Analytical Biochemistry 356 (2006) 1–11

www.elsevier.com/locate/yabio

ANALYTICALBIOCHEMISTRY

Review

Peptide, domain, and DNA aYnity selection in the identiWcation and quantitation of proteins from complex biological samples

Filippo Petti, Stuart Thomson, John D. Haley ¤

Translational Research, OSI Oncology, Farmingdale, NY 11735, USA

Available online 9 June 2006

Scalable methods for the analysis of binding interactionswith speciWc protein domains, peptides, and nucleic acid(DNA, RNA, and aptamer) have been used recently to elu-cidate protein interaction networks. The ease and potentialof these aYnity selection techniques coupled to mass spec-trometry (MS)1 to readily deWne the repertoire of potentialinteractions between modular protein domains, posttransl-ationally modiWed peptide motifs, and interactions requir-ing complex assembly on nucleic acids warrant a look atthe technical details and approaches.

The mammalian proteome comprises proteins encodedby approximately 30– 35,000 genes, typically with multiplesplice variants per gene and with multiple proteolytic and/or posttranslationally modiWed isoforms. These are presentin cells in a tissue- and cell type-speciWc manner over a widedynamic expression range. The identiWcation and quantita-tion of proteins from mammalian cells and tissues has beenhindered by the complexity of resolving this large number

* Corresponding author. Fax: +1 631 845 5671.E-mail address: jhaley@osip.com (J.D. Haley).

1 Abbreviations used: MS, mass spectrometry; TAP, tandem aYnity puri-Wcation; MS/MS, tandem mass spectrometry; SILAC, stable isotope label-ing with amino acids in cell culture; ICAT, isotope-coded aYnity tags;iTRAQ, isobaric tags for related and absolute quantitation; EGFR, epi-dermal growth factor receptor; PDGFR, platelet-derived growth factor re-ceptor; GST, glutathione S-transferase; EGF, epidermal growth factor;SDS, sodium dodecyl sulfate; qQ–TOF LC–MS/MS, hybrid quadrupoletime-of-Xight liquid chromatography tandem mass spectrometry; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis;GPCR, G protein-coupled receptor; MALDI–TOF, matrix-assisted laser/desorption ionization time-of-Xight; SCX, strong cation exchange; ESI–MS, electrospray ionization mass spectrometry; FKBP, FK506-bindingprotein; TFA, triXuoroacetic acid; DTT, dithiothreitol; cypA, cyclophilinA; GFP, green Xuorescent protein; ssDNA, single-stranded DNA; dsD-NA, double-stranded DNA; TBP, TATA box-binding protein; MCR,muscle creatine kinase; SRE, sterol responsive element; LDLR, low-densi-ty lipoprotein receptor; SREBP, sterol responsive element-binding pro-tein; SCAP, SREBP cleavage-activating protein; DMSO, dimethylsulfoxide; HVC, hepatitis C virus; IRES, internal ribosome entry sites.

0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2006.05.029

of diVerentially expressed proteins. Electrophoretic [1,2],chromatographic [3], and high-resolution mass spectromet-ric [4,5] approaches have been combined in an eVort todescribe and measure the protein complement of the cell,often under the diYcult conditions of biological perturba-tion [6,7]. IdentiWcation and quantitation of protein interac-tions has been very successful for less complex organisms,most notably in Saccharomyces cerevisiae where cross-com-parison of yeast two-hybrid, tandem aYnity tag proteininteractions, gene expression, and phenotypic screens allowa unique view into protein interactions [8] and the cellularresponses to external stimuli. However, the added complex-ity of the mammalian proteome has required the develop-ment of new approaches and methods to aid in theidentiWcation and measurement of proteins, protein com-plexes, and nucleic acid complexes, often under changingbiological states [9]. Recently, there has been considerableprogress in the use of peptides, protein domains, andnucleic acid polymers as aYnity matrices for the selectionof speciWc interacting proteins and complexes. Enrichedproteins are then identiWable through MS, either by peptidemass Wngerprinting [10] or by peptide fragmentation [11]coupled with automated computer-assisted protein data-base searching based on peptide masses or both peptideand fragment ion masses [10,12,13]. Clear criteria for pro-tein identiWcation by mass spectrometric methods havebeen deWned [14–17]. Both peptides and oligonucleotidesare readily synthesized by high-throughput synthetic meth-ods, and short protein domains can generally be recombi-nantly expressed in high yield and in a folded state. As aconsequence, the use of peptides, domains, and oligonucle-otides as speciWc aYnity matrices allows interrogation ofbiological samples with an ease and throughput not achiev-able with other methods. The range of biological conditionsthat can be rapidly evaluated using current technologymakes this strategy compelling for applications examiningdomain, peptide motif, and protein–DNA interactions.

2 Peptide, domain, and DNA aYnity selection / F. Petti et al. / Anal. Biochem. 356 (2006) 1–11

Furthermore, when coupled to isotope labeling techniques,changes in protein and peptide abundance and posttransla-tional modiWcation can be measured rapidly and reproduc-ibly. For that reason, we review recent progress andtechniques in the coupling of these types of aYnity selectionmethods with MS for the identiWcation and quantiWcationof interacting proteins, often in dynamic biological systemschanging in response to a given stimulus or perturbation.

The uses of aYnity chromatography as a means of sim-plifying proteomic analysis have been widely reviewed.Here we focus on new emerging techniques in protein andnucleic acid aYnity coupled to identiWcation and, impor-tantly, quantitation by MS. For example, this review doesnot include the many approaches to chemical compoundaYnity [18] and protein tag or tandem aYnity puriWcation(TAP)1 tag selection approaches to protein complex isola-tion [19–21], which have been described extensively. Anti-body aYnity approaches to the selection of individualproteins and complexes [22] and to the selection of groupsof proteins, such as those containing acetyl-lysine, methyl-arginine [23], or phosphotyrosine [24–26], have also beendocumented and reviewed extensively.

