high-resolution two-dimensional electrophoresis separation of proteins from metal-stressed rice...

Electrophoresis 2014, 00, 1–10 1

Florian Weiland1

Carla M. Zammit2

Frank Reith3

Peter Hoffmann1

1Adelaide Proteomics Centre,University of Adelaide,Adelaide, Australia

2Earth Sciences, Universityof Queensland, Brisbane,Australia

3School of Earth andEnvironmental Sciences,University of Adelaide,Adelaide, Australia

Received February 4, 2014Revised February 27, 2014Accepted March 7, 2014

Research Article

High resolution two-dimensionalelectrophoresis of native proteins

Blue native PAGE (BN-PAGE) is a powerful method to separate protein complexes whilepreserving their native state. However, the resolution of the method is limited as complexeswith similar molecular masses cannot be resolved. Here we describe native 2DE usingimmobilized pH-gradients in combination with BN-PAGE to resolve protein complexes bytheir pI and molecular mass. This method enables electrophoretic separation of proteinsbetween pI 3 and 10 and can resolve molecular masses up to 1.2 MDa. Visualized gel spotsat large molecular weight were identified using MS to confirm potential protein complexes.Several protein complexes could be identified, most prominent GroEL in complex withGroES, parts of the ribosomal machinery and membrane transport system. In summary,this method enables easy high-resolution electrophoretic separation of protein complexes.

Keywords:

BN-PAGE / 2DE / IPGs / Native DOI 10.1002/elps.201400060

1 Introduction

The detection and identification of protein complexes in theirnative state is one of the major challenges in Proteomics [1,2].The most widely used method for the analysis of oligomericprotein states is blue-native PAGE (BN-PAGE) (reviewedin [1, 3]). However, a major drawback of BN-PAGE is thelimitation of the molecular mass being the only dimension ofseparation. This results in difficulties to quantify changes inthese complexes due to the colocalization of similar masses.2DE allows the simultaneous resolution of thousands of pro-teins on one gel which overcomes these quantification dif-ficulties [4]. The application of native 2D approaches usingIEF as a first and BN-PAGE as a second dimension havebeen described previously. However, these approaches werelimited to the usage of carrier ampholytes to establish thepH-gradient [5] which results in pH-gradient instability overtime or could only resolve a limited number of proteins [6].Here we describe an improved method for native IEF in IPGsin combination with BN-PAGE. Applying this method, wewere able to achieve highly resolved native 2D gels whereprotein complexes and their charge isoforms between a pI of3 and 10 and with a molecular mass of up to 1.2 MDa can bevisualized.

Correspondence: Dr. Peter Hoffmann, Adelaide Proteomics Cen-tre, The University of Adelaide, Adelaide, South Australia 5005,AustraliaE-mail: [email protected]: +61-8-8313-4362

Abbreviations: BN-PAGE, blue native PAGE; emPAI, ex-ponentially modified protein abundance index; FDC-P1,factor-dependent continuous cell line, Paterson Laborato-ries 1; PPI, protein–protein interactions; TRAP, tripartite ATP-independent periplasmic

2 Materials and methods

2.1 Reagents and samples

IPG strips 3–10 NL (11 cm) and acrylamide were purchasedfrom Bio-rad (Hercules, CA, USA). EZQ protein quantifi-cation kit, Novex Sharp prestained and NativeMark un-stained were purchased from Life Technologies (Carlsbad,CA, USA). Protease inhibitor cocktail (P8340), tricine, im-idazole, sodium thiosulphate, formaldehyde, Tris-bufferedmineral salts medium, sodium gluconate, and Coomassiebrilliant blue G-250 were obtained from Sigma-Aldrich (St.Louis, MO, USA). Urea, sodium chloride, hydrochloric acid,ethanol, methanol, silver nitrate, sodium carbonate, andacetic acid were obtained from Merck (Darmstadt, Germany).CHAPS and PSC Protector reagent were purchased fromRoche (Basel, Switzerland). Pharmalyte 3–10, ammoniumpersulfate, thiourea, coverfluid, glycerol, and TEMED wereobtained from GE Healthcare LS (Uppsala, Sweden). Lowmelt agarose was from Probiogen (Berlin, Germany). Glycinewas purchased from AMRESCO (Solon, OH, USA). 2,2-Dithiodiethanol and 2-butanol were purchased from Fluka(St. Louis, MO, USA). PBS was obtained from SA Pathology(Adelaide, SA, Australia). Rapid Fixer was purchased fromAgfa (Mortsel, Belgium).

