(Ele 1999)advances in protein solubilisation for 2D electrophoresis

Advances in protein solubilisation for two-dimensional electrophoresisTwo-dimensional (2-D)electrophoresis remains the highest resolution technique for protein separation and is the method of choice when complex samples need to be arrayed for characterisation,as in proteomics.However,in current proteome projects the total number of proteins identified from 2-D gels is often only a small percentage of the predicted proteome.In addition,there is an almost complete lack of hydrophobic proteins on 2-D gels,especially those using immobilised pH gradients.Recently there have been a number of publications reporting reagents which improve protein solubili-sation prior to isoelectric focusing.The improved solubilization possible with these reagents has increased the total number of proteins able to be visualised on 2-D gels and also allowed the separation of hydrophobic proteins,such as integral membrane proteins.Keywords:Two-dimensional polyacrylamide gel electrophoresis /Membrane proteins /Solubility /Proteome /ReviewEL 3428ReviewBen HerbertProteome Systems Ltd.,North Ryde,Sydney,AustraliaContents1Introduction ........................6602Chaotropes ........................6613Surfactants ........................6614Reducing agents ....................6625Sequential extraction .................6626Concluding remarks (6637)References ...... (663)1IntroductionAs we reflect on the third Siena meeting it is appropriate to look at what progress has been made in the various proteomic technologies towards achieving the goals of proteomics,for example,to identify and characterise all proteins expressed by an organism or tissue [1].The scope of this review is sample preparation for 2-DE and I would like to focus on the advances that relate to en-hanced protein solubility and increased numbers of hydro-phobic proteins on 2-D gels.For a comprehensive review of many other aspects of sample preparation for electro-phoresis,see Rabilloud [2].Two-dimensional electro-phoresis (2-DE)remains the highest resolution method for arraying proteins prior to their characterisation by mass spectrometry;however,the number of proteins ac-tually identified on 2-D gels,even from species where the entire genome is sequenced,is very low.Wilkins et al.[3]reported that for three species,Bacillus subtilis,Escheri-chia coli and Saccharomyces cerevisiae ,with completely sequenced genomes,less than 5%of the proteins in SWISS-PROT had been identified on 2-D gels.In addi-tion,within the 5%of identified proteins,only between 5%for S.cerevisiae and 15%for E.coli were hydrophobic,compared to the theoretical hydrophobic contents of 16%and 29%,respectively.An initial appraisal of these figures may lead to the conclusion that 2-DE and proteomics is failing to deliver;however,there have been a number of recent advances to sample preparation and 2-D gel meth-odology [4±7]which are already allowing more proteins to be arrayed in micropreparative quantities.Pretreatment of samples for isoelectric focusing (IEF)in-volves solubilisation,denaturation and reduction to completely break the interactions between the proteins and to remove nonprotein sample components such as nucleic acids [2].Ideally,to avoid protein losses,one would achieve complete sample solubilisation in a single step and thus eliminate unnecessary handling,as is the case for soluble protein samples which can be readily tak-en up in the most commonly used IEF sample solution of 8M urea,4%3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),50±100m M dithiothreitolCorrespondence:Dr.Ben Herbert,Proteome Systems Ltd.,Locked Bag 2073,North Ryde,Sydney,NSW 1670,Australia E-mail:ben.herbert@ Fax:+61-2-9889-1805Abbreviations:ASB 14,amidosulfobetaine 14;GRAVY ,grand average of hydropathy;SB 3±10,N -decyl-N,N -dimethyl-3-ammo-nio-1-propane sulfonate;TBP ,tributyl phosphineWILEY-VCH Verlag GmbH,69451Weinheim,19990173-0835/99/0405-0660$17.50+.50/0(DTT)and40m M Tris.However,the`standard'IEF sam-ple solution is not ideal for many proteins and the chal-lenge for2-D PAGE,particularly in the context of proteo-mics,is the solubilisation and separation of insoluble samples such as membrane and membrane-associated proteins and proteins from highly resistant tissues like hair and skin.Enhanced protein solubility has been reported by introducing new reagents such as thiourea,sulfobe-taine surfactants and tributyl phosphine into the IEF sam-ple solution[4,5].To obtain a broad picture of a sample,the unfractionated proteins can be applied to a single2-D gel with a wide pH gradient,but with complex samples this approach only re-veals a small percentage of the anelle and plasma membrane fractions can be used to considerably reduce the complexity of cellular samples;however,they require considerable expertise and access to expensive equipment such as ultracentrifuges.In contrast,the sim-ple concept of sequentially extracting proteins by exploit-ing differential solubility has been used on many different tissues,usually with aqueous,organic solvent,and sur-factant-based extraction solutions[8±10].Recently Molloy et al.