A Simple Protocol for Protein Extraction of Recalcitrant Fruit Tissues Suitable for 2-De and MS...

Jun SongGordon BraunEric BevisKristen Doncaster

Agriculture and Agri-Food Canada,Atlantic Food and HorticultureResearch Centre,Kentville, Canada

Received October 18, 2005Revised January 16, 2006Accepted January 21, 2006

Research Article

A simple protocol for protein extraction ofrecalcitrant fruit tissues suitable for 2-DEand MS analysis

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Threephenol-free protein extraction procedures for 2-DE were compared and evaluated onapple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone pre-cipitation was found to be the most effective protocol. The results from SDS-PAGE and2-DE analysis showed high quality proteins. More than 500 apple polypeptides wereseparated on a small scale 2-DE gel. The successful protocol was further tested onbanana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues,respectively. To demonstrate the quality of the extracted proteins, several protein spotsfrom apple and banana peels were cut from 2-DE gels, analyzed by MS and have beententatively identified. The protocol described in this study is a simple procedure whichcould be routinely used in proteomic studies of many types of recalcitrant fruit tissues.

Keywords: Apple / Banana / 2-DE / Protein extraction / SDSDOI 10.1002/elps.200500921

1 Introduction

Proteomics is a global term for the study of proteinpopulations in tissue, cell or subcellular compartmentsrelated to changes in protein structure and abundance inresponse to development, treatment and environmentalstress [1]. In recent years, research on Arabidopsis tha-liana (L.) Heynh. and other plant tissues has demon-strated that proteomics is a very powerful tool in the studyof molecular mechanisms in plants [2–5]. One of the pri-mary advantages of proteomics research based on 2-DEis the ability to simultaneously investigate hundreds orthousands of proteins with high accuracy. The ability toprecisely determine molecular weight by MS and thedevelopment of genomic sequence databases for pep-tide mass matches make it possible to achieve highthroughput of plant protein identification [1, 6].

2-DE is especially useful for comparative studies betweenpairs of samples. Comparative proteomics has the poten-tial in postharvest research to help understand the physio-

logical and biochemical changes related to fruit ripeningand senescence as well as the effects of handling andstorage treatments on stored fruit quality. Although manyprotocols have been reported, optimum conditions for thepreparation of protein extracts from plant tissue that aresuitable for 2-DE have primarily been developed for youngvegetative tissues that have high protein content and lowamounts of contaminants [7–10]. Fruit are consideredrecalcitrant plant tissues for proteomic analysis, fromwhich it has been difficult to obtain high quality proteinsuitable for 2-DE analysis due to a low protein content andthe presence of interfering substances such as pigments,carbohydrates, polyphenols, polysaccharides and starch.Establishing a reliable and effective protein extraction pro-cedure is an essential first step in conducting proteomicresearch. Apples (Malus6domestica Borkh.) are amongthe most popular fruits consumed in the world, yet to ourknowledge there has been no successful protocol reportedfor the 2-DE separation of apple fruit proteins, to date.

The objective of this study was to compare two previouslypublished protein extraction protocols, widely applied onplant tissue, with a protocol we developed to extract andpurify protein from apple, a recalcitrant fruit tissue. Proteinyield, quality, the number of well resolved polypeptides after2-DE, and their suitability for MS analysis were evaluated.Ourprotocolwas tested furtheronbanana fruit, known for thedifficulty of protein extraction from both peel and flesh tissue.