Cupriavidus metallidurans CH34 was obtained fromDeutsche Sammlung von Mikroorganismen und Zellkul-turen and grown in Tris-buffered mineral salts medium [7]containing 2 g/L sodium gluconate at 30°C with shaking at120 rpm. C. metallidurans CH34 was initially grown on solidmedia and a single colony was picked and grown in liquid me-dia, until the completion of the exponential phase of growth.In 150 mL Erlenmeyer flasks, 10 �L of culture was addedto 40 mL of fresh media and grown to the beginning of theexponential phase. Cultures were left to grow until the end

C© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

2 F. Weiland et al. Electrophoresis 2014, 00, 1–10

of the exponential phase and the cells were harvested by cen-trifugation at 10 000 rpm for 10 min at 4°C. Cells were thenwashed three times with PBS (137 mM NaCl, 2.7 mM KCl,10 mM Na2HPO4, 2 mM KH2PO4) and stored at −80°C.

Factor-dependent continuous cell line, Paterson Labora-tories 1 (FDC-P1) [8] was grown from a 1 × 105 cell per micro-liter starting culture in 10% fetal calf serum supplementedwith mouse interleukin-3, 5% CO2 atmosphere at 37°C for2 days, the cell count was maintained between 1 × 105 and1 × 106 cells per microliter. Cells were harvested by centrifu-gation with 400 × g for 5 min at room-temperature (RT). Thecell pellet was washed twice with ice-cold PBS and stored at−80°C until further use.

2.2 Protein extraction

Cell pellets were resuspended in 200 �L extraction buffer(96.9% H2O, 1.1% PSC protector reagent, 1% CHAPS, 1%protease inhibitor cocktail) and cell lysis was carried out usinga Bioruptor UDC-200 using the following settings: Output:high, Sonication: 30 s, Pause: 60 s for 7.5 min with a subse-quent exchange of the ice-cold water in the ultrasonic bathand a second cycle of 7.5 min. Cell debris was centrifugeddown at 20 000 × g for 30 min at 4°C. The supernatant wasaliquoted and the protein concentration was determined byan EZQ protein quantification kit according to the manufac-turer’s recommendations. The remaining protein extract wasstored at −80°C until further use.

2.3 Native IEF

IPG strips 3–10 NL (11 cm) were rehydrated overnight in200 �L native rehydration solution (97.5% H2O, 1% pro-tease inhibitor cocktail, 1% CHAPS, 0.5% Pharmalyte 3–10)at room-temperature. Three-hundred micrograms protein ofC. metallidurans CH34 and 500 �g protein of FDC-P1 werefilled up to a volume of 98 �L with extraction buffer and2 �L Pharmalyte 3–10 were added. Samples were appliedby anodal cup-loading. IEF was performed at 15°C using anIPGphor II (GE Healthcare) with following parameters: 90 Vfor 2 h, 180 V for 2 h, 360 V for 2.5 h, ramp-up to 8000 V for1.5 h, 8000 V for 27 000 Vh. After the last step, the voltagewas set to 180 V until the strips were taken off. The currentwas limited to 50 �A per strip.

2.4. Denaturing IEF

The same methodology as under Section 2.3 was applied, de-viating in the use of 200 �L denaturing rehydration solution(6 M urea, 2 M thiourea, 1% CHAPS, 0.5% Pharmalyte 3–10, and 200 mM 2,2-dithiodiethanol) as rehydration buffer.Samples were mixed with denaturing sample buffer (7 Murea, 2 M thiourea, 4% CHAPS) to a total volume of 98 �L todenature the proteins, before IEF 2 �L Pharmalyte 3–10 were

added. IEF was performed at 20°C. All other conditions werethe same as in Section 2.3.