Attempts to simplify the proteome by aYnity selectioncan impart sample biases that must be reconciled with theoverall project experimental aims. As we will illustrate, inmany cases aYnity selection methods impose a bias that

can be both predictable and advantageous to an under-standing of the complex biology underlying the project.This simpliWcation can greatly assist interpretation of thebiological conclusions in what can be extremely complexsystems. In reviewing the approaches and methods used foraYnity isolation of target proteins, we have tried to includeseveral key details regarding the absolute amount and ratioof aYnity resin and cell lysate, the general conditions forcell lysis, the amount of target coupled to a unit amount ofresin, and (where possible) the expected yields of proteinderived from aYnity selection. These details are provided togive the reader an expectation of outcome associated with agiven approach.

AYnity selection methods can be coupled to MS- andtandem mass spectrometry (MS/MS)-based peptide andprotein quantitation strategies to allow determination ofrelative protein abundance between two or more experi-mental conditions. The most commonly used quantitativeproteomic approaches are shown in Fig. 1 and include themetabolic stable isotope incorporation approach SILAC(stable isotope labeling with amino acids in cell culture)[27], the protein labeling reagent ICAT (isotope-codedaYnity tags) [28,29], and the isobaric peptide labelingreagent iTRAQ (isobaric tags for related and absolutequantitation) [30]. These approaches to peptide quantita-tion have been reviewed in detail [31]. Recent progress also

Fig. 1. Experimental schema for three commonly used mass spectrometry-based methods for peptide and protein quantitation: the metabolic stable iso-tope incorporation approach SILAC (stable isotope labeling with amino acids in cell culture) [27] (A), the protein labeling reagent ICAT (isotope-codedaYnity tags) [28,29] (B), and the isobaric peptide labeling reagent iTRAQ (isobaric tags for related and absolute quantitation) [30] (C). SILAC allows themetabolic incorporation of heavy isotope-containing amino acids into organisms of cells in culture, followed by ratiometric and/or absolute quantitationof “heavy” (i.e., treatment) and “light” (i.e., control) peptides in MS spectra. In the ICAT approach, protein samples are labeled on cysteine residues withbiotinylated tags containing heavy or light isotopes. Isotope-tagged peptides can then be isolated on streptavidin-containing resins, followed again byratiometric and/or absolute quantitation of heavy and light peptides in MS spectra [28,29]. In the iTRAQ approach, protein samples are Wrst subjected toproteolysis, after which peptides are derivitized with either 114, 115, 116, or 117 mass tags on N-terminal and lysine amines and/or on tyrosine. Samplesare mixed and quantitation is performed by MS/MS measurement of these quantitative fragment ions (m/z 114, 115, 116, and 117) released during colli-

Control: 12C/14N-arginineExpt-1: 13C/14N-arginineExpt-2: 13C/15N-arginine

Metabolically label cells

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Affinity selectMix

Trypsin digestLC-MS/MS

Control: Peptide + 114m/z- labelExpt-1 : Peptide + 115m/z- labelExpt-2 : Peptide + 116m/z- labelExpt-3 : Peptide + 117m/z- label

Affinity selectTrypsin digest

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sion-induced dissociation [30]. These approaches to peptide quantitation have been reviewed in detail [31].

Peptide, domain, and DNA aYnity selection / F. Petti et al. / Anal. Biochem. 356 (2006) 1–11 3

has been made in the label-free quantitation of peptide andprotein abundance based on measurement of peak intensityand detector counts [32]. Peptide, domain, and DNAsequence-speciWc interactions can allow further in silicomodeling based on the experimental data sets that can thencomplement new experimental data. Predictive algorithmsare increasingly accessible via the Internet, for example,ScanSite (http://scansite.mit.edu), ProSite (www.expasy.org/prosite/), and InterPro (www.ebi.ac.uk/interpro/) databases.

ProWling domain interactions

The modular interactions of proteins with cellular mac-romolecules, including proteins, DNA, RNA, and carbohy-drates, often is mediated by speciWc folding units withinproteins termed domains. The identiWcation and categori-zation of protein domains according to the speciWc interac-tions they mediate has been an active area of investigationover the past two decades. Comprehensive listings ofdomains and domain interactions have been reviewed inthe literature [33] and publicly available internet sites (seeabove). The coupling of peptides and recombinantlyexpressed protein domains to solid-phase resins, for use asaYnity selection matrices, is an established methodology.Proper folding of small domains has been achieved with rel-atively simple prokaryotic and in vitro reticulocyte systems,avoiding the need for more time-consuming construction ofstable mammalian-expressing cell lines or baculovirus con-struction. The use of peptide and domain solid-phase resinsin the mass spectrometric proWling of protein interactionsand posttranslational modiWcations is an emerging tech-nique enjoying increasing use.

