The IBC conference on applications ofproteomics for drug target and biomarkeridentification was held from 30 June to 2 July 2003 in Basel, Switzerland.Withparallel sessions, a tremendous amount ofinformation on many subjects, includingbioinformatics, was presented in three days.Only a few selected presentations onprotein microarrays, role of biomarkers,and glycoproteomics are presented in thisreport. Proteomic technologies aredescribed in detail elsewhere [1].

Protein and tissue microarraysInvestigation of gene function involves themeasurement of target protein and the studyof protein–protein and protein–DNAinteractions using protein and antibodymicroarrays. John McCafferty (WellcomeTrust Sanger Institute, http://www.sanger.ac.uk) presented the keynote addresson the use of microarrays in gene and proteinexpression. Protein microarrays are requiredto show high sensitivity and specificity and tobe reproducible. Other important featuresinclude the detection of folded proteins andtheir functions. Protein microarrays have beenused mostly for research but have potentialas diagnostic tools. Gene expression can beanalysed in tissue sections.Tissue arrays areconceptually different from DNA and/orprotein chips; they have fewer data points butstill have high information content as theyretain cellular as well as subcellularinformation. Protein expression can beoptimised by cloning PCR products intoexpression vectors. Potent high-affinityantibodies can be obtained from phagedisplay libraries and are a source of drugleads.The process is amenable to automation.Data from image analysis is analysed byinformatics software.The Atlas of Gene

Expression (http://www.sanger.ac.uk/Teams/Team86/) project provides informationon the expression levels and localisation ofprotein products by using recombinantantibodies as probes to localise protein intissues via immunocytochemistry.

Thomas Joos (University of Tübingen,http://www.uni-tuebingen.de/) presented athorough review of protein microarray andmicrofluidic technology, a part of which hasbeen previously published from his institute[2]. Solid-phase assay systems are highlyparallel, miniaturised and sensitive and requirelow sample consumption.There are severalminiaturised ligand-binding assays, includingplanar microarrays and bead-based systems(Luminex xMap, http://www.luminexcorp.com). Planar wave guide (Zeptosens) andsurface acoustic wave (Advalytix) technologiesare used for detection. Several proteinengineering technologies are available but ahigh number of binders leads to a bottleneck,as their characterisation needs time.

Barry Schweitzer (Protometrix, http://www.protometrix.com) described ProtoArray™,acomprehensive protein microarray format thatcan be used to evaluate protein function andto identify agents that interact with proteins ofinterest. ProtoArrays™ are a cost-effective,miniaturised, high-throughput technology thatcan be used to screen up to several thousandproteins simultaneously for drug binding,molecular interactions or enzymatic activity.They have important applications in proteincharacterisation, drug target discovery anddrug development.The Yeast ProtoArray™ isthe world’s first proteome microarray. Itcontains almost 5000 Saccharomyces cerevisiaeproteins,which are double-spotted onto asurface-modified glass microscope slidealongside several hundred internal andexperimental controls.

In vivo proteomics for anticancerstrategiesThe development of anticancer therapieswith better discrimination between tumourcells and normal cells is the most importantgoal of modern cancer research, as mostchemotherapeutic agents do notpreferentially accumulate at tumour sites.Ligand-based tumour targeting based onhigh-affinity monoclonal antibodies enablesexcellent localisation to the tumourenvironment [3]. Dario Neri (Swiss FederalInstitute of Technology, http://www.eth.ch/)and his collaborators have developed amonoclonal antibody that is specific for theEDB (extra domain B) of fibronectin, amarker of angiogenesis, and is capablebecoming selectively localised to new bloodvessels, although this is a slow process as themarker is located in albumin. He argued thatin vivo biotinylation of tumour-bearing micecoupled with state-of-the-art proteomictechnologies is the most efficient methodavailable for the discovery of selectivemarkers of tumour neovasculature.Terminalperfusion of tumour-bearing cells was shownto be feasible.

Biomarkers andpharmacoproteomicsProteomic technologies are playing animportant role in the discovery of diseasebiomarkers that have diagnostic value as wellas the potential to be targets for drugdiscovery. Odille Carrette (GenevaUniversity Hospital, http://www.hug-ge.ch/)presented the results of a study usingprotein array technology that demonstratedthe presence in the cerebrospinal fluid (CSF)of sensitive polypeptide biomarkers forAlzheimer’s disease. Cystatin C and β-2-microglobulin were upregulated and VGF, a

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Proteomics for drug target andbiomarker identificationK.K. Jain, Blaesiring 7, 4057 Basel, Switzerland. e-mail: jain@pharmabiotech.ch

neuroendocrine secreted polypeptide, wasdownregulated.These biomarkers could beused for diagnosis, for assessment of theseverity and progression of the disease, andas a basis for new therapeutic approaches.

Howard Schulman (SurroMed,http://www.surromed.com/) describedmethods for the multidimensional proteomicand metabolomic analysis of serum and CSF. High-throughput LC-MS (liquidchromatography mass spectrometry) can beperformed on human CSF.The company’sDeepLook™ technology enables analysis ofmembrane proteins. Finally, bioinformatics isused for discovery of biomarkers from theintegrated datasets. He presented as anexample the identification of biomarkers forresponse versus non-response in a study ofanti-TNFα (tumour necrosis factor α) inrheumatoid arthritis patients.