2.5 Equilibration after native and denaturing IEF

IPG strips were incubated in 4 mL equilibration solution(50 mM NaCl, 50 mM imidazole, 10 mM CHAPS, 30% glyc-erol, 0.125% Coomassie brilliant blue G-250, pH 7.0) for30 min on an orbital shaker at 40 rpm. BN-PAGE was con-ducted immediately afterwards.

2.6 BN-PAGE gel casting

15 × 16 × 1 mm3 polyacrylamide gradient gels (T = 4–16%)incorporating a 15 mM imidazole, pH 7.0 buffer system [9]under omission of 6-aminohexanoic acid were cast. Imme-diately afterwards, the gels were overlayed with 1 mL of a2 + 3 mixture of gel buffer and 2-butanol and polymerizedovernight at RT.

2.7 BN-PAGE

Gel buffer and 2-butanol mixture was removed from theheadspace of the cassette and the gel edge was washed withH2O. IPG strips were applied onto the upper edge of thegel. Five microliters NativeMark or Novex sharp prestainedmaker, respectively, was mixed with 1 �L 95% H2O, 5% w/vCoomassie brilliant blue G-250 and 4 �L cathode buffer B/10(50 mM tricine, 7.5 mM imidazole, 0.002% Coomassie bril-liant blue G-250, pH 7.0) [9] and applied using a filter paperplaced on the cathodal side of the IPG strip. For 1D BN-PAGEof the two samples, 2 �L of total lysate of the respective cellline was mixed with 2 �L cathode buffer B/10 and 2 �L 95%H2O, 5% w/v Coomassie brilliant blue G-250 solution andapplied on a filter paper. The strip and filter paper(s) wereoverlayed with 1% agarose in cathode buffer B/10 which wascooled down to RT beforehand. Electrophoresis was carriedout using a SE 600 Ruby System (Hoefer) employing an-ode buffer (25 mM imidazole, pH adjusted to 7.0 with HCl)and cathode buffer B/10 [9]. Following settings were applied:100 V for 30 min, 500 V for 4 h, the current was limited to15 mA per gel; the temperature was set to 4°C.

2.8 Protein visualization

After BN-PAGE, the proteins were fixed into the gels using40% ethanol, 10% acetic acid and placed on an orbital shakerat RT overnight. The next day the proteins were visualizedusing the silver stain protocol according to Blum et al. [10].

2.9 Protein identification

Proteins were cut out manually, washed in 400 �L 50 mMammonium bicarbonate and destained using Rapid Fixer


Electrophoresis 2014, 00, 1–10 Proteomics and 2DE 3

Figure 1. Reproducibility ofnative 2D electrophoresis:500 �g of FDC-P1 total celllysate was separated usingIPG 3–10 NL and BN-PAGE(T = 4–16%). A, B, C indicatethe three replicate gels. Silverstain.

under vortex. Afterwards, the gel pieces were washed repeat-edly in 400 �L 50 mM ammonium bicarbonate until theyappeared clear. Subsequently the proteins were digested us-ing trypsin and analyzed on a nano-flow HPLC coupled LTQOrbitrap XL mass spectrometer (Thermo) following previ-ously published protocols [11]. Deviating from the protocolmentioned in [11] the protein searches were conducted usingdifferent databases: C. metallidurans was searched against theNCBInr_20130423 database, with the search space definedas “Cupriavidus metallidurans and mammals,” samples fromFDC-P1 were searched against proteins classified as mam-malian in the SwissProt_2013_04 database.

3 Results

Two model systems to test the feasibility of the native 2DEsetup were used. C. metallidurans CH34 (previously namedRalstonia metallidurans [12], Ralstonia eutropha and Alcali-genes eutrophus [13]) was chosen as the prokaryotic model.This bacterium is Gram-negative and plays a vital role in

Figure 2. Native 2D of C. metallidurans CH34. 300 �g protein load,IPG 3–10NL, BN-PAGE (T = 4–16%). S: BN-PAGE of 12 �g C. metal-lidurans CH34. M depicts the native molecular mass marker lanewith the corresponding masses noted on the right side. Numbers1–19 indicate the protein spots picked for identification by MS(see Table 1).

the formation of secondary bacterial gold grains [14]. FDC-P1 was chosen as the eukaryotic model system and is ahemopoietic precursor cell line established from long termcultures of mouse bone marrow DBA/2 cells grown in WEHI-3 (myelomonoeytic leukemia cell line) conditioned medium-supplemented medium [8].

