IBC’s Proteomics – Delivering New Routesto Drug Discovery conference (14–17 May2001, Philadelphia, PA, USA), providedbroad coverage of the application of innovative proteomic technologies fordrug discovery. The second part of thisconference review deals with the analy-sis of proteomic information, and detailsof technologies are described elsewhere1.

Analyzing the human proteomeThe first part of this conference dealt withthe role of proteomics in drug develop-ment and structural genomics2. ScottPatterson (Celera Genomics, Rockville,MD, USA) described proteomics usingliquid chromatography–MS (LC–MS), asopposed to two-dimensional (2D) gelelectrophoresis (GE), which offers higherthroughput and simpler automation.Integration of genomic, proteomic, transcript-level and biological data isachieved using bioinformatics. LeighAnderson (Large Scale Proteomics,Germantown, MD, USA) discussed theintegration of protein chips with the ex-isting technologies of 2D GE and MS.Protein chips are more sensitive than 2DGE or MS, have a wide dynamic range,high specificity and excellent quantifica-tion capability. The drawbacks are poorcorrelation with phenotype, and thatmRNA does not reflect protein amount.

Whereas classical 2D electrophoresis(2DE) has definite limitations with respect to resolution, reproducibility and protein-loading capacity, 2DE using immobilized pH gradients (IPGs) hasproved to be amazingly flexible in termsof the requirements of proteome analy-sis3. Angelika Görg (Technical Universityof Munich, Freising-Weihenstephan,

Germany) described the advances in analytical and micropreparative 2DE ofproteins using wide-range IPGs up to pH 12.0 for overview patterns, as well asnarrow, overlapping IPGs (betweenpH 2.5 and pH 12.0) with extended sep-aration distances for the detection of a maximum of spot numbers, avoiding thepresence of multiple proteins per spotand/or cross contamination of neigh-bouring spots for further investigation.

Chris Sutton (Kratos Analytical,Manchester, UK) described matrix-as-sisted laser desorption ionization (MALDI)as an essential tool for current proteomics,with applications in high-throughputprotein identification and biomarkeridentification. The next generation ofMALDI instruments is being designed forthe structural elucidation of protein se-quences and post-translational modifica-tions to enable our understanding ofbiological function. Andreas Köpke (WITAProteomics, Tetlow/Berlin, Germany) described a high resolution ‘all-in-one’system (both dimensions in one cham-ber without gel handling) of 2D GE for diagnostics and drug targeting. Non-equilibrium-pH gel electrophoresis(NEPHGE) can be applied within a pHrange of 2–11 to samples containing25–100 µg protein or <1 mg of tissue,and can separate up to 10,000 spots. AnMS platform consisting of MALDI-MSand electrospray ionization (ESI)-MS–MS-Q-TOF (quadrupole time-of-flight) hasbeen used for the detection of lungmetastases in clinical trials, in an effort todetermine the effectiveness of a com-pound in suppressing lung cancer. Anassay could detect metastases consistingof <2000 cancer cells.

Protein biochips and microarraysIn understanding protein structure–func-tion relationships, a knowledge of bind-ing stability, multi-molecular complexassembly and crucial amino acid residuesis essential. JoAnne Bruno (Biacore,Piscataway, NJ, USA) presented a chip-based, surface plasmon resonance (SPR)approach to monitoring protein–proteininteractions. This method has the advan-tage that measurements are label-freeand in real-time and, in many cases,samples do not require pretreatment before they are analyzed4. This powerfulapproach not only identifies bindingpartners to target molecules, but alsoprovides binding specificity, active con-centration, and quantitative affinity andkinetic data. SPR is useful for screeningfor ligands to orphan receptors, identify-ing functionally active proteins, character-izing protein–protein interactions, andexploring protein-complex assembly.

It is essential to develop analyticaltools capable of systematically reportingon the output of each and every gene inthe human genome. In the absence ofhybridization assays or a PCR-equivalent,high affinity and high specificity anti-bodies represent the most logical path to success. Antibody production is rendered genomically relevant throughparallel generation and screening, so asto reduce the time and cost associatedwith high quality antibodies destined forincorporation on arrays. Ian Humphery-Smith (Universiteit Utrecht, Utrecht, TheNetherlands) described peptide, proteinand antibody arrays as an integrated approach to following human tissues inhealth and disease. However, many genesare difficult to clone or are associated

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Proteomics: delivering new routes todrug discovery – Part 2Kewal K. Jain, Bläsiring 7, CH-4057 Basel, Switzerland, tel/fax: +41 61 692 4461, e-mail: jain@pharmabiotech.ch, web: http://pharmabiotech.ch

with problematic proteins. Therefore, anessential element of this procedure is aclosure strategy centred on ‘signaturepeptides’. Other aspects already inte-grated in this process include cruciallyimportant surface chemistries, expres-sion-vector design, robotics and three-dimensional (3D) biocomputing for datamanagement and pattern recognition.