Protein tyrosine kinases frequently are overexpressed andactivated in human cancers [34], and their academic andindustrial study has been widespread. Activated tyrosinekinases phosphorylate substrates, whose phosphotyrosineresidues in turn become binding motifs for the recruitment ofSH2 and PTB domain-containing proteins such as Grb2 andSHC [35,36]. The recruitment of SH2 and PTB domain-sig-naling adaptors is critical for signal transmission and signaltermination [37]. Several laboratories have used domain aYn-ity selection methods coupled to MS to isolate, identify, andquantitate proteins that interact with SH2 domains, forexample, from Grb2 [38,39]. The adaptor protein Grb2 con-tains one SH2 domain and two SH3 domains. Its SH2domain binds tyrosine-phosphorylated sequences [36,40],whereas the major function of the two SH3 domains is todirect complex formation with proline-rich regions of otherproteins. It is involved in growth factor receptor signaling,such as in the epidermal growth factor receptor (EGFR) andplatelet-derived growth factor receptor (PDGFR) signaltransduction pathways, via association of activated tyrosine-autophosphorylated receptors [41] with SH2 domain-con-taining proteins [35]. In addition, Grb2 interacts with othercellular phosphotyrosyl-binding proteins, such as IRS-1 andSHC, via its SH3 domains. The SH2 domain of human Grb2(aa 59–158) was expressed in Escherichia coli as a glutathione

S-transferase (GST) fusion, purifed on glutathione–Sepharose resin, and incubated with detergent lysates fromHeLa cervical carcinoma cells stimulated with epidermalgrowth factor (EGF) for various times. Proteins were elutedin sodium dodecyl sulfate (SDS) sample buVer, gel puriWed,trypsin digested, and subjected to hybrid quadrupole time-of-Xight liquid chromatography tandem mass spectrometry(qQ–TOF LC–MS/MS). Proteins were identiWed by databasesearching using parent and fragment ion mass information.Quantitation of peptide and inferred protein abundance wasachieved by metabolic incorporation of [12C]- or [13C]-labeledarginine, present within the tissue culture media, into HeLacell protein. Using this approach, 228 proteins were identiWed,and 28 of these were modulated by stimulation of the intrin-sic EGF receptor tyrosine kinase activity by EGF. The locali-zation and EGF induction of key proteins was veriWed byconfocal microscopy [38]. These studies were extended usinga second arginine stable isotope (13C/15N), allowing for a timecourse of protein phosphorylation by EGF receptor activa-tion to be measured. In this case, HeLa cell culture mediacontaining either 12C/14N, 13C/14N, or 13C/15N were used toisolate Grb2 SH2-binding proteins 0, 1, 5, 10, and 20min fol-lowing EGF stimulation. These methods allowed the identiW-cation of 202 proteins, 81 of which were induced or repressedgreater than 1.5-fold. Protein phosphorylation changes wereveriWed by immunoprecipitation and/or Western blot [39].

A complementary approach is to use peptide-bindingmotifs for domains of interest, such as those whosesequences bind speciWcally to SH2, SH3, or PDZ domains,to select and isolate interacting proteins, which can then beidentiWed and quantitated by MS. This was pursued suc-cessfully for SH2 and SH3 interactions [42,43]. Biotinyla-ted peptides comprising a tyrosine-phosphorylated SH2interacting motif, a proline-rich SH3 interacting motif, orrespective control nonbinding peptides (15 residues inlength) were prepared by solid-phase synthesis and addedto detergent cell lysates (30 nmol peptide/6 mg lysate).Interacting proteins were captured on peptide-loadedstreptavidin beads, eluted with SDS sample buVer, sub-jected to sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis (SDS–PAGE) separation, subjected to in-geldigestion, and identiWed by LC–MS/MS. The tyrosine-phosphorylated peptide led to the selection of Grb2, aknown interactor, whereas the proline-rich motif fromSOS-1 selected several proteins, including Grb2, Pacsin3,Snz9, CD2AP, and Snx18. Importantly, nonspeciWc bind-ing could be measured by stable isotope labeling of cellswith [12C]- and [13C]-labeled arginine and parallel incuba-tion with either trap peptide or nonspeciWc control peptide,followed by mixing of the two samples and discriminatingbetween speciWc and nonspeciWc binding by the ratiobetween 12C- and 13C-containing peptides [42]. Similarquantitation of nonspeciWc binding may be achieved byother protein- and peptide-labeling techniques such asICAT [28] and iTRAQ [30]. Coimmunoprecipitationexperiments conWrmed that the interactions occurredwithin cultured cells.

4 Peptide, domain, and DNA aYnity selection / F. Petti et al. / Anal. Biochem. 356 (2006) 1–11

A similar approach was used to deWne proteins thatinteract with the SH3 domain of Hck, an Src family proteintyrosine kinase expressed predominantly in granulocyticand monocytic cells. Src family proteins contain SH2, SH3,and tyrosine kinase domains required for protein functionwhere the aim of the study was to identify protein-bindingpartners speciWc to the SH3 domain of Hck [44]. The HckSH3 domain was expressed as a GST fusion in E. coli(NB42 cells) and puriWed on glutathione agarose beads.U937 cell lysates (containing »5 mg protein) were incu-bated with either GST–SH3 resin (containing »50�g SH3domain) or control GST resin in isotonic neutral pH buVerand 1% Triton X-100 for 2 h. Protein complexes werewashed extensively and subjected to SDS–PAGE, and pro-tein bands were visualized by Coomassie staining. SH3-spe-ciWc bands were excised, digested with trypsin, andsubjected to LC–MS/MS using an ion trap mass spectrome-ter. A total of 26 proteins speciWcally interacting with theSH3 domain of Hck were identiWed, and the more interest-ing of these proteins (WASP, WIP, and ELMO) were fur-ther investigated by coimmunoprecipitation and Westernblotting [44].