First, three native 2D gels using 500 �g FDC-P1 totallysate as sample, were produced to test the reproducibilityof the method (Fig. 1). Proteins with a pI of 3–10 could berepeatedly resolved in all three gels without major changes inthe spot pattern.

Figures 2 and 3 show that proteins from C. metalliduransCH34 with a pI between 3 and 10 could be resolved withmolecular masses up to 1.2 MDa. The possibility that theseobserved masses are artefacts of the BN-PAGE due to incom-patibility of the proteins or an effect of slow migration in thegel was tested by 2D gels combining denaturing IEF withBN-PAGE. This resulted in a clear change of the mass distri-bution of the majority of spots from 60 and 480 kDa, towardlower molecular masses between 20 and 240 kDa (see Fig. 4).

Figure 3. Native 2D of FDC-P1. 500 �g protein load, IPG 3–10NL,BN-PAGE (T = 4–16%). S: BN-PAGE of 32 �g FDC-P1. M depictsthe native molecular mass marker lane with the correspondingmasses noted on the right side. Numbers 1–15 indicate the pro-tein spots picked for identification by MS (see Table 2).



This change of the protein pattern in the molecular mass di-mension was expected in a comparison between native anddenaturing electrophoresis (discussed below) and is evidencefor the ability of the method preserving protein complexes.

The identity of the spots on the native gel as proteinswas tested by subsequent excision and mass-spectrometricidentification of 19 spots (see Fig. 2). The majority of resultsin Table 1 show several protein identifications per spot withtheir theoretical molecular masses an order of magnitudebelow the molecular mass of the corresponding marker onthe gel. The identified proteins belong mostly to the mem-brane transport system (Spots 2, 3, 11, 15, 16, 18, and 19,Fig. 2, Table 1), the ribosomal machinery (Spots 3, 11, and15, Fig. 2, Table 1), GroEL in complex with itself (Spots 7,10–13, Fig. 2, Table 1) and in complex with GroES (Spots 8and 9, Fig. 2, Table 1).

The native 2D setup was also tested using a eukaryoticcell line (FDC-P1, 500 �g). As shown in Fig. 3, a set of proteinswith similar attributes in terms of pI range as in the prokary-otic sample could be resolved. The molecular mass range ofthe majority of spots in the eukaryotic sample was expectedlywider (60–720 kDa) than in the prokaryotic sample. Fifteenspots from the gel shown in Fig. 3 were excised and thecontained proteins identified by MS (see Table 2).Prominentidentified colocalized proteins include ferritin heavy and lightchain, plectin in conjunction with actin, the actin-related pro-tein complex 2/3 and lactate dehydrogenase coresolved withactin.

4 Discussion

The reproducibility of the native 2D system was tested by run-ning three gels of the same samples (FDC-P1) under compa-rable conditions. As evident in Fig. 1, the major features ofthe three gels are identical. Minor gel to gel variations anddifferences in staining intensity are visible, both common in2DE [15]. As there were no major deviations between the gels,the reproducibility was deemed satisfactory.

The molecular weights of the identified proteins in spots7–13 using C. metallidurans CH34 as sample (Fig. 2) are

evidence that the method described here enables native elec-trophoretic separation. In these spots GroEL has been identi-fied, which forms a complex of 14 subunits and was isolatedpreviously as a 800 kDa complex [16]. The molecular massof GroEL on the 2D gel and its mass as a 14mer complexcorrespond (see Fig. 2, Spots 7–13). Spots 8 and 9 contain ad-ditionally GroES, which cooperates with GroEL resulting insubsequent ATP hydrolysis and formation of a stable GroEL-7ADP-GroES complex [16–20]. The sole identification of GroESin spot 6 indicates either an artefact or the presence of further,unidentified proteins.