Correspondence: Dr. Jun Song, Agriculture and Agri-Food Canada,Atlantic Food and Horticulture Research Centre, 32 Main St., Kent-ville, N.S., B4N 1J5, CanadaE-mail: [email protected]: 11-902-679-2311

Abbreviations: SB 3-10, N-decyl-N, N-dimethyl-3-ammonio-1-pro-pane sulfonate; TCEP, tris(2-carboxyethyl) phosphine hydrochloride

3144 Electrophoresis 2006, 27, 3144–3151

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2006, 27, 3144–3151 Proteomics and 2-DE 3145

2 Materials and methods

2.1 Fruit materials

Apples (Malus domestica, Golden Delicious) were har-vested at the preclimacteric stage from a commercialorchard in Nova Scotia. Fruit was transported to the labo-ratory and stored at 207C for 21 days. On day 8, fruit peri-carp tissue samples were taken from five individual fruit.Banana fruit (Musa spp. AAA group Cavendish subgroup)at ripening stage 5 [11] were purchased from a localsupermarket. Peel and flesh (mesocarp) tissues wereremoved from five individual fruit and pooled separately. Allfruit tissues were immediately frozen in liquid nitrogen, thenmacerated in a laboratory blender with liquid nitrogen athigh speed for 60 s followed by manual grinding to a finepowder with a mortar and pestle. The finely powdered tis-sues were stored at 2857C prior to protein extraction.

2.2 Chemicals

All chemicals used in this study were of the highest gradeavailable. They were purchased from Sigma-Aldrich(Oakville, ON, Canada) and GE-Healthcare (formerlyAmersham BioScience, Baie-d’Urfié, QC, Canada). Milli-Q water (Millipore, Bedford, MA, USA) with resistancegreater than 18 MO was used throughout. All chemicalsolutions were filtered through mixed cellulose estermembrane filters (0.45 mm).

2.3 Protein extraction procedures

Three extraction procedures, A, B, and C, were evaluatedin this study. Protocol A: Total protein was extracted at47C from 2.0 g of powdered tissue in 8.0 mL of thiourea/urea lysis buffer based on the description by Gallardoet al. [3]. The solution contained 5 M urea, 2 M thioureaand 2% CHAPS. After stirring for 10 min, 40 mM DTTwasadded to the solution. The solution was stirred for a fur-ther 10 min and centrifuged at 35 0006g for 10 min at47C. The supernatant was submitted to a second cen-trifugation and the total protein obtained.

Protocol B: Total protein was extracted following themethod of Mechin et al. [7]. Powdered fruit or peel tissue(0.2 g) was placed in a 2.0 mL microfuge tube and theprotein precipitated at 2207C for 45 min with 1.75 mL of aprecipitation solution (10% TCA in ice-cold acetone). Theprecipitated protein was centrifuged for 10 min at10 0006g at 297C. The supernatant was discarded andthe pellet rinsed with 2 mL of ice-cold acetone and storedat 2207C for 1 h to remove residual TCA then further cen-trifuged for 15 min at 10 0006g at 2207C. Acetone rinses

were repeated until a white pellet was obtained. The pro-tein pellet was dried under vacuum for 5 min and then dis-solved in varying volumes of buffer containing 5 M urea,2 M thiourea, 2% CHAPS, 2% N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, 20 mM DTT, 5 mM tris(2-carboxyethyl) phosphine hydrochloride, and two carrierampholytes 0.5% pH 4–6.5 and 0.25% pH 3–11 nonlinear.

Protocol C: Total protein was extracted with hot SDSbuffer followed by ice-cold TCA-acetone precipitation, asmodified from Jeffries et al. [12]. Powdered tissue (2.0 gapple pericarp) was suspended in 10–12 mL of 2% SDSextraction buffer (2% SDS, 60 mM DTT, 20% glycerol and40 mM Tris-HCl pH 8.5). After heating at 957C for 8 min,the samples were centrifuged at 80006g for 15 min at47C. Proteins were precipitated with three volumes of ac-etone containing 10% TCA and 20 mM DTT at 2207C for45 min then centrifuged at 18 0006g for 10 min at 277C.The pellet was washed with ice-cold acetone containing20 mM DTT, precipitated for 60 min at 2207C, then cen-trifuged at 20 0006g for 10 min and the supernatant dis-carded. The protein pellet was air-dried for 3–5 min thendissolved in rehydration buffer [13].