Bioinformatics for proteomicsThe use of protein and expressed se-quence tag (EST) similarity to help geneprediction is widespread. ESTs are an invaluable resource for protein identifi-cation and characterization in pro-teomics. They also allow proteins to beidentified in the absence of genome se-quence data. Jeanette Schmidt (IncyteGenomics, Palo Alto, CA, USA) discussedthe role of bioinformatics in organizingEST databases into full-length genes.More recent tools include GeneWise™(Compugen, Richmond Hill, Ontario,Canada), which considers every possiblegene prediction in a genomic sequenceand compares each one with the proteinprofile. The best combined score of boththe gene prediction and the protein pro-file is used to provide a simultaneousgene prediction and protein alignment5.

New tools have become available toanalyze, search and store 2D-gel imagesin databases using differential protein-expression patterns. Suzanne Mattingly(Scimagix, Redwood Shores, CA, USA)described how new capabilities of 2D-gel-analysis software and 2D-gel data-bases can be leveraged for drug discov-ery research. Recent advances in theanalysis and retrieval of 2D gels have included the automated registration of gels into an Oracle™ database, thus enabling rapid search and retrieval. Adata-reduction capability can improvethe recognition of patterns. Advantagesof database storage of 2D-gel data include: options to quickly process datafrom prospective studies; rapid evalu-ation of results to discover new proteins;decisions for early termination of a

compound with undesirable characteris-tics; and switching to more promisingoptions. The recognition of protein-expression patterns facilitates the under-standing of the mechanism of action ofdrugs. This approach can accelerate thedrug discovery process.

Marc Wilkins (Proteome Systems,Sydney, Australia) described a bioinfor-matic platform for the integration andanalysis of proteomic data to derivemeaningful results. Challenges pre-sented by the application of bioinfor-matics to high-throughput proteomics include the massive amount of data(>500 gigabytes per year) being gener-ated, and the complexity of the datasuch as that found in protein-expressionmaps. Projects need laboratory infor-mation management systems (LIMS)and constant updating of bioinformaticstools in-house. Some measures to address these bottlenecks are completeprocess automation and client-server delivery via a web browser and integrated image analysis. When EST sequences are used for protein identifi-cation, they are usually first processedinto contigs to reduce redundancy andgenerate longer sequences from theoverlapping ESTs. However, the processof generating contigs can accidentallygroup biologically meaningful isoformstogether. Protein identification andcharacterization of the dbEST databasecan be done by integrating clustering,translations, BLASTN and peptide-massfingerprinting6.

A variety of sequence and structuralanalysis methods are available to help assign biochemical function to proteins.Kim Fechtel (Genome Therapeutics,Waltham, MA, USA) described amethod for the automated analysis of genomic and protein sequence. ThePathoGenome™ data analysis processcarries out BLAST analysis of all publicgenomic data and annotates data fromother databases. The master catalogcontains curated data compiled into>50,000 evolutionary families, as well as

composite gene-product modules. Eachfamily is associated with precalculatedphylogenetic trees and secondary struc-ture motifs. These comprehensive geneor protein databases enable rapid cross-referenced predicted functional analysis,without the need for bioinformatic computation by the user.

Applications of proteomicsApplications of proteomics in basic research, drug discovery, profiling drugeffects, molecular diagnostics and vari-ous therapeutic areas were discussed.Stephen Pennington (University ofLiverpool, Liverpool, UK) described theintegration of proteomics into basic bio-medical science. With mRNA analysis directing proteomic strategy, protein-expression mapping is considered to bea viable alternative to 2D electrophor-esis7. This approach has been used tostudy the role of calcium signalling in theregulation of gene expression duringcell-cycle progression.

Heart diseaseIn the post-genome era, proteomics willplay an important role in the characteriz-ation of the global alterations in proteinexpression that underlies heart disease.The proteomic approach complementsgenomics-based and more traditionalsystems-based approaches, and will provide new insights into the cellularmechanisms involved in cardiac dysfunc-tion. This improved understanding ofthe molecular basis of cardiac dysfunc-tion should result in the development of novel therapeutics for the treatmentof heart disease and chronic heart failure. Michael Dunn (Imperial Collegeof Science, Technology and Medicine,Harefield, UK) presented the results ofhis studies on dilated cardiomyopathy(DCM), a disease of unknown etiology.The results of these studies have shownthat the expression of some 100 cardiacproteins is altered significantly in DCM,with the majority of these proteins foundto be missing in the diseased heart.

