Affinity purification-mass spectrometry

1 MINIREVIEWA ffinity purification- mass spectrometryPowerful tools for the characterization of protein complexesAndreas Bauer and Bernhard KusterCellzome AG, Heidelberg, GermanyMulti-protein complexes are emerging as important entitiesof biological activity inside cells that serve to create func-tional diversity by contextual combination of gene productsand, at the same time, organize the large number of differentproteins into functional units. Many a time, when studyingprotein complexes rather than individual proteins, the bio-logical insight gained has been fundamental, particularly incases in which proteins with no previous functional anno-tation could be placed into a functional context derived fromtheir molecular environment.

2 In this minireview, we sum-marize the current state of the art for the retrieval of multi-protein complexes by affinity purification and their analysisby mass spectrometry . The advances in technology madeover the past few years now enable the study of proteincomplexesonaproteomicscaleanditca nbeanticipatedthatthe knowledge gathered from such projects will fuel drugtarget discovery and validation pipelines and that the tech-nologyisalsogoingtoprovevaluableint heemergingfieldofsystems : TAP (tandem affinity purification).Protein complexesIn the postgenomic era, proteins are coming back into focusbecause it has been realized again that whole genomesequence information alone is not sufficient to explain andpredict cellular phenomena, as it is largely the proteins thatexecute and control the majority of cellular activities.

3 Whilethe human genome is estimated to contain approximately30 000 40 000 genes, the corresponding proteome is muchmore complex. Events such as alternative splicing of genesand post-translational modifications generate a highlydiverse set of proteins that could exceed a million distinctmolecular species within a given cell. This moleculardiversity could contribute to explaining many of thedifferences between evolutionary distant species that donot differ substantially in the total number of genes encodedin their respective genomes. However, within this diverse setof molecules, it is critical to maintain functional organiza-tion. It is becoming increasingly clear that an importantlevel of organization is provided by multi protein complexesbecause instead of proteins and substrates colliding in adiffusion-dependent manner, proteins generally interactwith each other and form larger assemblages in a time-and space-dependent manner [1].

4 At the same time, proteincomplexes provide functional diversity that is coded by thecontextual combination of gene products. Within a proteincomplex, each individual protein may have a particularspecialized function that contributes to the overall functionof the complex. In turn, this specialized function may well bedependent on the interaction with neighboring proteinsurfaces that may lead to modulation of protein activitythroughconformationalchangesorpo st-translationalmodi-fications. Prominent examples of protein complexes are theribosome, the spliceosome and the nuclear pore complexbut many more (and less macroscopic) protein complexeswith more subtle or diverse functions and exemplified by themany signal transduction pathways have been described[2,3].

5 The beauty of studying complexes is that it allows toplace proteins with hitherto unknown roles into a functionalcontext that is provided by their associated partners, someof which may have a known function. Even when analyzingproteins of known function, novel insight can be gainedfrom describing their molecular environment. Quite often,proteins participate to more than one complex or do so indifferent subcellular compartments which can help tounderstand cross-talk between seemingly unconnected cel-lular activities. The extent to which such functionalconnectivities are operating in cells may be best appreciatedby large-scale functional proteomics projects that buildcomprehensive interaction maps for proteins and proteincomplexes [4,5].

6 From a simplistic pharmacological point ofview, functional proteomics via the analysis of proteincomplexes would contribute to the identification of noveldrug targets, the reconstruction of pathways and help tounderstand the mechanism of action and side-effects oftherapeutic toB. Kuster, Cellzome AG, Meyerhofstrasse 1,69117 Heidelberg, Germany. Fax: + 49 6221 13757202,E-mail: PrP(C), cellular prion protein; N-CAM, neural celladhesion molecule; TAP, tandem affinity purification;TEV, Tobacco etch virus; CBP, calmodulin binding peptide;PMF, peptide mass fingerprinting; MS, mass : web page available at (Received 13 September 2002, accepted 12 December 2002)Eur. J. , 570 578 (2003) FEBS 2003 of protein complexesWithin the scope of this review, we will focus on summari-zing the current technical state of the art for the biochemicalretrieval of associated proteins and protein complexes fromcells and tissues as well as their analysis by mass spectro-metry, for it is this combination of biochemistry and massspectrometry that is driving progress in the field offunctional proteomics today.

7 Special attention will be paidto one major technical aspect dominating both the discoveryand analysis parts of functional proteomics which is thehandling of very complex protein mixtures. A protein ofinterest will typically represent only a tiny fraction of thetotal protein present in the source material. More than10 000 different genes might be expressed at the same timein a single cell or tissue and, as mentioned earlier, diversityon the protein level is much higher. In addition to thediversity on the level of primary protein sequence and thepresence of modifications, complexity is further increasedwhen considering the dynamic range of expression levels ofindividual proteins. While some proteins are present inseveral thousand copies per cell, others are just representedby a few molecules.

8 On top of the pure quantities theavailability of proteins for complex retrieval may be muchlower as it is easy to imagine that just a minor percentagemight be in a physiologically active state and possibly partof several protein an example, the cell cell adhesion molecule beta-catenin was originally described to be associated with theplasma membrane protein E-cadherin which mediatescell cell contact [6 8]. Unexpectedly, the protein wasrecently also found to bind to HMG box transcriptionfactors (TCF, LEF-1) which drive the expression ofdownstream target genes of the Wnt-signaling pathway[9 11]. Both interactions are largely independent of eachother and knowledge of both yields information ondifferent aspects of beta-catenin function.

9 While it isstraightforward to purify the stable and fairly abundantcell adhesion complex, the identification of the nucleartranscription factor complex is technically complicatedbecause the percentage of beta-catenin participating to thiscomplex is typically very of protein complexes by affinitychromatographyThe purification of protein complexes has been accom-plished by a multitude of different techniques ranging fromclassical methods such as size exclusion or ion exchangechromatography to different varieties of affinity chroma-tography. Following the arguments that have been madeabout sample complexity in the previous section, it becomesapparent that successful approaches will have to include atleast one highly discriminating separation step.

10 This istypically provided by affinity-based methods. The commontheme of these is the use of an inherent interaction (affinity)of two biomolecules. If one of the molecules is immobilizedon a solid support, the interacting molecule can be purifiedfrom a cell lysate along with associated proteins. Thereare many different such affinity reagents but we will confineourselves to examining those that have proven useful for theretrieval of protein complexes, notably recombinant pro-teins, epitope-tagged proteins and antibodies (Table 1). Formore specialized applications, peptides and nucleic acidshave also been , the discovery of interacting proteins isinfluenced by a number of parameters. Biochemical deter-minants include binding affinities between components ofTable 1.