[6]have expanded on the sequential extraction ap-proach by incorporating highly solubilising conditions in the IEF sample solutions[4,5].Denaturing IEF sample solutions are made up of three main types of reagents, chaotropes,surfactants and reducing agents,and in the main part of this review I will summarise the advantages of some of the new reagents which constitute the en-hanced,highly solubilising IEF sample solution.2ChaotropesChaotropic agents such as urea allow proteins to unfold and thus expose their hydrophobic cores.This is a-chieved by changing the hydrogen bond structure in the solution,thus decreasing the energy penalty for contact of the hydrophobic residues with the solution[2,11].In practice,because the hydrophobic residues of proteins are exposed by urea denaturation it is normal to have sur-factants,such as CHAPS,present to aid in protein solubi-lisation.At the1996Siena meeting,and subsequently in a number of publications[4,12],T.Rabilloud has shown that proteins can be lost,by adsorption to the gel matrix, when IEF is conducted in immobilised pH gradients (IPGs).Rabilloud et al.[4]introduced the use of thiourea in combination with urea to increase the solubility of pro-teins in IPGs and showed that thiourea had a major posi-tive effect on the number of proteins visualised in the sec-ond dimension with four different subcellular fractions, including integral membrane proteins.Thiourea is an effi-cient chaotrope,although it is poorly soluble in water and requires high concentrations of urea for solubility,the opti-mal conditions being solutions of2M thiourea in5±7Murea.The enhanced protein solubility obtained by using thio-urea has also been demonstrated by Pasquali et al.[13],who showed improved2-DE of up to2mg of total mem-brane preparations and integral membrane preparationsfrom murine mammary epithelial cells.Fialka et al.[14] combined subcellular fractionation of murine mammaryepithelial cells with enhanced solubility using thiourea tocreate a range of organelle maps.They were able to sep-arate and identify some transmembrane proteins such asE-cadherin and caveolin.However,neither of these pro-teins would be classified as highly hydrophobic by thegrand average of hydropathy(GRAVY)measurementused by Wilkins et al.[3]to classify hydrophobic proteinsin B.subtilis,E.coli and S.cerevisiae.Although the useof thiourea/urea mixtures alone is a major advance in thesolubility of proteins for2-DE,the combination of these chaotrope mixtures with new sulfobetaine surfactants hasprovided a whole range of powerful sample solutions for2-DE.3SurfactantsAs stated in Section2it is normal to have at least one sur-factant present in the IEF sample solution to solubilise the hydrophobic residues that are exposed as a result of de-naturation in rge amounts of ionic substan-ces are not compatible with steady-state IEF;therefore,the use of SDS is not recommended and we are restrictedto nonionic or zwitterionic surfactants.It is possible to useSDS in the initial sample solubilisation and then dilute itout with a large excess of a nonionic surfactant[2];how-ever,obtaining sufficient dilution of the SDS can become impossible when micropreparative protein loads are re-quired.Traditionally,surfactants such as the Triton X-100and Nonidet P-40have been used,as well as sugar-based surfactants such as octyl glucoside[2,15].In re-cent years the sulfobetaine CHAPS has become the sur-factant of choice and is generally used at between2%and5%in8M urea.These commonly used surfactantsare soluble in high concentrations of urea,and thus itwould appear appropriate to add thiourea to the samplesolution and combine the enhanced chaotropic powerwith urea-soluble surfactants.However,while soluble,these surfactants are not efficient at protein solubilisationin high concentrations of chaotropes and their solubilisingpower is further minimised in the presence of highly cha-otropic thiourea[4,16].In contrast,sulfobetaines withlong linear alkyl tails such as N-decyl-N,N-dimethyl-3-am-monio-1-propane sulfonate(SB3±10)are more efficientthan CHAPS although they suffer from poor solubility inhigh concentrations of urea[17±19].Proteomicsand2-DEAs a compromise between chaotropic power and surfac-tant efficiency,Rabilloud et al.