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Problems of investigation of human material include disease heterogeneity,changes specific to disease and superim-posed changes caused by heart failureand drug treatment. One approach tothe investigation of this problem is toapply proteomics to appropriate animalmodels of human disease. Ubiquitin C-terminal hydrolase is greatly increasedin both human and bovine DCM at the level of mRNA, as well as the expressed protein. The ubiquitin-protea-some pathway might be a possible target for therapeutic intervention inheart disease.

DiabetesStephen Fey (University of SouthernDenmark, Odense, Denmark) demon-strated the use of proteome analysis instudying insulin-dependent diabetesmellitus. In rat models of the disease, thepancreatic islets were challenged bycytokines, which strongly regulated protein expression (up or down). Theseislets were grafted into diabetes-pronemice, re-isolated, labelled and analyzedby 2DE, which confirmed the earlier results. The selected proteins were iden-tified by MALDI-MS. These studies arenow being extended to human pan-creatic islet cells. Although the genes involved in humans are different, theprotein pathways involved are the sameand play an important role in the genesis of the diabetes.

Infection biologyPeter Jungblut (Max Planck Institute ofInfection Biology, Berlin, Germany) dis-cussed the role of proteomics as a toolfor infection biology. Several immuno-logically relevant proteins have been detected by comparing virulent strainswith attenuated strains, studying secretedproteins, outer-surface proteins and ana-lyzing the immunoproteome. Severalvaccine candidates for Mycobacterium tuberculosis8 and antigens for Heliobacterpylori 9 have been identified by proteomicapproaches.

Toxicology and drug-safety assessmentProteomics has been applied to toxicol-ogy and drug-safety assessment. Variousstudies have been conducted to studytoxicity mechanisms and to assess the safety of new drug candidates by comparison with fingerprints of refer-ence compounds with known toxicity.The molecular fingerprint of a drug is its gene-regulation pattern in response to the perturbances evoked by drug ac-tion and visualized by gene-expression profiling at the mRNA or protein level.

Sandra Steiner (Large Scale Proteomics)presented several projects to illustratethe use of proteomics to enhance thepredictive power of safety assessmentand accelerate the drug developmentprocess. Various preclinical markers havebeen used to monitor therapeutic versustoxicity mechanisms. An example is the effect of HMG CoA reductase in-hibitors on cholesterol metabolism. Thistakes place through a tightly regulatedpathway affecting a small number ofproteins, and is upregulable by cholestyra-mine and lovastatin. The pattern ofgene-network regulation induced in he-patic proteins as a response to lovastatintreatment was analyzed by proteomicsin the livers of rats10. Lovastatin adminis-tration to the rats was associated withsigns of toxicity, as reflected by changesin a heterogeneous set of cellular-stressproteins involved in functions such as cytoskeletal structure, calcium homeo-stasis, protease inhibition, cell signallingor apoptosis10. These results present newinsights into liver gene-network regula-tions induced by lovastatin, and illustratea yet to be explored application of pro-teomics to discover new targets by analy-sis of existing drugs and the pathwaysthat they regulate.

Research and development Paul Cutler (GlaxoSmithKline, Harlow,UK) discussed the role of proteomics inpharmaceutical research and develop-ment. The applications include clinical ortumour markers, biodistribution studies,

toxicology, target identification, post-translational modifications and study ofresponse to experimental treatment. Anexample was given of the MALDI-TOFMS of serum samples of transgenic miceexpressing the human mutant ApoE*3-Leiden protein. ApoE*3 is a variant formof ApoE that shows reduced affinity forthe low-density lipoprotein (LDL) recep-tor, and results in the dominant expres-sion of type III hyperlipoproteinemia.This proteomic approach has enabledthe ApoE*3-Leiden variant to be posi-tioned in a 2DE separation of serum pro-teins, and has identified changes in theexpression of haptoglobin, indicatingthat this protein could be a marker forthe potential onset of atherosclerosis11.

Concluding remarksThis was the most comprehensive pro-teomics conference that the author hadever attended. The calibre of the presen-tations was high, with the participationof academic proteomics centres. In addi-tion to the presentations discussed,there were parallel sessions on samplepreparation and strategic corporate alliances, as well as ample opportunitiesfor audience–faculty interactions. Thesize of the conference reflects the ex-pansion in proteomic technologies and applications.

