Sample preparation focusing on plant proteomics: extraction, evaluation and identification of proteins from sunflower seeds

Abstract

In the present work 17 different procedures for extraction of proteins from sunflower (Helianthus annuus L.) seeds in natura were evaluated regarding extraction time and temperature, type of solvent, and analytical procedure. After each extraction procedure, total protein was determined, ranging from 260 ± 5 to 2727 ± 11 μg g⁻¹. Then, a 2-D PAGE was used for P4, P5, P13 and P17, which were considered the most efficient extraction protocols, to calculate the match between them. The highest (77 ± 3%) and the lowest (48 ± 1%) values were achieved when P13 × P17 and P4 × P17 protocols were compared, respectively. Additionally, the P4 and P5 protocols presented the highest number of spots (196 ± 11 and 194 ± 20) after 2-D PAGE protein separation. From both protocols (P4 and P5), 66 protein spots were visualized in both extraction protocols and also analyzed by the 2-D PAGE technique for verifying possible changes in protein expression. Thirty-six spots were differentially found at 90% (or 1.8 times) and related to their relative volume and/or intensity. From these protein spots, 24 (ca. 67%) were successfully identified, 17 being proteins from sunflower seeds. The others are homologous proteins from other plant organisms. All of these analyses contributed to choosing the protocol P4 as the best for protein extraction from sunflower seeds.

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Introduction

Working with biomolecules is not an easy task since many are labile and present extreme complexity.¹ Even with these drawbacks, biomolecules represent a considerable portion of analytes evaluated in studies focusing on the life-sciences, including those carried out on genomic, proteomic, metabolomic, or metallomic platforms, among others.^1,2 As the first step of any method refers to sample preparation, and due to the already commented characteristics of the biomolecules, this step must be performed as carefully as possible in order to ensure reliable results, avoiding wrong conclusions. Based on this fact, it is of utmost importance that sample preparation procedures for biomolecules (focusing on proteins in the present work) take into account the compromise between efficiency and the maintenance of the integrity of the analyte.¹ The sample preparation procedures most commonly used for proteins involve extraction, which can be performed manually^3–5 or, to a lesser extent, aided by microwave or ultrasonic energies.^6–8

Plant proteomics is an important area related to life-science, with the proteomic techniques being extremely important to elucidate several aspects of metabolic regulation of essential processes, besides others.^9,10 Since proteins can be up or down regulated during these processes, their expression analyses are currently targets of many studies, presently characterizing a considerable number of applications in the literature involving plant proteomics.^11–13 However, depending on the sample preparation procedure employed, the results can vary widely, emphasizing that a well optimized procedure is of utmost importance.

In view of the difficulty in working with proteins and their importance to plant proteomics, in this work sunflower was particularly chosen as the sample due to its complexity and the diversity of its by-products, such as seed kernel size and oil content, where proteomic studies can be used as aids in the improvement of the protein content of these products.^14,15 Besides, proteomic assays can also be applied in the evaluation of stress conditions, such as reactive oxygen species (ROS)¹⁶ and water stress.¹⁷ Thus, the present work reports the optimization of the sample preparation procedure for protein extraction from sunflower seeds. Seventeen protein extraction procedures were evaluated, using manual extraction as well as those employing microwave and ultra-sound energies. In all situations, the total protein concentration was evaluated and for the better extraction procedures, a 2-D PAGE and image analysis were employed to check on the possible changes in protein expression. Those proteins whose expressions had changed were then identified by ESI-QTOF MS/MS and some discussions related to their functions are included.

Material and methods

Reagents and sample

Reagents employed for electrophoretic separations, such as acrylamide, bisacrylamide, DTT, iodoacetamide, urea, thiourea, ampholytes, strips for isoelectric focusing and mineral oil, were purchased from Amersham Biosciences (Uppsala, Sweden). Mass spectrometry grade trypsin was purchased from Promega (Madison, USA). All other chemicals (including reagents for buffer preparation and gel stain) were obtained from J.T. Baker (Phillipsburg, NJ, USA) and Merck (Darmstadt, Germany). All solutions were prepared with deionized water (≥18.2 MΩ cm) using a Milli-Q water purification system (Millipore, Bedford, USA).

The sunflower seeds employed in this study (Helianthus annuus L.) and obtained after plant breeding (variety IAC Iarama) were obtained from the Campinas Agronomical Institute.