[4]suggested two IEF sample solution formulations which take advantage of the increased chaotropic power of thiourea.The first solution, 5M urea,2M thiourea,2%CHAPS and2%SB3±10is for proteins which require strong surfactants for solubility. The sulfobetaine SB3±10is not soluble in concentrations of urea greater than5M and thus the overall chaotrope concentration is relatively low.The second solution,7M urea,2M thiourea and4%CHAPS is for proteins which require a high concentration of chaotropes.To address the problem of surfactant-chaotrope compati-bility,Chevallet et al.[20]synthesised a range of novel sulfobetaines with more polar,but zwitterionic,head groups and long alkyl tails of more than12carbon atoms. The new surfactants were tested on membrane prepara-tions from bovine neutrophils and also on plasma mem-brane preparations from Arabidopsis thaliana.The most efficient surfactants were of the amidosulfobetaine type with either a linear alkyl tail,such as amidosulfobetaine 14(ASB14),or a mixed alkyl-aryl tail,such as C8F.The number refers to the number of carbon atoms in the tail. Because these surfactants have more polar head groups than sulfobetaines such as SB3±10they are soluble in high concentrations of urea,typically7±8M.When compared to the conventional IEF sample solution con-taining CHAPS,the new surfactants allowed the separa-tion and detection of many more membrane proteins. Specifically,C8F allowed the separation of some,quite hydrophobic proteins such as the PIP1isoform of a plas-ma membrane water channel protein from A.thaliana [20].PIP1has a GRAVY score of0.37,which rates it as one of the most hydrophobic proteins ever detected on2-D gels.In the case of the bovine neutrophil membranes the ASB14surfactant was highly efficient and resulted in many more proteins being visualised,including stomatin, a transmembrane protein[20].4Reducing agentsAs discussed above,reagents such as urea and surfac-tants are used to denature proteins and expose their hy-drophobic cores;however,to allow complete unfolding of many proteins it is necessary to reduce disulfide bonds. Reduction is usually achieved with a free-thiol-containing reducing agent such as b-mercaptoethanol or dithiothrei-tol(DTT)[2].However,free-thiol-containing reagents such as DTT are charged,especially at alkaline pH,and thus migrate out of the pH gradient during the IEF,which results in a loss of solubility for some proteins,especially those which are prone to interation by disulfide bonding, such as the keratins and keratin-associated proteins from hair and wool.Herbert et al.[5]replaced the thiol-contain-ing reducing agent DTT with an uncharged reducing agent,tributyl phosphine(TBP),which greatly enhanced protein solubility during the IEF and resulted in increased transfer to the second dimension with a range of samples including wool proteins.A further advantage of the mech-anism of phosphine reduction is that because phosphines do not contain a thiol they cannot be alkylated,which leads to a simplified IPG equilibration protocol incorporat-ing reduction and alkylation in a single ing an im-proved single-step IPG equilibration protocol[5],it is possible to combine TBP and an alkylating agent and ob-tain complete alkylation of cysteine.Because thiourea, sulfobetaine surfactants and TBP increase protein solubil-ity through different routes,they can be seen as comple-mentary reagents.Thiourea increases the chaotropic power of the sample solution,sulfobetaines such as SB 3±10,ASB14and C8F are efficient surfactants,and TBP ensures complete reducing conditions during the IEF; thus it is advantageous to combine all three in an en-hanced IEF sample solution.5Sequential extractionBy combining thiourea,sulfobetaines such as SB3±10 and the uncharged reducing agent TBP,it is possible to create a powerful IEF sample solution and thus solubilise proteins which would remain insoluble in conventional IEF sample solutions.However,the drawback of this en-hanced solubility is that for complex samples the2-D gels become swamped with overlapping proteins,especially when high loads are applied for micropreparative purpos-es.Molloy et al.