A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometryThe growing availability of genomic sequence information,together with improvements in analytical methodology,have enabled high throughput,high sensitivity protein identi-fication.Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE).Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis.We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne;Amersham Pharmacia Biotech,Amersham,UK).The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods.Omitting the sensitizing reagent allows higher loading without saturation,which facilitates protein identification and quantita-tion.We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization.We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.Keywords:Proteomics /Two-dimensional gel electrophoresis /Silver stain /Mass spectrometry /Protein identification /Matrix assisted laser desorption/ionization ±time of flight /Electrospray ionization ±time of flightEL 4190Jun X.Yan 1Robin Wait 2Tom Berkelman 3Rachel A.Harry 1Jules A.Westbrook 1Colin H.Wheeler 1Michael J.Dunn 11Department of Cardiothoracic Surgery,National Heart and Lung Institute,ImperialCollege School of Medicine,Heart Science Center,Harefield Hospital,Harefield,Middlesex,UK 2Kennedy Institute ofRheumatology,Hammersmith,London,UK 3Amersham Pharmacia Biotech,San Francisco,CA,USAThe increasing availability of genomic sequence informa-tion,together with improvements in protein characteriza-tion by mass spectrometry,have facilitated huge in-creases in the throughput of protein identification.Most commonly,sample components are separated by two-dimensional gel electrophoresis (2-D PAGE)and protein spots are visualized by staining silver,Coomassie blue or SYPRO fluorescent dyes [1±3].Individual spots are then excised from the gel,proteolytically digested,and the masses of the resulting peptides are determined by matrix assisted laser desorption/ionization ±time of flight ±mass spectrometry (MALDI-TOF-MS).The list of peptide masses thus obtained can then be used as a highly spe-cific query to interrogate a protein database [4±9].Recent advances in tandem electrospray ionization-mass spec-trometry (ESI-MS/MS),particularly the development of hybrid quadrupole /orthogonal acceleration TOF instru-ments (Q-TOF),enable routine de novo sequencing of low femtomole levels of peptides [10±13].The ability to determine 10±20amino acid lengths of sequence greatly facilitates cross-species protein identification and retrieval of homologous proteins from genomic and EST data-bases,even when an exactly matching sequence is not present.It is desirable to visualize protein spots in the gel at sensitivities which are roughly comparable to those of the subsequent MALDI-and ESI-MS analyses (usually in the range of nanograms per protein spot).Silver staining has been widely used for this purpose [14,15]since it re-quires relatively inexpensive equipment and reagents and remains one of the most sensitive methods for perma-nently staining proteins in polyacrylamide gels.For protein silver staining,a polyacrylamide gel is soaked in a solution containing soluble silver ions (Ag +)and sub-sequently developed by treatment with a reductant.Pro-tein molecules in the gel promote the reduction of silver ions to metallic silver (Ag 0),which is insoluble and visible.Initial deposition of metallic silver promotes further depo-sition by an autocatalytic process,resulting in exception-ally high sensitivity.There are many published versions of the silver staining process [16,17],which may incorpo-rate,in addition to silver impregnation and development,fixation steps,incubations with sensitivity enhancers (e.g.,glutaraldehyde or formaldehyde),stopping and preservation,and washing steps.The reagents used vary,but the silver reductant is always formaldehyde.The high sensitivity of silver staining comes at the cost of sus-ceptibility to interference from a variety of scources.Correspondence:Dr.Jun X.Yan,Heart Science Center,Hare-field Hospital,Hill End Road,Harefield,Middlesex,UB96JH,UK E-mail:jun.yan@ Fax:+44-(0)1895-828-9003666Electrophoresis 2000,21,3666±3672WILEY-VCH Verlag GmbH,69451Weinheim,20000173-0835/00/1717-3666$17.50+.50/0Exceptional cleanliness must therefore be practiced and reagent and water quality are critical.Silver staining pro-tocols have been developed specifically for visualizingproteins prior to in-gel digestion and mass spectrometric analysis [14,18].Subsequent MS constrains the choice of reagents that can be used during silver staining,because the proteins in the gel must not be chemically modified.Thus many common sensitization reagents (e.g.,glutaraldehyde and strong oxidizing agents)cannot be employed.Since silver staining is a multistep process utilizing