Protein extraction protocols

Initially, the embryos, which have higher protein contents than full seeds, were separated from the seed coats and 0.6 g was ground in a mortar with liquid nitrogen for 10 min for the extraction procedure. The powder obtained was then mixed with petroleum ether (6 mL) and gently agitated (ca. 15 min) for oil removal. This procedure was repeated three times and an adequate volume of each extraction mixture was then added.¹³

For the extractions, four parameters were evaluated: solvent (deionized water or Tris–HCl buffer containing KCl, DTT, PMSF and SDS); extraction time (10 or 20 min); temperature (25 or 50 °C) and analytical procedure (manual maceration, sonication or microwave radiation), resulting in a total of 17 extraction protocols (Table 1). For each extraction procedure, 6 mL of each solvent (water or buffer as in Table 1) were used. The buffer was prepared at the following concentrations: 50 mmol L⁻¹ Tris–HCl, pH 8.8, 1.5 mmol L⁻¹ KCl, 10 mmol L⁻¹ dithiothreitol (DTT), 1.0 mmol L⁻¹ phenylmethanesulfonyl fluoride (PMSF) and 0.1% m/v sodium dodecyl sulphate (SDS). Additionally, a Branson Bransonic^® 2510 MTH 2.8L UltraSonic Bath Cleaner (USA) and a Milestone Microwave Digestion System, model Start E (USA), were used from P8 to P13 and from P14 to P17, respectively. For the analytical procedure involving microwave radiation, the temperature was not a variable, since the objective of this procedure was to verify the influence of the heating generated from the microwave radiation for the extraction of proteins.

Table 1 Protein extraction protocols

Protocol	Remarks
P1	Deionized water, 25 °C, 10 min, manual maceration
P2	Deionized water, 25 °C, 20 min, manual maceration
P3	Deionized water, 50 °C, 10 min, manual maceration
P4	Deionized water, 50 °C, 20 min, manual maceration
P5	Buffer, 25 °C, 10 min, manual maceration
P6	Buffer, 25 °C, 20 min, manual maceration
P7	Buffer, 50 °C, 20 min, manual maceration
P8	Deionized water, 25 °C, 10 min, sonication
P9	Deionized water, 25 °C, 20 min, sonication
P10	Deionized water, 50 °C, 20 min, sonication
P11	Buffer, 25 °C, 10 min, sonication
P12	Buffer, 25 °C, 20 min, sonication
P13	Buffer, 50 °C, 20 min, sonication
P14	Deionized water, 50 °C, 10 min, microwave radiation
P15	Deionized water, 50 °C, 20 min, microwave radiation
P16	Buffer, 50 °C, 10 min, microwave radiation
P17	Buffer, 50 °C, 20 min, microwave radiation

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Taking into account the Osborne classification,¹⁸ the proteins extracted from P1 to P4, from P8 to P10, and P14 and P15 (Table 1) could be correlated with the proteins' albumin class (water-soluble). All the others protocols used could be correlated with the globulin class (salt-soluble).

After each procedure, the remaining insoluble material was then removed by centrifugation (Bio-Spin-R Ultracentrifuge, BioAgency, São Paulo, Brazil) for 5 min at 8700 × g (4 °C). The supernatant solutions were stored in Eppendorf® flasks and frozen at −18 °C.

For protein precipitation, 1 mL of 0.1 mol L⁻¹ ammonium acetate in methanol was added to 200 μL of the previously obtained protein extract. The precipitated material was then washed twice with ammonium acetate–methanol solution, two times with cold 80% (v/v) acetone and, finally, once with cold 70% (v/v) ethanol. Protein resolubilization was performed using 7 mol L⁻¹ urea, 2 mol L⁻¹ thiourea, 2% (m/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate (CHAPS), 0.002% (m/v) bromophenol blue and 0.5% (v/v) carrier ampholytes.¹³

Total protein concentration

After the application of each protocol, total protein concentrations were determined according to the Bradford method¹⁹ using bovine serum albumin as the standard. The analyses were carried out on a B582 spectrophotometer (Micronal, Brazil) and all measurements were performed in triplicate. In this case, the pellets were solubilized in 50 mmol L⁻¹ Tris–HCl buffer at pH 8.8, prior to the measurement.

