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Two-Dimensional Difference in GelElectrophoresis for Biomarker Discovery

Haleem J. Issaq, Timothy D. VeenstraLaboratory of Proteomics and Analytical Technologies, Advanced Technology Program and

Frederick National Laboratory for Cencer Research, Frederick, MD, USA

Ph

O U T L I N E

Introduction 19

1

Gel Electrophoresis: Historical Perspective 192

Two-Dimensional Differential In-GelElectrophoresis 192

Strengths and Weaknesses of 2D-PAGEand 2D-DIGE 193

roteomic and Metabolomic Approaches to Biomarker Discoveryttp://dx.doi.org/10.1016/B978-0-12-394446-7.00012-1 191

Application of 2D-DIGE to BiomarkerDiscovery 19

4

Conclusions 195

Acknowledgment 195

References 195

INTRODUCTION

Over the last two decades, there has been anincrease in the efforts to develop technologiescapable of separating and quantifying largenumbers of proteins expressed within a cellsystem (i.e., the proteome) with the hope of iden-tifying proteins that can be used as diseasemarkers. The complexity of the proteome hasmade developing methods for efficient separa-tion and sensitive detection of proteins a criticalcomponent of this effort. The most widely used

analytical methods for the separation of proteinsare two-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis (2D-SDS-PAGE) and two-dimensional differential in-gelelectrophoresis (2D-DIGE), both of which canseparate hundreds of proteins in a single experi-ment. Advances in electrophoresis, such as DIGEand the introduction of preformed pH gradientimmobilized gel strips with various isoelectricranges and mass spectrometry (MS) technolo-gies, have enabled the detection of separatedproteins at much greater speed and sensitivity

Copyright � 2013 Elsevier Inc. All rights reserved.

12. TWO-DIMENSIONAL DIFFERENCE IN GEL ELECTROPHORESIS FOR BIOMARKER DISCOVERY192

than previously possible. In 2D-PAGE and2D-DIGE, the area and intensity of the spotsindicate the levels of protein expression in thesample. This measure is used to quantitativelycompare protein concentration levels betweentwo different samples. Also, in-gel digestion ofproteins with trypsin prevents any losses dueto sample manipulation and simplifies the iden-tification of proteins since peptides are easier toanalyze by MS than proteins. This chapter isa brief introduction to DIGE and its applicationto protein biomarker discovery.

GEL ELECTROPHORESIS:HISTORICAL PERSPECTIVE

To properly understand the advances made in2D-PAGE, one needs to go back to 1930, whenArne Tiselius introduced the moving boundarymethod as an analytical tool for studying theelectrophoresis of proteins.1 Since his pioneeringwork, for which he received the Nobel Prize,various forms of electrophoresis have beenemployed to separate complex protein mixtures.Early studies used a single run of gel electropho-resis that did not result in the complete separa-tion of complex protein mixtures. Scientistsrecognized that a combination of two orthogonalelectrophoretic processes on a gel at right anglesshould give a much greater degree of resolutionthan is possible with either separately.2e4 Theirprediction was proven true and has formed thebasis for the development of multidimensionalmethodologies for the separation of complexprotein mixtures not only by gel electrophoresisbut also by chromatography and capillary elec-trophoresis. As early as 1962, Raymond andAurell3 demonstrated the significant nonlineareffects of gel concentration on the electrophoreticmobility of proteins by employing two-dimensional (2D) electrophoresis using differentacrylamide gel concentrations to separate serumproteins. Two years later, Raymond4 demon-strated the superiority of flat slab gels compared

to cylindrical tube gels. The flat slab providedmaximum surface area for cooling the gel andthe resulting patterns were easier to quantify.Also, a large number of samples can be pro-cessed using a single 20� 20 cm gel plate, allow-ing the direct comparison of samples processedunder identical conditions. An additional advan-tage of flat bed electrophoresis is that it permitsthe application of 2D separations.

Current 2D electrophoretic separations of pro-teomes are based on the method of O’Farrell,5

which was introduced in 1975 for separatingcellular proteins under denaturing conditions.This method enabled the resolution of hundredsof proteins on a single gel plate. The principleemployed is very simple: proteins are resolvedin the first dimension according to their isoelec-tric point and in the second dimension accordingto their molecular mass. Today, 2D-PAGE andits newer version, 2D-DIGE, are the analyticalmethods of choice by biologists and biochemists.