2.4 Protein assay

Protein concentration was measured using the RC/DC™

protein assay kit (Bio-Rad Laboratories, Hercules, CA,USA) to compensate for interfering compounds accord-ing to the manufacturer’s protocol, using BSA as astandard. The protein concentration was expressed asmg/g fresh weight. Three replicates were conducted toevaluate the protein extraction protocols and protein yieldwas presented as mean 6SD.

2.5 SDS-PAGE

The purity and overall quality of protein extracts wereevaluated with Laemmli buffer SDS-PAGE [14]. Protein(10 or 16 mg) was suspended in 15 mL of loading bufferand transferred to a 1.0 mm thick 8–16% acrylamidegradient gel (Bio-Rad Laboratories). Electrophoresis wasconducted using a Bio-Rad mini-Protean™ II apparatus(Bio-Rad Laboratories) at 84 V for 2 h. The gel was thenstained with silver (Sigma-Aldrich) or Bio-Safe™ CBB G-250 (Bio-Rad Laboratories). All SDS-PAGE gel evalua-tions were repeated in triplicate.

2.6 2-DE

Proteins were first separated by IEF using a Multiphor II™

system (GE Healthcare) using Immobiline™ Drystrip gels(11 cm) with a nonlinear pH gradient (3–11), according to


3146 J. Song et al. Electrophoresis 2006, 27, 3144–3151

the manufacturer’s instructions. Equal amounts of protein(10 mg per sample) and 7 mL of 2-D protein standard (Bio-Rad Laboratories) were added to DeStreak™ rehydrationbuffer (GE Healthcare) to give a final volume of 200 mL,which was used to rehydrate strips overnight at 207C.Rehydrated strips were isoelectrofocused for 30 000 Vh at207C. 2-DE (600 V, 20 mA, 30 W for 30 min, then 600 V,50 mA, 30 W for 5 min and finally 600 V, 50 mA, 30 W for68 min) was conducted with two strips placed on a pre-casted 8–18% acrylamide Excel™ Gel 8–18%(24 cm618 cm, GE Healthcare) on the Multiphore II™ sys-tem at 157C. Three microlitres of protein standard wasapplied on a sample application piece placed between thestrips. Resulting gels were stained with silver (Sigma-Aldrich) according to Shevchenko et al. [15]. All SDS-PAGEgels for 2-DE were repeated in triplicate.

2.7 MS analysis and protein identification

2-DE gel images were captured with a digital camera, andimage analysis was performed using PDQuest 2-D analy-sis software (Version 7.3.1, Bio-Rad Laboratories). Select-ed polypeptide spots were manually excised from gels,treated with DTT to break disulfide linkages, alkylated withiodoacetamide, then digested with trypsin. The resultantpeptides were extracted in washes of ammonium bicar-bonate solution, ACN and 10% formic acid. Extractionsolvent was removed under vacuum and the peptideswere resuspended in 30 mL of 5% MeOH, 0.5% formicacid. HPLC was performed on an LC Packings Ultimatenanoflow system (Dionex, Sunnyvale, CA, USA). Samples

(3 mL) were injected directly onto a PepMap reversedphase C18 column (0.0756150 mm) supplied by LC Pack-ings (Dionex). The flow rate after splitting was 320 nL/min.MS was performed on a hybrid quadrupole linear ion trap(Q-TRAP LC/MS/MS, Applied Biosystems, Foster City, CA,USA) equipped with a nanospray ion source. Spectra wereacquired using the Information Dependent Acquisitionmode. The ion spray voltage was 1750 V, the curtain gaswas set to 15 (arbitrary units) and the declustering potentialwas 60 V. The raw MS/MS data were searched againstNCBI non-redundant entries (NIH, Bethesada, MD, USA)using the MASCOT algorithm (Matrix Science, London,UK). The data were also searched against the AppleEST database (http://www.mainlab.clemson.edu/,sook/download/malus.est updated on 2005:05:24) using the ProID algorithm (Applied Biosystems).