Two-dimensional gel electrophoresis

The protein separation by 2-D PAGE was performed according to the manufacturer's (GE Healthcare, Uppsala, Sweden) recommendations.²⁰ A protein mass of 500 μg was loaded onto immobilized pH gradient strips (13 cm length, 4–7 pH range).¹³ The referred masses were applied to the IPG strips and rehydrated at room temperature for at least 12 h. Isoelectric focusing was then carried out in an Ettan IPGphor II (GE Healthcare), totalizing 14 300 V h. The second dimension separation was carried out at 25 mA per gel and 250 W in an SE 600 Ruby system (GE Healthcare) with lab cast 1 mm SDS polyacrylamide gels, and at final concentrations of 12.5% (m/v). The buffer system consisted of a solution of 25 mmol L⁻¹ Tris–HCl, pH 8.3, 192 mmol L⁻¹ glycine and 0.1% (m/v) SDS. After separation, the gels were stained with colloidal Coomassie brilliant blue.²¹

Image analysis of 2-D gels

The gels were scanned using an ImageScanner II (Amersham Biosciences, Uppsala, Sweden) with a densitometer operating at 300 dpi resolution, 100% zoom and 12 or 16 bits per pixel depth, according to the manufacturer's recommendations.²² The images obtained were saved as ^*tif, and the Image-Master 2D Platinum 6.0 software (GeneBio, Geneva, Switzerland) was employed for gel image analysis.

For improving the accuracy, image treatments were carried out by the same analyst and concomitantly applied for all gels in the same electrophoretic run (3 gels). All values were represented as average ± standard deviation.¹³

Initially, a contrast image of the gel was taken for improving visualization. Then, a small region of gel containing clearly visualized spots was selected, and the parameters 3-smooth, 60-saliency, 48 min area were applied for spot detection, in agreement with the manufacturer.²² Then, these parameters were applied to spot detection in all gel regions. All the parameters were also employed for analyzing the entire gel, using the automatic detection tool of the program. In this way, all values were normalized by the program for future data acquisition in terms of intensity and volume of the spots. After the automatic detection of the spots, manual editing was carried out to either eliminate false positive spots, to consider false negative spots previously not considered by the program, or to provide corrections related to the spot regions (to analyze peak overlap, increasing resolution as well as to better define the spot area—elliptical or circular).²³ The following equation was used for differentiating a spot from the background:^24,25

I_spot > I_background + 3s_background (1)

For the matching process, only three landmarks were necessary and were manually defined for the matching process. Thus, the gel presenting the highest number of spots was considered as the master gel. Finally, the spots of gels of each selected experimental procedure were statistically evaluated in terms of relative intensity and volume (normalized values), and the data obtained were analyzed by a statistical program. A more detailed explanation regarding image gel treatment can be found in the literature.¹³

Tryptic digestion

In-gel digestion of protein spots that showed changes in expression was performed. For this task, the spots (ca. 2 mm) were manually cut from the gel and placed in a micro-SPE plate containing peptide affinity resin—Montage® In-Gel digest_ZP kit (Millipore). The digestion and vacuum elution protocols were performed according to the manufacturer's recommendations. Basically, a dye removal step using acetonitrile was first employed, followed by the tryptic digestion (using ca. 166 ng of enzyme for each spot). Then, a clean-up step was carried out using 130 μL of 0.2% (v/v) TFA solution. Finally, purified peptides were eluted from the resin using 20 μL of 0.1% (v/v) TFA in 50% (v/v) acetonitrile solution. For vacuum elution, a Multiscreen® Vacuum Manifold (Millipore) was used.

Protein identification

For nESI-QTOF MS/MS analysis, the peptides obtained by enzymatic digestion were dried and resolubilized in deionized water. An aliquot (4.5 μL) of the resulting peptide mixture was separated using a C18 column (Waters BEH C₁₈, 100 mm × 100 μm) RP-nanoUPLC (nanoAcquity, Waters) coupled to a Synapt HDMS mass spectrometer (Waters) with a nano-electrospray source at a flow rate of 1.0 μL min⁻¹. The gradient was 2–90% acetonitrile in 0.1% (v/v) formic acid over 40 min. The instrument was operated using Data Dependent Analysis (DDA), where the equipment acquires one spectrum per second, and when multi-charged species were detected, the three most intense species were fragmented in the collision cell (collision energy set according to precursor's m/z and charge). Spectra were acquired using MassLynx v.4.1 software.