numerous reagents,the quality of which is critical,it is often advantageous to purchase a dedicated kit in which the reagents are quality-assured specifically for sil-ver staining.We report here a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne;Amersham Pharmacia Biotech)and we show that it is compatible with subsequent in-gel digestion,MALDI,and ESI analysis.The method is based on that of Heukes-hoven and Dernick [19],but omits the use of glutaralde-hyde in the sensitization step and formaldehyde in the sil-ver impregnation step.The detailed protocol is shown in Table 1.Staining was performed in glass dishes and par-ticular care was taken to avoid contamination by keratin and other extraneous proteins.Electrophoresis 2000,21,3666±3672Silver staining compatible with mass spectrometric analysis 3667Table 1.The modified silver staining protocol using Silver Stain PlusOne kit Step Solution (250mL per gel)Time (min)1.Fix 25mL acetic acid,100mL methanol,125mL milli-Q water 152.Fix25mL acetic acid,100mL methanol,125mL milli-Q water153.Sensitization a)75mL methanol,10ml sodium thiosulfate (5%),17g30sodium acetate 165mL milli-Q water 4.Wash 250mL milli-Q water 55.Wash 250mL milli-Q water 56.Wash 250mL milli-Q water57.Silver a)25mL silver nitrate (2.5%),225mL milli-Q water 208.Wash 250mL milli-Q water 19.Wash 250mL milli-Q water110.Develop6.25g sodium carbonate,100m L formaldehyde,250mL milli-Q water 11.Stop 3.65g EDTA,milli-Q water 1012.Wash 250mL milli-Q water 513.Wash 250mL milli-Q water 514.Wash250mL milli-Q water5a)Omitting the use of glutaraldehyde in the sensitization step and formaldehyde in the sil-ver impregnation step.Working solutions are freshly made immediately prior tostaining.Figure 1.Gel image of normal rat left ventricle using IPG pH 3±10NL 2-D PAGE (12%T)with 100m g total protein loading and silver staining (Owl silver stain kit).P r o t e o m i c s a n d 2-D ENormal human and rat heart left ventricle tissues were used.Sample preparation and2-D PAGE were performed essentially according to Weekes et al.[20].We routinely use100m g total protein loading for analytical gels(pH 3±10NL)and the Owl silver stain kit(Owl Separation Sys-tem,Portsmouth,UK)for visualization.A typical image of one of these gels is shown in Fig.1.To investigate the sensitivity of the PlusOne kit,100,200,300and400m g total protein loading were used.The corresponding gel images are shown in Fig.2.The patterns obtained using the two different kits(Figs.1and2d)are very similar. This,therefore,facilitates comparisons between semipre-parative and analytical gels stained with conventional pro-tocols(e.g.,the Owl kit).Excellent patterns were achieved at higher protein loadings(200,300,and400m g;Fig.2a±c),whereas many spots display negative staining at these loadings when more sensitive staining methods,such as the Owl kit,are used(data not shown).Note that the high-er background obtained from the higher protein loading gels were removed using transform to autoscale the gel image in PDQuest2-D software version6.1(Bio-Rad, Hercules,CA,USA).A400m g total protein loading for IPG3±10strips appears to be optimal in that adequate concentrations of most spots are obtained for MS analy-3668J.X.Yan et al.Electrophoresis2000,21,3666±3672Figure2.Gel images of normal rat left ventricle using IPG pH3±10NL2-D PAGE(12%T)and modi-fied silver staining described in Table1(modified PlusOne silver stain kit)with total protein loading of(a)400m g,(b)300m g,(c)200m g and(d)100m g.sis,while minimizing excessive background and the for-mation of large spot clusters.Higher loadings are possi-ble,however,when using narrow-range IPG strips(data not shown).We investigated the compatibility of the PlusOne modified protocol with ESI-and MALDI-MS.While MALDI is rela-tively tolerant of salts and other contaminants[21,22], the ESI technique is much more susceptible to such inter-ference.Thus,it is necessary to validate the compatibility of the modified stain with both ionization methods.Figure 3shows a gel image of human heart left ventricle(400m g total protein loading)from which20proteins spots were excised for MS characterization.A modified sample prep-aration method was used,which incorporates a destain-ing step[15]to remove silver prior to in-gel digestion with trypsin[14].Aliquots(0.5m L)of the digest supernatant were applied directly to the MALDI target,after which,if necessary,the remainder of the sample was extracted, desalted,and analyzed by ESI-MS/MS.These data are summarized in Table.2.If the results from MALDI mass mapping were ambiguous,ESI-MS/MS was used to gen-erate amino acid sequence data suitable for sequence tag or similarity searching using BLAST.Figure4shows the MALDI mass spectrum of spot19which,when sub-mitted to database searching,retrieved