The suppressor of cytokine signaling (SOCS) proteinsserve as negative regulators of a number of cytokines andhormones. The SOCS box of these proteins was found tointeract with elongins B and C to form a complex contain-ing cullins and Rbx1 with E3 ubiquitin ligase activityresponsible for regulating proteosomal degradation. TheSH2 domain of the SOCS proteins was predicted to link theSOCS–elongin–ubiquitin ligase complex to Janus kinases(JAKs), required for cytokine signaling, to inhibit signaltransmission. SOCS-6 SH2 domain interactions were inves-tigated by aYnity selection MS using His-tagged SOCS-6and SOCS-7 SH2 domains recombinantly expressed in E.coli, refolded, and covalently coupled via an N-hydroxysuc-cinamide linkage to Sepharose resin [45]. A control resin,useful in measuring nonspeciWcally bound proteins, wasprepared by quenching activated resin with ethanolamine.Triton X-100 (1%) extracts were prepared from approxi-mately 6£ 108 cells and precleared with 200 �l of blankSepharose resin to preabsorb proteins preferentially inter-acting with resin. Precleared extracts were incubated with50 �l of SH2 domain resin (3 mg SH2 domain/ml resin).SH2-bound proteins were eluted with SDS sample buVerand subjected to SDS–PAGE, Coomassie-stained bandswere excised and trypsin digested, and peptides wereidentiWed by electrospray ion trap MS and database search-ing. Binding of the SOCS-6 and SOCS-7 SH2 domains toIRS-2, IRS-4, PI-3 kinase p85� and p85� subunits, andtubulin was observed both by MS and Western blottinganalyses [45].

PDZ (PSD-95/discs large/ZO-1) domains are protein–protein interaction domains, approximately 90 residueslong, that bind to the C-terminal residues of their targetproteins, frequently within transmembrane receptors andion channels. These interactions can exist at high aYnity,with the consensus binding sequence containing a hydro-

phobic residue at the C terminus. PDZ domains typicallyare found in combination with other interaction elementsand participate in directing the speciWcity of receptor tyro-sine kinase-mediated signaling [33,46]. EBP50 contains twoPDZ domains located within the N-terminal domain. Sev-eral PDZ domain constructs (aa 1–97 PDZ-1, 138–248PDZ-2, 1–248 PDZ 1 + 2) were expressed in E. coli as GSTfusion proteins [47,48] and bound to glutathione agarose.Triton X-100 lysates of tissues were prepared by homogeni-zation and centrifugation (100,000g) and incubated withaYnity resin. Binding was achieved by incubation ofapproximately 13 �l of aYnity resin with approximately5 mg of lysate in a 1-ml volume at 4 °C for 90 min. Boundproteins were eluted with 2 M NaCl, mixed with SDS sam-ple buVer, and subjected to SDS–PAGE. Coomassie-stained bands were excised, digested, and subjected to iontrap LC–MS/MS and peptide database searching [48].

PDZ domain-containing proteins have also beenobserved to interact within the C-terminal residues of Gprotein-coupled receptors (GPCRs). Domain aYnity selec-tion methods have been used to isolate proteins interactingwith the C-terminal PDZ recognition element within the 5-HT2c receptor [49]. The 90-amino acid C-terminal domainwas recombinantly expressed as a GST fusion protein,immobilized on glutathione–Sepharose resin, and incu-bated with tissue extract (50 �g GST–C terminal 5-HT2cRdomain/20 mg of tissue membrane extract) for 3 h. Enrichedproteins were puriWed by two-dimensional gel methods andvisualized by silver staining. Proteins were excised, digestedwith trypsin, and subjected to peptide analysis usingmatrix-assisted laser/desorption ionization time-of-Xight(MALDI–TOF) and quadrupole TOF methods. Quadru-plicate samples were analyzed. To account for nonspeciWcprotein binding, two control reactions were performed.These employed GST tag alone and GST–C-terminaldomain with an alanine mutation in a residue critical forPDZ domain binding. Proteins speciWcally bound by nativeGST–C terminal region were visualized by silver stainingand identiWed by MS, including the PDZ domain proteinsVeli3 and Dlgh3.

Domain aYnity selection methods have been used todeWne the binding speciWcity of poorly understooddomains such as FF domains (domains with two con-served phenylalanines) [50]. FF domains have beenobserved in nuclear factors regulating transcription andsplicing and in cytosolic RhoGTPases, but their functionhas not been well described. FF domains from CA150, aregulator of RNA polymerase II, were expressed as GSTfusions either singly or in multiple copies (4–6) as theycommonly appear in FF domain-containing proteins.GST–FF domain fusions were expressed in E. coli (BL21)and GST–FF domain-containing resins incubated withcell lysates (prepared in isotonic neutral pH buVer and 1%Triton X-100) for 1–2 h. Isolated proteins were separatedby SDS–PAGE, excised, reduced, alkylated, digested withtrypsin, and subjected to LC–MS/MS using an ion trap.GST resin alone was used as a control for nonspeciWc

Peptide, domain, and DNA aYnity selection / F. Petti et al. / Anal. Biochem. 356 (2006) 1–11 5

binding, a common confounding variable in aYnity isola-tion experiments. A total of 36 proteins speciWc to theGST–FF domain resins were identiWed by databasesearching, and half of these proteins were particularly richin serine or acidic residues and subsequently shown to becritical for FF domain binding [50].

Ubiquitinylation of proteins can be critical to bothlicensing of protein activity and targeting of proteins fordegradation by the 26S proteosome [51]. In this case, theubiquitin chain, a small polypeptide of approximately9 kDa, is ligated to target proteins by a large family of ubiq-uitin ligases. To identify HeLa cell proteins ubiquitylatedin vitro, extracts were incubated with His-tagged ubiquitinand an ATP regenerating system. Western analysis of thereaction mixture using anti-His antibody clearly indicatedconjugation of His–ubiquitin to proteins within the lysate.Ubiquitylated proteins subsequently were isolated by metalchelate chromatography, reduced, carboxamidomethy-lated, and digested with both LysC and trypsin. Peptideswere analyzed by two-dimensional LC combining strongcation exchange (SCX) with a reverse-phase C18 columnprior to online MS and MS/MS analysis using a quadru-pole TOF instrument. A total of 113 proteins were identi-Wed, including components of the 19S proteosomeregulatory subunit, proteins associated with DNA damageresponse, RNA splicing, cytoskeletal assembly, and endocy-tosis [52].