3 Results

3.1 Total protein content and SDS-PAGE analysis

Total protein content and 1-D SDS-PAGE evaluation ofapple protein obtained by the three different extractionprotocols are shown in Table 1. Total amounts of proteinextracted from apple fruit varied depending on the proto-col used. Overall, protocol A gave only 94.5 6 24.3 mg/gprotein, protocol B gave 296.5 6 12.7 mg/g and protocolC gave the highest amount of protein with 303.0 6 6.5 mg/g. Among the three protocols, protocol C gave the leastvariation between repeated extractions.

Figure 1. SDS-PAGE evaluationof apple and banana proteinextracts obtained with three dif-ferent protocols. (A) Ten micro-grams of protein extracted fromapple with protocol A were loa-ded onto a 8–16% poly-acylamide gels and visualizedwith silver. (B) Sixteen micro-grams of protein extracted fromapple with protocol B and Cwere loaded on to 8–16% poly-acylamide gel and visualizedwith CBB. The size of proteinstandards are indicated on theleft. (C) Sixteen micrograms ofprotein extracted from bananapeel and flesh were loaded onto8–16% polyacylamide gels andvisualized with CBB. The size ofprotein standards are indicatedon the left.



Table 1. Protein yield from apple and banana tissue usingthree phenol-free extraction protocols

Fruit tissue Protein yield (mg/g fresh weight)

Protocol A Protocol B Protocol C

Apple pericarp 94.5 6 24.3a) 296.5 6 12.7 303.066.5Banana peel nt nt 408.565.0Banana flesh nt nt 320.864.7

a) Mean 6 SD, n = 3nt: not tested

The overall protein profile and purity of protein extractionwas demonstrated by SDS-PAGE (8–16% polyacryl-amide gel) analysis (Figs. 1A and 1B). Due to the lowamount of protein obtained from protocol A, gels wereloaded with only 10 mg protein and visualization required

silver staining. Protocol A produced fewer bands ofpoorer quality than either protocol B or C. For protocol A,most peptides in the range of 50–150 kDa were absent.There were no differences between protocol B and C inthe range of 37–150 kDa peptides, and the protein con-centration was high enough to be visualized by CBBstaining. Also, protocol C gave very intense bands in themiddle range of 20–37 kDa. It was obvious that proto-col C yielded the most extensive protein profile withoutapparent protein degradation or contamination.

The low yield and variable concentrations of protein fromprotocol A and B made them unsuitable for recalcitrantfruit tissues and they were excluded from further 2-DEanalysis. Protocol C was also evaluated for its ability torecover the protein content of banana peel and flesh. Ityielded 408.5 6 5.0 and 320.8 6 4.7 mg protein/g of tis-sue, respectively (Table 1). SDS-PAGE analysis indicatedclean protein samples and intense bands between 10 and

Figure 2. 2-DE of apple protein extracts. Ten micrograms protein from apple tissue was extractedwith protocol C and dissolved in DeStreak™ solution. After isoelectric focusing, the proteins werefurther separated on SDS-PAGE (8–16%) polyacylamide gels and visualized by silver staining. Arrowsindicate proteins identified by LC-MS/MS and listed in Table 2: (1) Malate oxidoreductase (malic en-zyme); (2) UDP glucose pyrophosphorylase; (3) protein similar to unknown protein from Brassicaoleracea var. gemmifera.



200 kDa from peels and between 20 and 200 kDa fromflesh (Fig. 1C). Proteins in banana peel and flesh werefurther evaluated by 2-DE analysis.

3.2 2-DE analysis

2-DE was conducted to assess the purity, quality andquantity of proteins extracted from apples and bananas byprotocol C. It gave well resolved apple proteins on small2-DE gels (1167 cm). Individual polypeptide spots wereeasily identified and showed little streaking. In apples, morethan 500 spots were detected (Fig. 2). Selected spots wereexcised from the gel and identified by LC-MS/MS (Table 2).Malate oxidoreductase (malic enzyme), UDP glucose pyro-phosphorylase and an unknown protein from Brassica oler-acea var. gemmifera were identified. These proteins corre-spond to spot numbers 1 to 3 respectively on Fig. 2.