a highly signifi-cant match to ubiquinol cytochrome C reductase.Figure 5a shows a MALDI mass spectrum obtained from spot4, which,when searched,did not find any unambiguous hits.Electrophoresis2000,21,3666±3672Silver staining compatible with mass spectrometric analysis3669Figure3.Gel image of human left ventricle using IPG pH3±10NL2-D PAGE(12%T)with400m gtotal protein loading.The gel was stained using the PlusOne silver stain kit.Protein spots(1±20)labeled on the image were subjected to trypsin digestion and MALDI-TOF-MS or ESI-MS(Table2).This protein was identified by ESI-MS/MS of a doubly charged ion at m/z 777.4,from which 13residues of amino acid sequence were deduced (Fig.5b),which exactly matched the sequence of ATPsynthase a -chain.We conclude that the modified silver staining kit is com-patible with both MALDI-and ESI-MS.Although the sensi-tivity is somewhat lower than other versions,this kit ena-bles higher protein loading,thus facilitating identification by MS.In our laboratory the modified protocol has pro-vided consistent results over a 12-month period and,since the resulting patterns are similar to those produced by the Owl silver stain kit,we have been able to correlate results from semipreparative and analytical gels.Spots of interest are easily located for excision and further charac-terization.A loading of 400m g protein on IPG 3±10strips provides adequate concentrations for successful MALDI analysis of the majority of visible spots.For very low abu-dance proteins,use of the modified stain in conjunction with high protein loadings on narrow pH range IPG strips avoids excessive background staining and spot cluster-ing.We thank the Cardiovascular Disease Group,Rhone-Poulenc Rorer (Collegeville,PA,USA)for providing the rat heart tissue,and Tim Harwood,Amersham PharmaciaElectrophoresis 2000,21,3666±3672Silver staining compatible with mass spectrometric analysis3671T a b l e 2.c o n t i n u e dS p o t E s t i m a t e d I d e n t i f i c a t i o n (a c c e s s i o n n u m b e r )T h e o r e t i c a l A m i n o a c i d P e p t i d e s e q u e n c e i n f o r m a t i o n o b t a i n e d b y E S I -T O F -M S /M S a n a l y s i sN o .p I /M r (D a )p I /M r (D a )c o v e r a g e (%)w i t h M A L D I -T O F -M S 195.7/46200U b i q u i n o l -c y t o c h r o m e c r e d u c t a s e c o m p l e x c o r e p r o t e i n I 5.94/5261939.31.m /z 787.5(3+)A V E L L G D I V Q N C S L E D S Q I E K p r e c u r s o r ,h u m a n (P 31930)2.m /z 901.5(3+)A G Y G P L E Q L P D Y N R3.m /z 1044.3(3+)...F Q G T P L A Q A V E G P S E N V R 4.m /z 1180.7(2+)A V E L L G D I V Q N C S L E D S Q I E K5.m /z 1351.7(2+)Y I I D Q C P A V A G Y Y P I E Q L P D Y N R 205.9/40700A c t i n ,a -c a r d i a c ,h u m a n (P 03996)5.24/4200930.21.m /z 565.9(2+)G Y S F V T T A E R2.m /z 652.7(3+)V A P E E H P T L L T E A P L N P K3.m /z (2+)P y r -E Y D E A G P S I V H RP r o t e i n s p o t s 1±20w e r e e x c i s e d f r o m t h e 2-D g e l s h o w n i n F i g .3a n d a n a l y z e d b y M A L D I -a n d E S I -M S .T h e r e s u l t i n g p e p t i d e m a s s m a p s w e r e s e a r c h e d a g a i n s t S W I S S -P R O T /T r E M B L r e l e a s e 35,u s i n g P r o t e i n P r o b e (M i c r o m a s s ),o r a g a i n s t a n o n r e d u n d a n t d a t a b a s e m a i n t a i n e d b y t h e N a t i o n a l C e n t e r f o r B i o t e c h n o l o g y I n f o r m a t i o n (N C B I )(h t t p ://w w w .n c b i.n l m .n i h .g o v )u s i n g t h e M a s c o t [23]s e a r c h e n g i n e (h t t p ://w w w .m a t r i x s c i e n c e .c o .u k ).A n i n i t i a l m a s s t o l e r a n c e o f 100p p m w a s u s e d ,b u t w a s r e d u c e d t o 50p p m i f e x c e s s i v e n u m b e r s o f h i t s w e r e r e t r i e v e d .A m i n o a c i d s e q u e n c e s o b t a i n e d f r o m E S I -M S /M S w e r e s e a r c h e d a g a i n s t a n o n r e d u n -d a n t d a t a b a s e i n N C B I u s i n g t h e B L A S T p r o g r a m [23].M S /M S s h o w e d t h a t s p o t 1c o n t a i n e d t w o -c o m i g r a t i n g p r o t e i n s .S p o t s 6,8±13,a n d 15±17w e r e n o t a n a l y z e d b y M S /M S b e c a u s e t h e s e a r c h r e s u l t s f r o m t h e M A L D I d a t a w e r e u n a m b i g u o u s .T h e s e q u e n c e c o v e r a g e o f 6,8,9a n d 12a p p e a r s l o w ,b e c a u s e t h e o b s e r v e d p r o -t e i n s p o t s c o r r e s p o n d t o t r u n c a t e d f o r m s ,w h e r e a s t h e c o v e r a g e w a s c a l c u l a t e d f r o m t h e f u l l -l e n g t h s e q u e n c e.Biotech,for conducting this collaboration.JXY acknowl-edges Aventis for their financial support.RAH and JAW thank Proteome Sciences Inc.for their financial support. 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