All mass spectra were processed into peak list format using Mascot Distiller (Matrix Science, London, UK). Protein identification was performed by searching the NCBI database.

MS/MS search parameters included oxidation of methionine as a variable modification, carbamidomethylation of cysteine as a fixed modification, ±0.1 Da peptide and fragment mass tolerance, and a maximum of one missed cleavage. The significance threshold was set at P < 0.05, which corresponds to a minimum score of 37.

Results and discussion

Total protein concentration

Four parameters were evaluated with the extraction protocols applied: extraction time, solvent for extraction, temperature of extraction and analytical procedure employed. After application of each protocol, quantification of total protein concentrations was carried out on the precipitate obtained. The results of total protein concentrations in the embryos of sunflower seeds are shown in Table 2. The analytical curves obtained using the Bradford method always showed linear correlations (R² ranged from 0.975 to 0.995).

Table 2 Total protein concentrations (μg g⁻¹) determined by the Bradford method (n = 3) from different protein extraction protocols (defined in Table 1)

Protocols	Total protein concentration (μg g^−1)
P1	260 ± 5
P2	327 ± 2
P3	532 ± 13
P4	651 ± 9
P5	2534 ± 15
P6	2561 ± 8
P7	2727 ± 11
P8	304 ± 3
P9	343 ± 2
P10	438 ± 5
P11	1306 ± 7
P12	1468 ± 6
P13	1964 ± 6
P14	308 ± 14
P15	321 ± 10
P16	1308 ± 13
P17	1742 ± 7

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According to the results, the parameters that most influenced the protein content were the solvent for extraction and the analytical procedure employed. Then, comparing the extraction solvent in the same analytical procedure, improvements of ca. 319% in the quantities of protein extracted were observed, comparing the protocols P4 and P7, 348% between P10 and P13 and 343% between P15 and P17. These results demonstrate the high efficiency in using Tris–HCl buffer as an extraction solvent. However, despite the differences in total protein concentrations obtained with solvents used in all analytical procedures applied, deionized water as an extraction solvent showed interesting results when using the technique of 2-D PAGE, as discussed later.

By comparing the different analytical procedures used and selecting buffer as the extraction solvent, manual maceration showed the best results when compared with ultrasonic energy and microwave radiation. From these results, a decrease of 28% in the extracted amount of protein was observed by comparing protocols P7 and P13, and 36% between P7 and P17. The literature reports a few studies using ultrasonic energy and microwave radiation in the extraction of proteins from plant samples. Magalhães et al.⁵ used ultrasonic energy to extract metalloproteins from chestnuts (Aesculus hippocastanum L.), and good results were obtained for protein extraction using a combination of grinding and sonication. However, this strategy was not suitable to preserve metal ions in the protein structure. Additionally, some studies have focused on ultrasonic energy^26–28 and microwave radiation^29,30 in proteomics, principally for accelerating enzymatic digestion for protein identification. Therefore, the interaction between ultrasonic energy, due to the high energy delivered to the system, and some proteins may have led to chemical degradation of their structures, reducing the range of separated proteins. Regarding microwave radiation, this problem can be attributed to heat-induced degradation.³¹ In addition, microwave radiation is also used in hydrolysis processes, resulting in the release of amino acids.^32,33

Thus, low molar mass proteins or proteins of low concentration could be more affected by microwave radiation. Furthermore, the increase in temperature (25 to 50 °C) improves the efficiency of protein extraction in relation to the same analytical procedure and solvents used, when combining manual maceration and ultrasonic energy. As examples, improvements of ca. 105% in the amount of protein extracted by comparing protocols P1 and P3, and 34% between P12 and P13 were detected. Thus, an increase in the temperature proved to be an auxiliary tool in the extraction of protein from the sample matrices, as the sunflower embryos have a high concentration of fatty acids and lipids.^34,35

Regarding extraction time, it was clear that longer extraction times gave higher extraction efficiencies. However, as there was only a small increase in the total protein concentrations, 20 min were considered sufficient for sunflower seed protein extraction.

In conclusion, protocols P4, P5, P13 and P17 were selected for protein separation using 2-D PAGE. The choice of procedure P5 against P7 was based on the fact that there was no significant difference in total protein contents between the protocols used and, additionally, procedure P5 is simpler to apply than P7.