2-DE gels of protein extracted from banana peel and fleshwith protocol C are shown in Fig. 3. Overall, there were 510and 394 polypeptide spots visualized in peel and fleshsamples, respectively. One protein that was common to allgels was excised and identified by LC-MS/MS as Rubisco(Table 2). Examples of MS/MS spectra for protein identifi-cation of peptides extracted from Spots No. 1 and 2 (Fig. 2)are shown in Figs. 4A and B.

4 Discussion

The extraction of high-quality protein from recalcitrant,low protein content plant tissue such as apple andbanana fruit is a well-known challenge. Until now, mostproteomic studies of plants have focused on youngvegetative tissues. Little information is available for fruittissue. It was our intent to develop and evaluate a proteinextraction protocol for apple fruit that provided proteinyields and quality suitable for 2-DE and MS analysis.

We compared two commonly used protein extractionprotocols (A and B) from [3] and [7] with a protocol (C) thatwe developed for apple fruit. Protocol C uses a heatedextraction buffer containing 2% SDS, 60 mM DTT,20% glycerol, and 40 mM Tris-HCl buffer (pH 8.5). SDS isone of the most widely used surfactants for protein solu-bilization [16, 17]. It has been reported that the high pro-tein binding capacity of SDS, particularly at an elevatedtemperature of 957C, improves the solubilization of mem-brane proteins and the recovery of soluble proteins [17].Hot SDS can also inhibit protease activity during cell dis-ruption and protein extraction, thus avoiding potentialartifacts [11, 18]. However, SDS has not been utilized inprotein extractions because it has an anionic charge thatmay cause proteins to precipitate in IEF gels [16, 17].

Therefore, we used heated SDS to increase the solubilityof proteins in the initial extraction procedure and thenreduced or eliminated the negative effects of SDS byprecipitating the proteins with a TCA/acetone precipita-tion procedure adapted from other reports [7, 19, 20]. Inaddition to removing SDS, TCA/acetone precipitation isbelieved to remove other interfering compounds such aspolyphenolics and polysaccharides. In our protocol, welimited the TCA/acetone precipitation procedure to45 min in order to minimize the negative effects of low pHon proteins caused by TCA. Finally, the TCA was removedby washing the protein pellet, at least three times, withice-cold acetone [1, 7]. Large amounts of protein wereobtained with protocol C with minimal variability (Table 1).

We found that similar yields of proteins could be obtainedwith TCA/acetone precipitation alone compared to SDSextraction combined with TCA/acetone. However, SDS-PAGE analysis revealed that TCA/acetone precipitationalone caused the loss of some low-molecular-weightproteins (Fig. 1B). The combination of SDS extractionwith TCA/acetone precipitation enhanced the overallquantity and size range of proteins present in the finalsample.

Both SDS-PAGE and 2-DE were used to validate qualita-tively and quantitatively the protein extraction protocols.A rehydration buffer [12] was used to dissolve all proteinsamples and seemed to improve protein quality and sol-ubility and reduce streaking in 2-DE gels. DeStreak™

rehydration solution was also found to satisfactorily dis-solve proteins prior to the rehydration of Immobiline™ IEFstrips. In addition, we found that there was no apparentloss of the protein quantity or quality after more than sixmonths of storage at 2807C in either DeStreak™ solutionor the rehydration buffer (data not shown). Ten micro-grams of fruit protein was loaded during rehydration onto11 cm IEF strips. Two IEF strips were subjected to 2-DEsimultaneously on a Multiphor™ II system followed by sil-ver staining to visualize the proteins. In our study, discreteprotein spots were detected upon 2-DE with goodrepeatability. Using 18 or 24 cm long Immobiline™ IEFdrystrips and large-format gels would potentially permitincreased protein loads, improve the separation resolu-tion on 2-DE gels and further increase the number of pro-tein spots that could be visualized.