References1 Jain, K.K. (2001) Proteomics: technologies and

commercial opportunities (7th edn) JainPharmaBiotech Publications, Basel, Switzerland

2 Jain, K.K. (2001) Proteomics: delivering newroutes to drug discovery – Part 1. Drug Discov.Today 6, 772–774

3 Görg, A. et al. (2000) The current state of two-dimensional electrophoresis withimmobilized pH gradients. Electrophoresis 21,1037–1053

4 Nelson, R.W. et al. (2000) Biosensor chipmass spectrometry: a chip-based proteomicsapproach. Electrophoresis 21, 1155–1163

5 Birney, E. and Durbin, R. (2000) UsingGeneWise™ in the Drosophila annotationexperiment. Genome Res. 10, 547–548

6 Lisacek, F.C. et al. (2001) Strategy for proteinisoform identification from expressedsequence tags and its application to peptidemass fingerprinting. Electrophoresis 22,186–193

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7 Jenkins, R. and Pennington, S.R. (2001)Protein expression mapping: Towards aviable alternative to 2-DE. Proteomics 1, 13–29

8 Mattow, J. et al. (2001) Identification ofacidic, low molecular mass proteins ofMycobacterium tuberculosis strain H37Rv bymatrix-assisted laser desorption/ionization

and electrospray ionization massspectrometry. Proteomics 1, 494–507

9 Bumann, D. et al. (2001) Proteome analysis of the common human pathogen Helicobacter pylori. Proteomics 1, 473–479

10 Steiner, S. et al. (2000) Proteomics to displaylovastatin-induced protein and pathway

regulation in rat liver. Electrophoresis 21,2129–2137

11 Skehel, J.M. et al. (2000) Phenotypingapolipoprotein E*3-Leiden transgenic miceby two-dimensional polyacrylamide gelelectrophoresis and mass spectrometricidentification. Electrophoresis 21, 2540–2545

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The second annual IBC High ContentScreening Symposium (11–13 June 2001,Miami Beach, FL, USA) was sponsored by Cellomics (Pittsburgh, PA, USA) andBeckman Coulter (Fullerton, CA, USA)and brought together scientists fromacademia and industry with interests in cell-based screening. Presentationscovered the capabilities of high contentscreening (HCS), its existing and poten-tial applications, and how it can be improved in the future. In particular,there was significant input fromCellomics application scientists regard-ing ArrayScan II™ (AS; Cellomics) whodiscussed the different applications ofthis technology platform, in addition toproduct demonstrations and interactivediscussions.

HCS versus HTSSeveral companies are now providingtechnology that enables scientists tomake multiple measurements of the cel-lular phenotype on a cell-by-cell basis.Compared with single-measurementwell-based assays, multiparameter cell-based HCS assays yield data with muchhigher biological information content.Whereas HTS is used as a fast primaryscreen to identify hits for further testing,

HCS can be used to identify leads fromhits. The ability to obtain quantitativedata from multiple endpoints, from bothindividual cells and cell populations,greatly enhances the information ob-tained from whole-cell screens in drugdiscovery. This could provide new in-sights into cell function and mode-of-action studies that were previously labourintensive and difficult to interpret.

ArrayScan™ technologyKeith Olson (Cellomics) reviewed thenew software of the AS system, whichenhances user interactions and improvesassay throughput. The addition of Image-Pro™ software places greater flexibilityfor image-analysis algorithm develop-ment in the hands of the end user. Thesub-population analysis feature enablesusers to select or reject cells based on nuclear size, shape, and intensity, orto gate on (that is, select a subpopula-tion of) cells that are above, below or between thresholds for a particularmarker. Responders or non-respondersto a stimulus (e.g. a cytokine) can be analyzed separately and compared withthe entire cell population.

The advantages of the AS technologyhighlighted were: (1) images are acquired

by a cooled CCD camera via standard inverted microscope optics (for mostHCS applications the resolution is suffi-cient so three-dimensional detail ob-tained by laser-confocal approaches isunnecessary); (2) spatial information onindividual cells (e.g. changes in cellshape or size), organelles (e.g. nuclearmorphology) or cellular targets (e.g.transcription factor translocation to nu-cleus) can be obtained; (3) experimentsare performed in standard clear-bottom96- or 384-well microtitre plates; (4)cells can be live or fixed; (5) AS uses a UV arc lamp source, therefore, it can simultaneously quantify molecules thatfluoresce over a wide spectral range.

Bright future for fluorescencetechnologyFluorescent markers that are currentlyused by cell biologists could be adaptedfor use on the AS platform, according toRichard Haugland (Molecular Probes,Eugene, OR, USA). These include mark-ers for organelles, nitric oxide and reac-tive oxygen species, intracellular Ca2+ (forG-protein-coupled receptor activation),enzyme substrates, fluorescent resonanceenergy-transfer (FRET) and enzyme-cou-pled assays. Haugland also described a

High content screening – from cells todata to knowledgeCatherine Liptrot, Senior Scientist, AstraZeneca, Cancer Research Area, 3F30 Mereside, Alderley Park, Macclesfield, Cheshire, UK SK10 4TG,tel: +44 1625 230147, fax: +44 1625 516033, e-mail: Catherine.Liptrot@astrazeneca.com