMBRAPA, Empresa Brasileira de Pesquisa Agropecuária; ESI-Q-TOF, electrospray ionization-quadrupoletime of flight; FDA, U.S. Food and Drug Administration; HDL-c, 1291



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(16) Ye, X. J.; Ng, T. B. Antitumor and HIV-1 reverse transcriptase inhibitory activities of a hemagglutinin and a protease inhibitor from mini-black soybean. Evidence-Based Complement. Alternat. Med. 2011, 2011, 1−12. (17) Ferreira, E. S.; Silva, M. A.; Demonte, A.; Neves, V. A. βConglycinin (7S) and glycinin (11S) exert a hypocholesterolemic effect comparable to that of fenofibrate in rats fed a high-cholesterol diet. J. Funct. Foods 2010, 2, 275−283. (18) Fassini, P. G.; Noda, R. W.; Ferreira, E. S.; Silva, M. A.; Neves, V. A.; Demonte, A. Soybean glycinin improves HDL-C and suppresses the effects of rosuvastatin on hypercholesterolemic rats. Lipids Health Dis. 2011, 10, 165−171. (19) Zhang, H.; Bartley, G. E.; Zhang, H.; Jing, W.; Fagerquist, C. K.; Zhong, F.; Yokoyama, W. Peptides identified in soybean protein increase plasma cholesterol in mice on hypercholesterolemic diets. J. Agric. Food Chem. 2013, 61, 8389−8395. (20) Gouda, K. G. M.; Gowda, L. R.; Rao, A. G. A.; Prakash, V. Angiotensin I-converting enzyme inhibitory peptide derived from glycinin, the 11S globulin of soybean (Glycine max). J. Agric. Food Chem. 2006, 54, 4568−4573. (21) Martinez-Villaluenga, C.; Rupasinghe, S. G.; Schuler, M. A.; Mejia, E. G. Peptides from purified soybean β-conglycinin inhibit fatty acid synthase by interaction with the thioesterase catalytic domain. FEBS J. 2010, 277, 1481−1493. (22) American Dietetic Association (ADA). Position of the American Dietetic Association: functional foods. J. Am. Diet. Assoc. 2009, 109, 735−746. (23) Mussatto, S. I.; Mancillha, I. M. Non-digestible oligosaccharides: a review. Carbohydr. Polym. 2007, 68, 587−597. (24) Amorim, A.; Freitas, A. L.; Mallassa, M. R.; Passos, S. P. Paraná − Destaques econômicos. Embrapa apresenta cultivares de soja. Analise Conjuntural 2008, 30, 19−29; http://www.ipardes.gov.br/webisis. docs/bol_30_1i.pdf (accessed Sept 24, 2013). (25) Trujillo, E.; Davis, C.; Milner, J. Nutrigenomics, proteomics, metabolomics, and the practice of dietetics. J. Am. Diet. Assoc. 2006, 106, 403−413. (26) Ferguson, P. L.; Smith, R. D. Proteome analysis by mass spectrometry. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 399−424. (27) Guijarro-Díez, M.; García, M. C.; Marina, M. L.; Crego, A. L. LCESI-TOF MS method for the evaluation of the immunostimulating activity of soybeans via the determination of the functional peptide soymetide. J. Agric. Food Chem. 2013, 61, 3611−3618. (28) Herman, E. M.; Helm, R. M.; Jung, R.; Kinney, A. J. Genetic modification removes an immunodominant allergen from soybean. Plant Physiol. 2003, 132, 36−43. (29) Peterson, G. L. Determination of total protein. Methods Enzymol. 1983, 91, 95−121. (30) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680−685. (31) Alves, F. P.; Oliveira, M. A.; Mandarino, J. M. G.; Benassi, V.; Leite, R. S.; Seibel, N. F. Composiçaõ centesimal de grãos de soja de oito diferentes cultivares. Resumos expandidos. VI Jornada Acadêmica da Embrapa Soja, 2011; ISSN 0021-8561; http://www.cnpso.embrapa.br/ download/Jornada2011.pdf (accessed April 26, 2013). (32) Zarkadas, C. G.; Gagnon, C.; Gleddie, S.; Khanizadeh, S.; Cober, E. R.; Guillemette, R. J. D. Assessment of the protein quality of fourteen soybean [Glycine max (L.) Merr.] cultivars using amino acid analysis and two-dimensional electrophoresis. Food Res. Int. 2007, 40, 129−146. (33) Natajaran, S. S.; Xu, C.; Bae, H.; Caperna, T. J.; Garrett, W. M. Characterization of storage proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds using proteomic analysis. J. Agric. Food Chem. 2006, 54, 3114−3120. (34) Natarajan, S. S. Natural variability in abundance of prevalent soybean proteins. Regul. Toxicol. Pharmacol. 2010, 58, S26−S29. (35) Esteves, E. A.; Martino, H. S. D.; Oliveira, F. C. E.; Bressan, J.; Costa, N. M. B. Chemical composition of a soybean cultivar lacking lipoxygenases (LOX2 and LOX3). Food Chem. 2010, 122, 238−242. (36) Oliveira, M. A.; Carrão-Panizzi, M. C.; Mandarino, J. M. G.; Leite, R. S.; Filho, P. J. C.; Vicentini, M. B. Quantification of the sugars,

high-density lipoprotein cholesterol; IPG, immobilized pH gradient; kDa, kilodalton; KTI, Kunitz trypsin inhibitor; LDLc, low-density lipoprotein cholesterol; MALDI-TOF, matrixassisted 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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Four Conventional Soybean [Glycine max (L.) Merrill] Seeds Exhibit

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