Jennifer Sutton (Protea Biosciences,http://www.proteabio.com/) discussedstrategies for identifying disease-relatedprotein biomarkers using solution-basedLC-MS. Advantages of this technique includeincreased sensitivity, which means that thatless material is required, the ability toanalyse complex protein mixtures, improveddynamic range, and the ability to identifypost-translational modifications.Thecompany’s strategy for biomarkeridentification is to analyse biological fluids in the initial phase and to identify themarkers from the disease tissues in thesecondary phase. In the final phase, anunderstanding of the marker is obtained atthe molecular level and protein–proteininteractions are investigated.

GlycoproteomicsGlycoproteins have a predominant role incell–cell and cell–substratum recognitionevents in multicellular organisms.There isincreasing recognition of the importance ofpost-translational modifications such asglycosylation as a means of diversifyingproteins and as potential modulator of theirfunction in health as well as in disease.Theterm ‘glycome’ is defined, in analogy to thegenome and proteome, as the whole set of glycans produced in a single organism.

Ten Feizi (Imperial College, London,http://www.ic.ac.uk/) started the session on glycoproteomics with a review of oligosaccharide receptors, whichmediate critical processes such as proteinfolding and trafficking.The challenge is to discover carbohydrate-recognisingproteins.The characterisation ofcarbohydrate ligands requires sensitivehigh-throughput technologies to analyseprotein–carbohydrate interactions in orderto detect oligosaccharide sequences boundwithin the glycomes. A carbohydratemicroarray system was described that can beused to generate the large repertoires ofimmobilised oligosaccharide probes requiredfor the detection of protein–carbohydrateinteractions [4].The arrays are obtained fromglycoproteins, glycolipids, proteoglycans,polysaccharides, whole organs or fromchemically synthesised oligosaccharides.Carbohydrate-recognising proteins singleout their ligands not only in arrays ofhomogeneous oligosaccharides but also inarrays of heterogeneous oligosaccharides.In addition to their roles in proteinexpression systems, mass spectrometry and bioinformatics, such arrays could formthe basis of methods for the identification ofoligosaccharide-recognising proteins in theproteome and for the mapping of thecomplementary recognition structures in the glycome. Such knowledge can beapplied not only to inhibit or facilitateprotein–carbohydrate interactions in vivo butalso to direct glycosylated drugs to specifictarget cells or tissues.

Nicolle Packer (Proteome Systems,http://www.proteomesystems.com/) showedhow specific protocols and bioinformaticsmethods using the hardware and softwarefrom the Company’s proteomics platformProteomIQ have been applied to glycomics.These technologies cover all areas of glycananalysis, from glycoprotein samplepreparation to the analysis of glycans fromglycoprotein isoforms separated by gelelectrophoresis. Mass spectrometric data onglycan fragments are interpreted by specificbioinformatic software integrated with thecompany’s glycan database, GlycoSuite DB,

to automatically generate thecorresponding oligosaccharide structures.

Ralph Riggin (Eli Lilly and Co,http://www.lilly.com/) presented acharacterisation strategy for glycoproteinbiopharmaceuticals during development.Oligosaccharide analysis involves thereduction of glycoprotein to oligosaccharideand protein, the precipitation of protein toleave oligosaccharide, and finally direct analysisby HPLC/electrospray mass spectrometry [5].One example of the application of thistechnique was the determination of thesialic acid content of a glycoprotein by usingLC/MS and obtaining mass data for theglycoprotein. Oligosaccharide formulas andthe number of sialic acid residues wascalculated for each oligosaccharide.The sialicacid content of the glycoprotein was thencalculated from the relative percentagecontent of each oligosaccharide.

Concluding remarksIBC has maintained its tradition ofpresenting the best proteomic conferences.The technologies presentations were of highstandard.There was an opportunity to learnabout cutting-edge technologies fromacademic researchers as well as from thosein the commercial sector. Perhaps the onlypossible criticism of the conference was theinadequate coverage of the commercialaspects of proteomics, which are importantfor the growth and survival of companiesdeveloping proteomic technologies.

References1 Jain, K.K. (2003) Proteomics: Technologies,

Markets and Companies, Jain PharmaBiotechPublications, Basel

2 Templin, M.F. et al. (2002) Protein microarraytechnology. Drug Discov. Today 7, 815–822

3 Santimaria, M. et al. (2003)Immunoscintigraphic detection of the ED-Bdomain of fibronectin, a marker ofangiogenesis, in patients with cancer. Clin. Cancer Res. 9, 571–579

4 Fukui, S. et al. (2002) Oligosaccharidemicroarrays for high-throughput detection andspecificity assignments of carbohydrate-proteininteractions. Nat. Biotechnol. 20, 1011–1017

5 Huang, L. and Riggin, R.M. (2000) Analysis ofnonderivatized neutral and sialylatedoligosaccharides by electrospray massspectrometry. Anal. Chem. 72, 3539–3546

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