In a complementary approach, the polyubiquitin-bind-ing proteins Rad23 and Dsk2, which interact with the pro-teasome via their ubiquitin-associated domains, were usedto discover protein targets for ubiquitin ligase and isopepti-dase enzymes in yeast cells both with and without the pro-teosome regulatory protein Rpn10 [53]. GST fusionproteins of receptors, Rad23 and Dsk2, were coupled tocyanogen bromide-activated Sepharose 4B resin (100 mMsodium bicarbonate, 0.5 M sodium chloride, pH 8.3). Theresins were subjected to both wild-type and mutant rpn10�yeast cell lysates, expressing His-tagged ubiquitin, for90 min at 4 °C. The resin was then washed with lysis buVer(containing 300 mM sodium chloride and 0.5% Triton X-100), followed by two washes with 2 M sodium chloride.SpeciWcally bound proteins were eluted in urea buVer(buVered 8 M urea and 20 mM imidazole). The eluate wasthen mixed with equilibrated nickel magnetic bead slurryfor 60 min, washed, denatured, and subsequently digestedwith trypsin directly on the beads. The proteolyticallydigested samples were processed by multidimensional chro-matography coupled to electrospray ionization mass spec-trometry (ESI–MS) inline and searched against theSaccharomyces database using SEQUEST. The approachidentiWed 127 proteins that are probable candidate sub-strates for the 26S proteasome and speciWcally 54 proteinsthat were uniquely recovered from the rpn10� cell lysates.Proteins found in the mutant rpn10� cells included cellcycle regulator Sic1 as well as the transcriptional activatorGcn4. By and large, the approach should be germane tomapping ubiquitin ligase substrate networks [54,55] in

mammalian systems where speciWc regulators have beenremoved by gene knockout or attenuated by si-RNAi andsh-RNAi approaches.

Protein interactions with the cytoplasmic regions of theprotein NCAM were determined recently by aYnity selec-tion using recombinantly expressed cytosolic domain [56].The cell adhesion protein NCAM is expressed in multiplesplice forms with diVering capacities to transduce signalsthrough the FGF receptor pathway. NCAM cytoplasmicdomains were expressed in E. coli (BL21) as His-taggedfusion proteins, immobilized on CNBr-activated Sepharose4B, and used to isolate interacting proteins from rat brainlysates [56]. Interactions with phospholipase C-�, LANP,TOAD-64, PP1, syndapin, and PP2A could be observedand veriWed by Western blot using antibodies speciWc to theidentiWed proteins.

Peptide and cyclic peptide interactions

Short synthetic peptide and cyclic peptide aYnity resinshave been used successfully to identify binding proteins.Examples include the bioactive cyclic peptides FK506,didemnin, and trapoxin that were shown to bind andrequire FK506-binding protein (FKBP) [57], EF-1 alpha[58], and histone deacetylase [59], respectively. Cyclosporinewas shown by a conventional protein puriWcation strategyto interact with the cellular protein cyclophilin [60]. The useof cyclosporine aYnity resins greatly simpliWes the isolationand identiWcation of cyclosporine-binding proteins by MS(G. Williams and J. Haley, unpublished). For example, abioactive cyclosporine A analogue can be prepared bydirect chemical modiWcation at position 3 (Fig. 2). Usingthe resulting functional group, the ligand was readilyimmobilized onto a solid support. The solid support chosenwas AY-Gel (Bio-Rad, Hercules, CA, USA), a crosslinkedagarose resin that has a size exclusion limit of 2–5million MW and is stable over a pH range of 2 –12 and tohigh levels of chaotropes. The capacity of the resins isapproximately 13�mol/ml, which equates to approximately15 mg of cyclosporine ligand per milliliter of packed solidsupport. The resins were stored in 20% aqueous ethanol as

Fig. 2. Cyclosporin aYnity resin coupling used in aYnity capture of pro-teins interacting with the cyclic peptide cyclosporine (G. Williams andJ. Haley, unpublished).

O

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MeLeu23

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6 Peptide, domain, and DNA aYnity selection / F. Petti et al. / Anal. Biochem. 356 (2006) 1–11