TWO-DIMENSIONALDIFFERENTIAL IN-GELELECTROPHORESIS

The advantage of 2D-PAGE as a separationtechnique is not only the separation of largenumbers of proteins but the determination oftheir relative abundances. For example, proteinsextracted from two serum samples (healthy anddiseased) are loaded on a separate gel plate.After separation and staining, the protein spotsare aligned and scanned to measure their indi-vidual intensities. Although many advances insoftware alignment tools have been made, ithas been challenging to ensure direct spot-to-spot intensity comparison between two separategels because slight differences in gels’ composi-tion, pH gradient, and applied voltage affectreproducibility, making it difficult to compareprotein expression levels between two samples.To overcome the reproducibility issues, it wouldbe more accurate if equal amounts of proteins

STRENGTHS AND WEAKNESSES OF 2D-PAGE AND 2D-DIGE 193

from two different samples are mixed, spotted,and separated on the same gel plate. In 1997,Ünlü et al.6 realized the advantages and limita-tions of 2D-PAGE and developed what is knownas two-dimensional differential in-gel electro-phoresis (2D-DIGE) to eliminate reproducibilityissues and achieve better protein-to-proteincomparison and quantitation. In a typical exper-iment, equal amounts of proteins extracted fromtwo different biological samples, healthy anddiseased, and an internal standard (a pooledsample formed from mixing equal amounts ofthe proteins extracted from the two test samples)are covalently labeled, each with a cyanine fluo-rescent dye that has a different excitation andemission wavelength. The same protein fromdifferent samples labeled with any of the dyeswill co-migrate to the same position on the gelbecause the dyes were designed to ensure thatproteins common to both samples have thesame relative mobility regardless of the dyeused to tag them.6 The control sample shouldcontain every protein present across all samplesin an experiment. This requirement means thatevery protein in the experiment has a uniquesignal in the internal standard, which is usedfor direct quantitative comparisons within eachgel and to normalize quantitative abundancevalues for each protein between gels. Scanningthe gel at the specific excitation and emissionwavelengths of each dye, using a fluorescenceimager, allows visualization of the differentiallylabeled proteins (Figure 1) without further pro-cessing. The images are merged and analyzedusing software that enables differences betweenthe abundance levels of proteins to be compared.

STRENGTHS AND WEAKNESSES OF2D-PAGE AND 2D-DIGE

Gel electrophoresis is an excellent techniquethat has undergone several advances, resultingin enhanced resolution, detection, quantitation,and reproducibility. 2D-PAGE can be used for

complex protein mixture fractionation and theconfirmation of a protein identity by comparingthemigration timewith that of a known standardand comparison of MS spectra of a test protein toits known protein standard. As a separation,detection, and quantitation technique, 2D-DIGEis very useful for measuring protein expressionlevels and has played an important role in diseasebiomarker discovery. The 2D-PAGE and2D-DIGE approaches are easily accessible tomost laboratories and possess high resolvingpower for the detection of hundreds of proteinson a single gel plate. Besides detection and quan-titation, gel electrophoresis can provide informa-tion about the charge, molecular weight, andconformational state of a protein. However,sample-to-sample and day-to-day reproduc-ibility has been an issue with 2D-PAGE. Resolu-tion in 2D-PAGE has been greatly improved bythe introduction of immobilized pH gradientstrips (IPGs), which enable the analyst to tailorthe pH gradient for maximum resolution usingultrazoom gels with a narrow pH gradient range.With advances in 2D-PAGE, it is not unusual toresolve two proteins that differ by 0.001 pI units.

The introduction of 2D-DIGE contributedimmensely to solving problems of reproduc-ibility and quantitation. The use of imagers andcomputers allows not only fast data mining,acquisition, and analysis but also spot detection,normalization, protein profiling, backgroundcorrection, and data reporting. The advantageof 2D-DIGE is that the experiment is performedunder the same experimental conditions (pHgradient and field strength) using a single gelplate, which means that inconsistencies betweengels are eliminated, which ensures more accuratequantitation than if samples are run on separategels.7 Also, 2D-DIGE requires 50% fewer gels,making it more economical. In addition, lesstime is required to detect the protein spotsbecause the labeling reaction in 2D-DIGE isfaster than visualization using staining methods.Also, 2D-DIGE is the method of choice when theabsolute protein expression is required.8

FIGURE 1 Schematic of 2D-DIGE procedure. (Courtesy of Amersham Biosciences.)