Two-dimensional gel electrophoresis: a matching study

After defining the extraction protocols, 2-D PAGE was applied for verifying the proteomic map after each one. To avoid a high background due to excess “staining agent”, the gels were washed 3 times with deionized water under gentle agitation (15 min). The selected parameters for image acquisition (see Image analysis of 2-D gels section) were those proposed by the manufacturer.²² However, the parameters were adjusted in a visual manner to obtain the highest number of spots that contrasted with the background (see also the equation of Image analysis of 2-D gels section). This procedure was previously applied by our research group.¹³ For the separation of proteins, 2-D PAGE was applied for the extracts from protocols P4, P5, P13 and P17, using 500 μg of protein mass and pH ranging between 4 and 7, for each protocol. The images obtained are shown in Fig. 1. A total of 196 ± 11 (80 ± 2% match), 194 ± 20 (83 ± 6% match), 160 ± 16 (85 ± 2% match) and 149 ± 17 (82 ± 12% match) spots were obtained for the P4, P5, P13 and P17 protocols, respectively. These results indicate no major differences between the protocols evaluated, in terms of the total number of spots in the 2-D gel. Moreover, good matches (>80%) between replicates indicate that aligned and undistorted gels were obtained.

Fig. 1 Gel electrophoresis profiles obtained from P4 (A); P5 (B); P13 (C) and P17 (D) protocols.

When comparing matches between methods, some differences were obtained (see Table 3). The solvent for extraction is a significant factor for differentiating the methods. Matches between 45% and 55% were obtained when comparing different procedures. As an example, the match between the extraction using manual maceration with deionized water (P4) and extraction using manual maceration with buffer (P5) was 53 ± 4, with both methods showing similar numbers of spots in the 2-D gel (196 ± 11 and 194 ± 20, respectively).

Table 3 Matches between the extraction methods (P4, P5, P13 and P17) used in the 2-D gel separation

Methods compared	P4 × P5	P5 × P13	P5 × P17	P4 × P17	P4 × P13	P13 × P17
Match (%)	53 ± 4	69 ± 1	73 ± 2	48 ± 1	52 ± 2	77 ± 3

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When different procedures with the same solvents were compared, the match values were above 65%. As an example, the match between the extraction using ultrasonic energy with buffer (P13) and extraction using microwave radiation with buffer (P17) was 77 ± 3%. These results indicate the influence of the solvent on the extraction of proteins.

Considering the P4 and P5 protocols, an absence of spots after overlaying the images of the gels between the methods is detected (Fig. 2). Although both methods showed a similar total number of spots in the 2-D gel, their proteomic profiles were quite different. Spots marked in green, which refers to the P5 method, were not correlated with spots present in the gel for P4 method. Thus, the most intense spots were more preferentially extracted using water as the extraction medium compared with buffer, with which proteins with spots of lesser intensity were extracted. One possible reason for this is the fact that most of the spots identified by mass spectrometry (see the Mass spectrometry protein identification section, Table 5) present acidic character, which could be extracted more efficiently with water. Then, depending on the solvent used, significant changes in the proteomic profile of the sunflower embryos samples can be seen.

Fig. 2 Overlay of images of the gels between the methods: P4 – manual maceration with deionized water (red) and P5 – manual maceration with buffer (green).

Taking into account the total protein content, the P5 protocol showed much higher values compared to the P4 protocol. As the number of spots detected in the gels was similar for both methods, a plausible explanation for this difference is that the P5 method possibly extracted a wide range of proteins in terms of molar mass, where the separation method using 2-D gel electrophoresis was not effective in the separation of these proteins.

As the P4 and P5 protocols showed the highest number of spots among the evaluated methods, these were selected for comparison in terms of differential spots. Proteins were considered as differentials, in terms of extraction efficiency, when the ratio between spot volume and/or intensity for methods 4 and 5 changed from 90% variation. This defined cut-off was the average value when comparative proteomic studies on protein expression are considered.^36–38 From the total number of spots obtained from protocols P4 (196 ± 11) and P5 (194 ± 20), 66 were correlated, taking into account three replicates of each protocol, which represents ca. 34% of the spots considering both protocols. From this total, 36 spots (4 to 7 pH gel range) were differential (Fig. 3 and Table 4). Considering the intensity, 15 spots were more intense when water was used than buffer (12 spots), and considering the volume, 19 spots presented higher volumes in water than buffer (14 spots). Thus, the P4 protocol was more efficient for extraction, as shown by the separation of proteins using the 2-D PAGE technique, and it was selected for identification of the differential spots. As P4 and P5 protocols are apparently complimentary, they may also be run sequentially for improving the amount of extracted proteins.