preservative prior to use. Tissue was extracted using TPERreagent (Pierce, Rockford, IL, USA) at a ratio of 20:1(extraction buVer:tissue, v/w) on ice in the fume cupboardusing a PolyTron homogenizer in the presence of proteaseinhibitors (1:50, Sigma, St. Louis, MO, USA). Approxi-mately 100�l of cyclosporine resin was incubated withapproximately 20 ml (400 mg) of tissue extract in milddetergent buVer at 4 °C for 2 h and washed extensively withTris-buVered saline, followed by elution of bound proteinswith 5£100 �l of 0.1% triXuoroacetic acid (TFA) and 5%methanol. Eluted proteins were dried under vacuum; resus-pended in 1 M guanidine HCl, 100 mM NH4CO3, and0.5 mM dithiothreitol (DTT); and subject to digestion withtrypsin (Promega, Madison, WI, USA) or GluC (RocheApplied Science, Indianapolis, IN, USA) for approximately18 h at 37 or 25 °C, respectively. Digests were desalted usingC18 ZipTips (Millipore, Billerica, MA, USA) eluted with50% acetonitrile and 0.1% TFA. The peptide digests wereanalyzed by MALDI–TOF MS using �-cyano-4-hydroxy-cinnamic acid (20 mg/ml 4-HCCA, 30% acetonitrile, 0.1%TFA) as matrix mixed 1:1 with sample, yielding spectrawith a high signal/noise ratio (Fig. 3). Peptide spectra werecompared with protein databases (GenBank NR andSwiss–Prot) using Knexus and Mascot search tools, wherespectra were internally calibrated using trypsin or gluCautodigestion products. Major peaks were found to corre-spond with cyclophilin A (cypA), and to a lesser extentcyclophilin B, with a mass accuracy of less than 30 ppm(Fig. 2), a Z score of 98% (P» 0.02), and 68% sequence cov-erage (total sequence [not adjusted for the MS range col-lected]). Peptide identiWcations were conWrmed by fragmention spectra obtained by LC–MS/MS using an orthogonalquadrupole TOF instrument where peptides were chro-matographed on a reverse-phase C18 column (0.1£150 mm, Michrom Bioresources, Auburn, CA, USA) usinggradient elution (2–60% acetonitrile) at a Xow rate ofapproximately 400 nl/min. Cyclosporin aYnity resins pro-

vided an eVective tool in examining cypA protein com-plexes (G. Williams and J. Haley, unpublished). The aYnityapproach yielded MS and MS/MS spectra with acceptableprotein coverage and high detector counts.

A similar synthetic peptide aYnity approach was used todeWne proteins interacting with the tumor suppressor pro-tein p21Cip1/Waf1 [61]. p21Cip1 was known to interactthrough its C-terminal domain with PCNA, an interactionrequired for cell cycle control. Here a biotinylated, C-termi-nal, 20-amino acid fragment was used to isolate interactingproteins, which were further puriWed by two-dimensionalgel electrophoresis, digested with trypsin, and subjected toMALDI–TOF MS. Several interacting proteins, includingp48CAF-1, Hsp70, BiP, cadmodulin, nucleolin, and PCNA,were identiWed by database searching using peptide massinformation. Protein identiWcations were further reWnedusing peptide sequence information obtained by LC–MS/MS analysis of the unfractionated tryptic digests.

The cellular targets of peptides with anticancer activitywere identiWed using a similar aYnity selection approach.Bioactive antiproliferative peptides were identiWed byscreening approximately 109 random green Xuorescent pro-tein (GFP) 20-mer peptides by retroviral transduction. Toidentify the target proteins of active peptides, biotinylatedor control peptides were incubated with A549 cell extracts(prepared by lysis in buVered 1% Triton X-100). Cellextracts (5 mg/ml) were precleared with agarose beads (5 mllysate/250 �l beads) prior to aYnity selection with biotinyl-ated peptide immobilized on streptavidin agarose beads(1 ml precleared cell extract/250�l peptide resin, equivalentto a 50-�M free peptide solution). Reactions were per-formed in quadruplicate. Captured proteins were digestedwith LysC and trypsin and were subjected to LC–MS/MSusing both ion trap and quadrupole TOF mass spectrome-ters. The three bioactive peptides analyzed were shown tointeract with importin-� (not observed in alanine mutantcontrol peptides), and their antiproliferative activity was

Fig. 3. Peptide MS spectra from cyclosporine aYnity selection. Cyclophilin A was isolated by cyclosporine aYnity capture, reduced, alkylated, digestedwith trypsin, and subjected to MALDI–MS using a dihydrobenzoic acid matrix (G. Williams and J. Haley, unpublished). CypA, peptides derived fromcyclophilin A identiWed at a mass accuracy of 30 ppm.

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1946.6477

1988.63531154.4754

1379.5823

1601.5175

1614.5002848.39761055.4666 2791.6181

737.3569 1274.5807 1949.67711541.5045880.5072 2808.60771797.53661620.53311157.4894842.5139 1402.5662 1970.6099 2774.63871636.5660

CypA CypA

CypA

CypA

CypA

[M+H]+

CypA

CypA CypA

CypA

649.0 1119.4 1589.8

Mass (m/z)

2060.2 2530.6 3001.0

% In

ten

sity

Peptide, domain, and DNA aYnity selection / F. Petti et al. / Anal. Biochem. 356 (2006) 1–11 7

proposed to occur through interference with nuclear trans-port [62].

DNA aYnity selection

AYnity methods for the isolation and characterizationof DNA- and RNA-binding proteins are potentially power-ful techniques if nonspeciWc binding events are minimizedand considered in the analysis of the data sets derived fromthe work. Both DNA and RNA oligonucleotides up toapproximately 100–150 bases in length are readily synthe-sized by current solid-phase methods, where parallel syn-thesis allows the simultaneous interrogation of wild-typeand mutant templates within a single experiment. Panels ofnucleic acid probes can be prepared in 96-, 384-, and 1536-well plates, amenable to large-scale experiments. Biotin tagsare easily incorporated, allowing high-aYnity isolation ofprobe-bound proteins by streptavidin, neutral avidin, ormonomeric avidin resin interaction. Protein–nucleic acidinteractions can be interrogated in a facile manner by use ofmultiple probes and controls in replicate experiments, mak-ing this type of methodology attractive to those studyingthe assembly of transcription complexes, DNA replicationcomplexes, DNA repair complexes, and RNA–proteinsplicing and transport complexes. In these experiments,mutant oligo controls or template “walking” controls canbe readily incorporated to facilitate data interpretation.Perhaps the most general complication in the use of nucleicacid polymers in the aYnity selection of interacting pro-teins is the relative abundance of nuclear and cytosolicnucleic binding proteins that can confound these experi-ments. Thus, proper controls and methods to minimizenonspeciWc binding are required. These necessitate substan-tial preabsorption, for example, with bulk nonspeciWc sin-gle-stranded and double-stranded DNA (ssDNA anddsDNA, respectively), with RNAs or tRNAs, and withnucleic acid polymers such as poly(dI/dC). Despite thesesteps, unexpected interactions can still confound data inter-pretation. For example, DNA repair proteins and com-plexes may be detected in dsDNA capture experiments, duein part to the presence of an exposed DNA terminus. Nev-ertheless, to some extent, these interactions can be predictedand subject to analysis. Examples are presented to aid inthe interpretation of such data.