In Spot 2 (see Fig. 2) further proteins of C. metallidu-rans CH34 at a molecular mass of 1.2 MDa were identified.Rmet_3665 has a predicted yscS-like domain [21], which ispart of the type III secretion system [22] and homologue toBacillus subtilis and Salmonella typhimurium proteins requiredin formation of the rivet portion in flagellar complex assembly[23,24]. The second protein which was identified is a tripartiteATP-independent periplasmic (TRAP) type transport compo-nent. These transporters mediate an ATP-independent up-take of organic acids by employing a substrate binding protein[25,26]. A further protein is membrane fusion protein HmvB,which has an identical sequence to “Heavy metal cation tri-component efflux, membrane fusion protein HmvB (CzcB-like).” CzcB is a cadmium, zinc cobalt resistance protein andis involved in the efflux of heavy metal cations across the cellmembrane [27]. Additionally, a putative heat shock protein(HslJ-like) was identified. HslJ proteins are contrary to theirnomenclature not induced by heat stress, but appear to be in-volved in the resistance to novobiocin in Escherichia coli [28].Together these proteins appear to be involved in the mem-brane transport system. Reflecting the molecular mass of1.2 MDa where these proteins have been resolved, an interac-tion of these proteins and/or formation of protein complexesis likely.

Proteins identified in Spot 3 (Fig. 2) at a molecular massof around 1 MDa are involved in the ribosomal machinery.Trigger factor binds to the 50S ribosomal protein in E. coli [29],of which the respective C. metallidurans protein has beenidentified as well in this spot. Additionally, trigger factor wasidentified in Spot 15. Complex formation of this protein with

Figure 4. Comparison of na-tive 2DE (A) and combina-tion of denaturing IEF andBN-PAGE (B) using C. met-allidurans CH34. Three hun-dred micrograms protein load,IPG 3–10NL, BN-PAGE (T = 4–16%). (A): S: 1D BN-PAGE of C.metallidurans, (A + B): M: Na-tive molecular mass marker.(B): *, **, ***: Denaturedprotein mass markers (Novexsharp prestained): 260, 160,and 110 kDa, respectively.


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Electrophoresis 2014, 00, 1–10 Proteomics and 2DE 7Ta

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Electrophoresis 2014, 00, 1–10 Proteomics and 2DE 9

30S ribosomal protein S7, which is colocalized in this spot,has been shown previously and the usage of trigger factor asmeans to aid assembly of protein complexes has been sug-gested [30]. Together with the identification of the nucleosidediphosphate kinase in this spot, which provides GTP for pro-tein synthesis [31], this indicates the identification of a partof the ribosomal machinery.

Malic enzyme was identified in Spot 14 (Fig. 2). This pro-tein forms under native conditions a homotetramer [32, 33]which sums up to a molecular mass of 340 kDa; howeverthe observed molecular weight on the gel is around double(slightly below the 720 kDa marker). This indicates a homooc-tamer formation of this protein. If this is a native conditionspecific for this bacterium, an increase in mass caused byfurther unidentified proteins within this spot or an artefactdue to dye-binding to the complex and subsequent bias theelectrophoretic behavior of the protein [34–36] cannot be de-termined based on the conducted analysis and would needto be further investigated using, for example, density gradi-ent centrifugation. However, the accuracy of the molecularmass estimation of protein complexes using BN-PAGE wasconfirmed previously [35] and therefore the resolved mass islikely to be correct.

The native 2D setup was further tested using the eu-karyotic model system. In the native 2D gel using FDC-P1as sample (see Fig. 3), the following proteins were identi-fied: Spot 2 in Fig. 3 contains ferritin light and heavy chain.Ferritin is a complex consisting of 24 subunits of light andheavy chain proteins [37] and is an iron-storage protein. Themass of the bound iron can reach up to 60% of the ferritinmass [37], which may explain the variable molecular mass ofthe protein complex as seen on the 2D gel. However, the low-est molecular mass of around 500 kDa of the protein complexcorresponds to the mass of 420 ± 40 kDa described in theliterature [37, 38].