The clear separation of a large number of protein spots withminimal streaking on 2-DE gels and the successful identifi-cation of protein spots by mass-spectra comparisons toprotein mass databases for apple and banana demon-strates that extraction protocol C was suitable to obtain thequantity and quality of proteins required for proteomic



Figure 3. 2-DE of banana protein extracts. Ten micrograms of protein from banana fruit was extracted with protocol C anddissolved in DeStreak™ solution. After isoelectrofocusing, the proteins were further separated by SDS-PAGE (8–16% polyacylamide gel) and visualized with silver stain. (A) Peel tissue. (B) Flesh. Arrows indicate ribulose bisphosphatecarboxylase identified by LC-MS/MS and listed in Table 2.

Table 2. Putative identification of protein spots in apple and banana fruit extracted with protocol C

Sample andspot No.

Protein identification GenbankAccession

Mascotscore

Match/% Cov.

Mr (kDa) pI Matched sequences

Exp. Theo. Exp. Theo.

Apple1 Malate oxidoreductase

(malic enzyme)(Arabidopsis thaliana)

gi)28059162 322 7/14 64.2 62.3 6.32 6.73 GKEYEDLLEEFOSAVK,LLNDEFYIGLR,ILGLGDLGBQGOGIPVGK

2 UDP glucose pyrophosphorylase(Pyrus pyrifolia)

gi)6136112 978 21/47 52.5 51.8 5.78 5.99 SDVASLSQISENEK,VLQLETAAGAAIR,ATSDLLLVQSD-LYTLQDGFVTR,LEIPDNAVIANK

3 Unknown(Brassica oleracea var.gemmifera)

gi)1469219 157 3/15 29.9 28.9 5.05 4.94 TVLVDNEDFLK,DPDGYTFELIQR,GNAYAQIAIGTDDVYK

Banana peel Ribulose bisphosphatecarboxylase large unit(Musa acuminata)

gi)20086560 250 6/13 53.2 48.3 6.5 6.3 AVYECLR,DTDILAAFR AQAETGEIK,LNYYTPDYEVK

% Cov: percent of coverage; Exp: experimental value; Theo: theoretical value found at NCBI.Protein spots excised from gels stained with silver subjected to digestion with trypsin and identified following MS analysis(LC-MS/MS).



Figure 4. Total ion chromatogram of a protein spot excised from a 2-DE gel, trypsin-digested and analyzed by LC-MS/MS.(A) Mass spectrum of peptide LLNDEFYIGLR with m/z 1351.71 from protein spot 1 in Fig. 2; (B) Mass spectrum of peptideVLQLETAAGAAIR with m/z 1311.83 from protein spot 2 in Fig. 2.



studies of ripening apples and bananas. Also, our proto-col may be suitable for other fruits with low protein con-tent and larger amounts of interfering contaminants.

Protein protocols utilizing phenol have been reported tobe suitable for the extraction of low concentrations ofprotein in vegetative plant tissues rich in componentswhich inhibit electrophoresis [8, 21]. Recently, classicalTCA/acetone precipitation and phenol extraction wereevaluated with banana, apple and potato plant tissuesand were considered to be useful as standard protocols[10]. Phenol extraction of protein from tomato and bananafruit was reported and found to be comparable to proteinprecipitation with acetone [22]. Further work is beingconducted to compare our hot SDS protein extractionprotocol with phenol protocols [21, 22] for protein extrac-tion from apple fruit and other fruit tissues.

We thank Dr. Greg Bezanson and Mr. Tim Ells for theircritical review of this manuscript. We are grateful toDr. David Byers and Mr. Elden Rowland, Proteomics CoreFacility, DalGEN Microbial Genomics Center, DalhousieUniversity for providing excellent ESI-MS/MS analysis.Contribution No. 2313 of the Atlantic Food & HorticultureResearch Centre, Agriculture & Agri-Food Canada.

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