Fig. 3 Spots with changes in expression of %V and/or %I (ca. 90% variation) considering P4 and P5 protocols. The numbers are related to the identification of proteins (see also Tables 4 and 5). The letters B (P5) and W (P4) indicate buffer and water, respectively. In addition, some images of differential spots are shown.

Table 4 Spots with changes in protein expression of 90% (1.8 times, in bold) related to relative volume and/or intensity

Spots	Intensity ratio^a	Volume ratio^a
a Average ± standard deviation (n = 3).
1	1.93 ± 0.10	4.02 ± 0.91
2	3.44 ± 0.05	5.14 ± 0.09
3	2.45 ± 0.06	3.56 ± 0.13
4	1.27 ± 0.07	2.46 ± 0.55
5	4.21 ± 0.05	6.71 ± 1.04
6	5.99 ± 0.05	5.72 ± 0.56
7	1.85 ± 0.23	1.68 ± 0.56
8	3.01 ± 0.09	5.14 ± 0.32
9	2.11 ± 0.06	2.93 ± 0.42
10	1.21 ± 0.08	1.83 ± 1.07
11	1.61 ± 0.12	2.36 ± 0.61
12	8.41 ± 0.04	16.54 ± 0.79
13	4.58 ± 0.03	10.94 ± 0.45
14	1.68 ± 0.04	2.28 ± 0.07
15	2.03 ± 0.04	1.37 ± 0.66
16	2.82 ± 0.05	6.38 ± 0.63
17	1.16 ± 0.04	1.92 ± 0.19
18	3.36 ± 0.08	7.89 ± 1.49
19	2.32 ± 0.10	5.21 ± 1.13
20	3.07 ± 0.02	3.77 ± 0.14
21	2.69 ± 0.03	2.82 ± 0.39
22	2.66 ± 0.05	3.78 ± 0.49
23	2.26 ± 0.03	2.34 ± 0.26
24	1.87 ± 0.04	4.95 ± 0.13
25	1.18 ± 0.03	1.97 ± 0.10
26	3.33 ± 0.05	3.24 ± 0.05
27	6.41 ± 0.06	8.12 ± 1.23
28	1.83 ± 0.02	2.01 ± 0.24
29	4.39 ± 0.05	7.57 ± 0.69
30	1.12 ± 0.02	2.32 ± 0.23
31	2.12 ± 0.03	2.83 ± 0.25
32	2.11 ± 0.05	2.41 ± 0.27
33	5.81 ± 0.02	9.22 ± 0.13
34	2.34 ± 0.04	3.32 ± 0.40
35	1.27 ± 0.05	4.89 ± 0.49
36	1.69 ± 0.03	2.42 ± 0.23

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Table 5 Proteins identified in sunflower seed embryos using 2-D PAGE and mass spectrometry