12. TWO-DIMENSIONAL DIFFERENCE IN GEL ELECTROPHORESIS FOR BIOMARKER DISCOVERY194

APPLICATION OF 2D-DIGE TOBIOMARKER DISCOVERY

Two approaches are used in the search forbiomarkers: collected samples are analyzed (a)individually or (b) pooled then analyzed. If bloodsamples are used, the analysis is carried out pref-erably by first depleting the most abundantproteins (HSA and IgG) using immunoaffinity,followed by labeling the proteins in the twosamples and internal standard by three differentcyanine dyes (Cy2, Cy3, and Cy5). The labeledproteins are then spotted on the same gel plateand separated. The intensity of the spots iscompared and the differentially expressed spotsdigested into tryptic peptides. The peptides areextracted from the gel and identified by high-pressure liquid chromatography (HPLC) tandemmass spectrometryandvalidatedbyWesternblot.

Today, 2D-PAGE and 2D-DIGE play animportant role in disease biomarker discovery.9

Petermann et al. discussed in a series of studiespublished in the journal Cancer in 1948 the roleof plasma proteins in different types of cancerusing electrophoresis.10e12 They concluded that“none of the abnormalities found in these anal-yses is characteristic of cancer in general or ofgastric cancer in particular.” In 1972, McIntire,using gel electrophoresis, reported that seruma-fetoprotein is a biochemical marker for hepato-cellular carcinoma.13 The number of proteinsseparated by electrophoresis before the develop-ment of 2D gel electrophoresis was small andincluded the high molecular weight proteins.Iwaki et. al.14 analyzed the proteome of urinesamples obtained from bladder cancer patientsand control subjects using 2D-PAGE. Threeproteins were identified as novel tumor marker

REFERENCES 195

candidates for bladder cancer. However, theseresults have not been validated. A 2D-DIGEstudy was carried out for the identification ofnuclear matrix proteins to investigate their diag-nostic and prognostic roles in invasive bladdercancer.15 The study used 3 normal, 9 nontumortissue specimens, and21muscle-invasive bladdercancers. More than 800 protein spots weredetected, of which 30 proteins were differentiallyexpressed by bladder tumor cells. Banerjee et al.16

used 2D-DIGE followed by spot picking andanalysis of proteins/peptides by MS to searchfor protein biomarkers for brain cancer. Theyreport that they identified at least ten differentnovel proteins/peptides that were differentiallyexpressed. In another 2D-DIGE comparativestudy of tissues taken from 15 colorectal cancerpatients and normal controls, 17 proteins thathad significant differential expression were iden-tified.17 Raimondo et al. used 2D-DIGE to searchfor protein biomarkers for kidney cancer.18 Atotal of 100 proteins were identified by MS outof 2,500 spots, 23 proteins overexpressed and 77underexpressed in kidney cancer samples.

CONCLUSIONS

Gel electrophoresis is possibly one of theearliest separation techniques used to searchfor protein disease markers. An electrophoreticapproach to protein profiling is simple andeconomical, while possessing high resolvingpower that enables the detection of hundredsof proteins on a single gel plate. The introductionof 2D-DIGE increases the accuracy in comparingprotein expression levels between two differentsamples and has been increasingly frequentlyused in the search for protein biomarkers ofdifferent diseases.

AcknowledgmentThis project has been funded in whole or in part with federalfunds from the National Cancer Institute, National Institutes

of Health, under Contract HHSN261200800001E. Thecontent of this publication does not necessarily reflect theviews or policies of the Department of Health and HumanServices, nor does mention of trade names, commercialproducts, or organizations imply endorsement by the UnitedStates Government.

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4. Raymond S. Acrylamide gel electrophoresis. Annals NYAcad Sci 1964;121:350e65.

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a diagnostic/prognostic biomarker for the brain cancerglioblastoma multiforme by 2D-DIGE-MS technique.Mol Cell Biochem 2012;367:59e63.

17. Hamelin C, Cornut E, Poirier F, et al. Identification andverification of heat shock protein 60 as a potentialserum marker for colorectal cancer. FEBS J 2011;278:4845e59.

18. Raimondo F, Salemi C, Chinello C, et al. Proteomicanalysis in clear cell renal cell carcinoma: identificationof differentially expressed protein by 2-D DIGE. MolBiosyst 2012;8(4):1040e51.