An isoform of the putative ferritin complex was iden-tified in Spot 3 which is located between the 480 and720 kDa marker. Additionally, plectin and actin were iden-tified in this spot, which have been shown to noncovalentlylink with each other and are involved in the cross-linkingof the intermediate filaments of the cytoskeleton [39, 40].This, too, gives evidence for the native environment of thehere described electrophoresis system. If this colocalizationof all twelve identified proteins in Spot 3 is due to ac-tual interaction with each other or to insufficient resolu-tion of the wide pH-gradient would have to be investigatedfurther.

Spot 7 contains proteins of the actin related protein 2/3complex. This complex is responsible for the growth of actinfilaments, consists of seven proteins and is not stable underdenaturing conditions [41]. The here derived molecular massof approximately 700 kDa is nearly three times the experi-mentally established mass of 220 kDa [42]. However, as seenin Fig. 3, the protein spot exhibits a vertical streaking with astarting point at approximately 300 to 400 kDa which is closeto the established mass. As mentioned above, the reasons forthis variable mass would need further investigation.

The proteins identified in Spot 9, lactate dehydrogenase,and actin, have been reported previously to interact with eachother in rabbit muscle, leading to conformational changes inF-actin [43].

Apart from colocalization of proteins known to inter-act, further evidence for a separation system keeping proteincomplexes intact would be the yield of a 2D protein spotpattern with an increased spot number in the high molecularweight range as in comparison with a denaturing system. Thiswould be due to the preservation of protein–protein interac-tions (PPI) and protein complexes and the resulting changein molecular mass in the native system. As seen in Fig. 4,the combination of a denaturing IEF and BN-PAGE led to analtered distribution of the proteins with the majority of pro-tein spots shifted toward the lower molecular mass region ofthe gel. Prominent high molecular mass protein complexessuch as GroEL are missing and no protein spot has a molec-ular mass of over 720 kDa. A denatured protein marker withknown molecular masses was used in comparison to nativemarkers to investigate possible changes in migration behaviorof denatured proteins in BN-PAGE. As seen from Fig. 4B, themolecular masses between the denatured and native markercorrespond, indicating no changes in protein migration be-havior. Additionally, Fig. 2 shows a 1D BN-PAGE band of thesame C. metallidurans CH34 sample as used for the 2D gel.The molecular mass of major protein bands correspond be-tween the 1D BN-PAGE and prominent protein spots in thecombination of native IEF and BN-PAGE. This is evidence forthe preservation of native conditions while IEF and equilibra-tion using the here described setup. In Fig. 3 a 1D BN-PAGElane was also applied, but due to the too high amount of pro-tein loaded (32 �g) for silver stain, no distinct features can beobserved.

In summary, the obtained results demonstrate that thepresented method for native 2DE is suitable to separate pro-teins while preserving PPI and protein complexes. Further-more, the methodology is easy to use and the cost is in thesame range as traditional, denaturing 2DE with no need toinvest into additional equipment. Drawbacks of the methodare the required optimization of detergent and Coomassiebrilliant blue concentrations to preserve the PPI and proteincomplexes, which is depending on the entities to study, butthis problem is inherent to BN-PAGE as well. The influenceof the pH and the high electrical fields used for IEF onto thestability of protein complexes and PPI would need furtherinvestigation as disruptive qualities cannot be excluded. Ad-ditionally, protein complexes up to 10 MDa, as in BN-PAGEusing gradient gels with a starting T-value of 3%, cannotbe resolved in the here described native 2DE setup, as IPGstrips classically have a T-Value of 4.5 and 5% [44, 45]. De-spite these drawbacks, the ability to additionally resolve pro-tein complexes by their charge offers the possibility to studycomplexes in which exchanges/modifications of their con-stituents do not significantly alter the molecular weight ofthe complex but their pI. This gain in resolution allows theseparation of singular protein complexes and therefore easyquantification of changes in the complexosome.



The authors would like to thank the support of theAustralian Research Council; The Commonwealth Scientific andIndustrial Research Organization; The University of Adelaide;Manuela Klingler-Hoffmann for providing the FDC-P1 cell lineand Michelle Hooi for the help in conducting the mass spectro-metric analysis.

The authors have declared no conflict of interest.

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high-resolution two-dimensional electrophoresis separation of proteins from metal-stressed rice...

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