One of the most complete studies using this type ofapproach to examine DNA–protein interactions was thatby Ranisch and coworkers deWning and quantitating theassembly of preinitiation transcription complexes contain-ing RNA polymerase II [63]. In this case, an approximately400-bp promoter template was prepared by PCR ampliWca-tion in which the 5� primer was biotinylated. DNA templatewas incubated with nuclear extracts for 60 min in buVer asdescribed previously [63]. At this point, the bound templatewas digested with PstI under standard conditions to releasethe fragment of interest. After the beads were removed bycentrifugation, the supernatant was collected, leavingbehind any proteins still nonspeciWcally bound to avidin

resin. Most important, nuclear extracts contained eitherfunctional or nonfunctional TATA box-binding protein(TBP), a protein required for full complex assembly. Byselectively labeling the two capture reactions (those with orwithout functional TBP) with ICAT tags containing diVer-ent masses [28], the relative abundance of peptides can bemeasured. This allows researchers to both infer proteinabundance and distinguish between speciWc RNA polymer-ase II preinitiation complexes and proteins interacting inthe absence of functional TBP. This control step allowed amore functional interpretation of the 326 proteins identi-Wed in the study. The investigators identiWed 49 proteinsthat were changed in abundance by at least 1.9-fold by theinclusion of functional TBP in the nuclear extract, and theywent on to identify novel proteins in transcription initiationassemblies such as YDR079c-a [64]. The key step in thesestudies was the use of ICAT to allow for protein quantita-tion, where only those proteins enriched by the addition ofTBP were highlighted in the analysis of the quantitativeICAT labeling data [65]. Thus, nonspeciWcally bound pro-teins, unaltered in abundance by the inclusion of TBP,could be ignored [63]. A similar strategy using quantitativeproteomics to discern key regulatory proteins, as opposedto proteins nonspeciWcally interacting with the template,has since been applied to the study of multiple transcriptionunits. For example, Six4 was identiWed as a key regulator ofmuscle creatine kinase (MCR) transcription, binding to theMCR positive control element Trex [66].

The interaction of serum response factor with biotinyla-ted DNA containing a sterol responsive element (SRE) wasreadily detected in DNA aYnity/MS methods [67].HEK293 cells were serum starved for 24 h and restimulatedwith EGF 20� prior to lysis in hypotonic buVer containing0.2% NP-40. Biotinylated PCR products were bound tomagnetic streptavidin beads (200 pmol DNA/mg beads).Protein binding was achieved by incubation of 600�gwhole cell lysate with 1 mg SRE beads for approximately15 min prior to washing (3£) and separation by SDS–PAGE. SYPRO Ruby- or Coomassie-stained bands wereexcised, digested with trypsin, and subjected to nanosprayMS/MS using orthogonal TOF instruments. Several tran-scription factors were shown to interact with the SRE,including SRF, Elk-1, and possibly four novel proteins(DDX-1, CGI-99, RS3, and RS4). Similar experiments wereperformed using a recognition element for AP-1, whereinteraction with the transcription factor YB-1 observed byDNA aYnity/MS/MS may explain YB-1 repression of AP-1-dependent transcription [68]. A detailed investigation ofthe parameters related to the stability and MS of DNA–protein complexes was reported recently [69].

Ancillary protein–protein interactions associated withthe DNA binding also can be monitored by DNA aYnityMS experiments. For example, a compound that speciWcallyactivated the transcription of the low-density lipoproteinreceptor (LDLR) (L. Scheafer, R. Kirsch, and J. Haley,unpublished) was found to activate sterol responsive ele-ment-binding protein (SREBP) cleavage-activating protein

8 Peptide, domain, and DNA aYnity selection / F. Petti et al. / Anal. Biochem. 356 (2006) 1–11

(SCAP). Transcription factor SREBP is a key positive regu-lator of LDLR transcription that is released from the endo-plasmic reticulum by SCAP activation [70]. SREBP–DNAcomplex interactions were investigated using a biotinylatedSREBP-binding site and adjacent sequences from theLDLR promoter. In this case, liver-derived HepG2 cells(4£108 cells Wnal) were exposed to either dimethyl sulfoxide(DMSO, 0.2%) or selective SCAP-activating compound for5 h prior to harvest. Nuclear extracts were prepared [71] andstored at ¡80 °C. Equal amounts of complementary oligo-nucleotide comprising the LDLR proximal promoter weremixed in 100 mM KCl to a Wnal concentration of 1�M,heated to 94 °C, and slowly cooled to room temperature.Approximately 500�g of nuclear extract was made (20�g/ml with poly(dI/dC)–poly(dI/dC) and 5�g/ml with soni-cated chromosomal DNA) and incubated at 25 °C for 5 minto reduce nonspeciWc interactions with nucleic acid. Biotin-ylated double-stranded capture oligonucleotide was added(0.1–10 nmol/ml) and incubated at 25 °C for 30 to 60 min.Biotin–DNA-bound proteins were bound to neutral avidinresin (Pierce) equilibrated with 20 mM Hepes (pH 7.6),0.5 mM DTT, 2 mM MgCl2, and 100 mM KCl, includingprotease and phosphatase inhibitors at 4 °C. Bound DNA–protein complexes were washed extensively (10 column vol-umes) with equilibration buVer, washed three times withTris–KCl, and eluted with 0.1% TFA and 5% MeOH(5£50�l). Sample volume was reduced under vacuum tonear dryness. Samples were resuspended in 8 M guanidineHCl, 10 mM DTT, and 50 mM NH4CO3; carboximidome-thylated; diluted to 1 M guanidine HCl with 50 mMNH4CO3; and cleaved with modiWed trypsin (20 ng/ul) for18 h at 37 °C. Resulting peptides were desalted by micro C18step chromatography (C18 ZipTip, Millipore). The peptideswere analyzed by MALDI–TOF MS, and peptide assign-ments were conWrmed by electrospray LC–MS/MS using an