Spot	NCBI	Protein name	Score	pI/mass	Coverage (%)
1	gi|9858781	BAC19.13 [Solanum lycopersicum]	67	4.98/59659	2
2	gi|15231255	Chaperonin, putative [Arabidopsis thaliana]	61	5.60/63708	4
3		Not identified
4		Not identified
5	gi|12053693	Putative dehydrin [Helianthus annuus]	224	6.63/25798	37
6	gi|12053693	Putative dehydrin [Helianthus annuus]	116	6.63/25798	12
7	gi|12053693	Putative dehydrin [Helianthus annuus]	202	6.63/25798	37
8	gi|1263291	Alcohol dehydrogenase 2b [Gossypium hirsutum]	68	6.23/41760	2
9	gi|4589716	Aspartic proteinase [Helianthus annuus]	65	5.78/55987	3
10		Not identified
11		Not identified
12	gi|4589716	Aspartic proteinase [Helianthus annuus]	96	5.78/55987	11
13		Not identified
14	gi|20502888	Heat shock protein 80 [Solanum tuberosum]	89	4.55/13891	19
15		Not identified
16		Not identified
17		Not identified
18	gi|3334471	10 kDa late embryogenesis abundant protein [Helianthus annuus]	68	5.36/10031	10
19	gi|27526481	Basic 2S albumin [Helianthus annuus]	65	8.62/34159	4
20	gi|3334471	10 kDa late embryogenesis abundant protein [Helianthus annuus]	75	5.36/10031	10
21		Not identified
22	gi|112676	11S globulin seed storage protein G3 [Helianthus annuus]	117	8.22/56001	9
23	gi|18252506	Cysteine synthase [Glycine max]	104	5.69/34364	13
24		Not identified
25	gi|3122228	Heat shock 22 kDa protein [Glycine max]	71	6.34/23970	5
26	gi|121485004	Cytosolic phosphoglycerate kinase [Helianthus annuus]	122	5.82/42335	12
27	gi|112676	11S globulin seed storage protein G3 [Helianthus annuus]	162	8.22/56001	11
28	gi|1235898	17.7 kDa heat shock protein [Helianthus annuus]	115	6.19/17662	14
29	gi|112676	11S globulin seed storage protein G3 [Helianthus annuus]	74	8.22/56001	7
30		Not identified
31	gi|112676	11S globulin seed storage protein G3 [Helianthus annuus]	56	8.22/56001	5
32	gi|109631584	Glutathione peroxidase [Helianthus annuus]	122	6.58/22078	37
33	gi|166029871	Mn-superoxide dismutase I [Helianthus annuus]	79	7.16/25331	15
34	gi|255542606	Short chain dehydrogenase, putative [Ricinus communis]	101	6.67/32084	8
35		Not identified
36	gi|3913794	RecName: full = glutathione peroxidase 1 [Helianthus annuus]	73	4.92/19063	11

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Mass spectrometry protein identification

Protein spots that presented differences in extraction (ca. 90% variation) were digested with trypsin and the digest analyzed by mass spectrometry (nESI-QTOF MS/MS) for protein characterization purposes. Results are shown in Table 5. From 36 protein spots, 24 were successfully identified, 17 proteins being from sunflower seeds. The others are homologous proteins identified in other plant organisms.

Considering the small number of entries in the Helianthus annuus L. protein database, the number of sunflower proteins identified was very significant. Table 5 shows the input code in the database (NCBI), score, molar mass and isoelectric point (based on the protein identified) and sequence coverage of the peptides matched.

In relative sunflower seed proteins, spots 5, 6 and 7 were identified as putative dehydrin. This protein is involved in response mechanisms in some abiotic stress such as dehydration and low temperatures.^39,40 Spots 9 and 12 were identified as aspartic proteinase, where this protein is involved in lipid metabolism processes.³⁹ Spots 18 and 20 were identified as an abundant protein in late embryogenesis. This protein is involved in the growth/cell division processes and is accumulated in the last stages of embryogenesis in seeds, related to processes of response to osmotic and water stresses.^41,42 Spot 19 was identified as albumin, involved in cellular transport.³⁹ Spots 22, 27, 29 and 31 were identified as globulin seed storage protein (11 S helianthinin). This protein is considered to be the major group of storage proteins, being reported to account for 60% of the total proteins in the mature seed.^39,43 Cytosolic phosphoglycerate kinase (spot 26) is involved in metabolic/energy processes, specifically in the carbohydrate metabolism.³⁹ Spot 28 was identified as a 17.7 kDa heat shock protein, being an important protein involved in stress responses, to hyperosmotic pressures, heat, high light intensity and hydrogen peroxide. Furthermore, this protein participates in the process of protein folding.³⁹

Spots 32 and 36 were identified as glutathione peroxidase, and spot 33 as Mn-superoxide dismutase. These enzymes are involved in mechanisms of the oxidative stress response.⁴⁴

Conclusions

The main target of this work was successfully attained, demonstrating how important a detailed assessment of the procedures for sample preparation is when focusing on plant proteomics, since both the extraction solvent and the analytical procedure were decisive for preserving the protein structure after extraction. The optimization of the extraction procedure allowed 24 proteins to be accurately identified by mass spectrometry, either from sunflower seeds or by homology, after separation and analysis of protein expressions by 2-D PAGE. This work also pointed out the necessity of working with gentle procedures for extracting the proteins, since excellent results were obtained using the P4 protocol, which involves the use of deionized water at 50 °C for extraction, with manual maceration of the sunflower seed embryos. Additionally, procedures P4 and P5 may also be run sequentially, for further improving the amount of extractable proteins.

Acknowledgements

The authors thank the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil) for financial support and fellowships. We also thank Prof. Carol H. Collins for language assistance.

Notes and references

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