[6]have adapted the concept of differen-tial solubility[8±10]by incorporating the enhanced solubi-lising conditions as the final step of a sequential extraction of E.coli.The first step is cell lysis and protein extraction using Tris base.The resulting pellet is extracted using conventional IEF sample solution,8M urea,4%CHAPS and DTT and the pellet remaining after this extraction is rich in membrane proteins and represents only11%w/w of the starting material.The final extraction is with5M urea,2M thiourea,2%CHAPS,2%SB3±10and2m M TBP.Eleven membrane proteins were identified on the 2-D gel from the final extraction,representing many of the outer membrane proteins of E.coli.Five of the identified proteins had not previously been identified from2-D gels and two of these were only previously known as open reading frames.It appears that many of the outer mem-brane proteins(OMPs)from E.coli were partitioned into the final pellet in a highly enriched state and were solubi-lised in the enhanced solubilising solution.Note that while the proteins in the final extract are clearly insoluble in conventional IEF solutions they are not rated as hydrophobic according to the GRAVY scale[3,6],asthey all have negative GRAVY scores.It seems that at the extremes of the GRAVY scale the values are predic-tive of protein solubility because the protein is mainly composed of either hydrophilic or hydrophobic residues. However,in the mid ranges of the GRAVY scale it is quite possible to have hydrophilic proteins,as measured by GRAVY,which are insoluble in the conventional IEF sam-ple solutions[6].The insolubility of a`hydrophilic'protein may be due to the presence of hydrophobic,possibly transmembrane,domains that are,on average,out-weighed by the majority of hydrophilic residues.Given the efficiency of some of the novel sulfobetaine sur-factants synthesised by T.Rabilloud et al.[20]it will be in-teresting to repeat the sequential extraction of Molloy et al.[6]using the new surfactants in the final step and look for more hydrophobic proteins.In another study,Molloy et al.[21]have used the novel sulfobetaine ASB14[20]as part of an enhanced solubilising solution to separate pro-teins extracted from E.coli with mixtures of chloroform/ methanol.The ASB14solution gave greater protein solu-bility,compared to a solution using SB3±10and CHAPS, probably because of the increased efficiency of the ASB 14and the fact that7M urea can be used with ASB14, compared with only5M urea with SB3±10.Out of the thir-teen proteins identified,eight had not been previously identified on2-D gels and five of these new proteins had positive GRAVY scores and are thus rated as hydropho-bic.6Concluding remarksThe past few years have seen2-DE undergo a revitalisa-tion which is strongly linked to the interest in proteomics. The high throughput nature of mass spectrometry,espe-cially MALDI-MS for peptide mass fingerprinting,has placed a large demand on2-DE to deliver large numbers of micropreparative gels of`unknown'proteins.It is clear that conventional technology,using urea,CHAPS,and DTT solubilisation on a single broad-range pH gradient is becoming exhausted.The same proteins are solubilised and identified many times,leaving behind the majority of the proteome,either insoluble or hidden behind the abun-dant proteins on a broad pH range2-D gel.The work summarised in this review has made significant progress on the protein solubility issue and has allowed many pre-viously insoluble proteins to be separated by2-DE.In ad-dition,the sequential extraction approach has proved to be a rapid and effective method of partitioning and con-centrating insoluble proteins such as membrane proteins.A combination of organelle fractionation with sequential extraction will prove to be very powerful.For example,se-quential extraction of purified organelles will provide cellu-lar localisation data as well as simplifying the2-D patterns by separating the soluble fraction from the membrane fraction.The next steps in the development of2-DE for proteomics will be continued refinements to prefractiona-tion and enhanced protein solubility,combined with2-DE using multiple pH gradients to cover the expected p I range of most proteomes.Received February17,19997References[1]Wilkins,M.R.,Sanchez,J.-C.,Gooley,A.A.,Appel,R.D.,Humphery-Smith,I.,Hochstrasser,D.F.,Williams,K.L., Biotechnol.Genet.Eng.Rev.1995,13,19±50.[2]Rabilloud,T.,Electrophoresis1996,17,813±829.[3]Wilkins,M.R.,Gasteiger,E.,Sanchez,J.-C.,Bairoch,A.,Hochstrasser,D.F.,Electrophoresis1998,19,1501±1505.[4]Rabilloud,T.,Adessi,C.,Giraudel,A.,Lunardi,J.,Electro-phoresis1997,18,307±316.[5]Herbert,B.R.,Molloy,M.P.,Walsh,B.J.,Gooley,A.A.,Bryson,W.G.,Williams,K.L.,Electrophoresis1998,19, 845±851.[6]Molloy,M.P.,Herbert,B.R.,Walsh,B.J.,Tyler,M.I.,Traini,M.,Sanchez,J.-C.,Hochstrasser,D.F.,Williams,K.L.,Gooley,A.A.,Electrophoresis1998,19,837±844. 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