orthogonal TOF instrument. Repeated DNA capture/MSexperiments identiWed transcription components, includingSREBP-1 and SREBP-2, YY1, CBP/p300, ARC complexproteins, several RNA polymerase II complex proteins,TFE3, and NF-Y subunits. Two of these proteins, SREBPand YY1, were highly relevant to the regulation of LDLRtranscription. The interaction of SREBP, a positively actingfactor required for LDLR transcription, with the LDLRpromoter aYnity resin could be veriWed by immunoblotexperiments (data not shown), where SCAP-activating com-pound led to an increase in DNA-bound SREBP. Interest-ingly YY1 peptides were detected only in MS experimentsfrom control cells; no YY1 peptides could be identiWed incells treated with the SCAP-activating compound. Com-pound-induced changes in transcription factor complexescontaining YY1 were veriWed by immunoblot using twodiVerent anti-YY1 antibodies (Fig. 4A). The DNA aYnityprotein extracts were visualized by SDS–PAGE andSYPRO staining, where equal loading of the captured mate-rial was observed (Fig. 4B). YY1 has been shown to be astrong negative regulator of LDLR transcription by inhibit-ing the activity of SREBP and was subject to perturbationby the chemical inducer of SCAP activity and LDLR tran-scription. The binding of SREBP and the modulation ofYY1 could be readily observed by a DNA aYnity/MSapproach without the need for gel separation of proteinsbound to the DNA–avidin resin. However, many DNArepair complex proteins, including proteins belonging to theRAD51 complex, could also be identiWed, presumably byinteraction with the free dsDNA terminus. This backgroundof interacting factors complicated analysis of this type ofdirect DNA binding experiment.

A study examining the speciWc requirements for detec-tion of protein binding to AP-1, PU-1, RARa, Pura, andGABP DNA recognition sites was performed [72] using

Fig. 4. DNA aYnity capture of SREBP–YY1 complex perturbed by SCAP inhibition. (A) Immunoblot of protein isolated by LDLR promoter DNA con-taining the binding site for SREBP. Two diVerent antibodies for YY1 show decreased interaction between the SREBP–DNA complex and YY1 in thepresence of a SCAP inhibitor. SREBP and YY1 interactions with the DNA template initially were characterized by MALDI–MS and LC–MS/MSapproaches. cyto, cytosolic. (B) SYPRO Ruby-stained loading control for the immunoblot experiments in (A) showing equal loading of protein for SDS–PAGE (L. Scheafer, R. Kirsch, and J. Haley, unpublished).

+ -

SCAP inhibitor - + - + - + - +

cyto nuclear cyto nuclear

YY1 Ab-1 YY1 Ab-2

A B

Peptide, domain, and DNA aYnity selection / F. Petti et al. / Anal. Biochem. 356 (2006) 1–11 9

nuclear extracts further puriWed by phosphocellulose chro-matography. This latter step, together with negativeselection of the extracts with nonspeciWc biotinylated dou-ble-stranded oligonucleotides, was found to be critical inobtaining speciWc binding with reduced interference bynonspeciWc nucleic acid-binding proteins. Essentiallynuclear extracts were prepared by the method of Dignam[73], bound to phosphocellulose (P11), and eluted using anNaCl gradient, followed by dialysis. P11 fractions wereincubated with double-stranded oligonucleotide attachedto streptavidin magnetic beads (200 pmol double-strandedoligonucleotide/mg beads) in extract buVer containing oli-gonucleotide and poly(dI/dC) (0.1 mg/ml each) at 4 °C andeluted with extract buVer containing 0.5 M NaCl. Proteinswere separated by SDS–PAGE, Coomassie stained, excised,subjected to in-gel trypsin digestion, and analyzed by MS.

Protein–RNA interactions have similarly been investi-gated by aYnity MS methods. For example, polycistronicmessenger RNAs are transcribed from the hepatitis Cvirus (HCV), giving rise to multiple translation productsthrough ribosome recognition and binding to internalribosome entry sites (IRES). To better understand theprotein assemblies that regulate use of IRES, biotinylatedRNA comprising the HCV IRES sequence was used as anaYnity template for protein interaction studies by MS[74]. Essentially, cells were lysed in hypotonic buVer,nuclei were removed by centrifugation (1000g), and asupernatant S10 fraction was obtained (10,000g, 20�). Bio-tinylated, in vitro transcribed IRES RNA was incubatedwith S10 extracts and aYnity puriWed using streptavidinresin. RNase inhibitor was included in the binding andwash buVers. A fraction of the bound protein was elutedwith SDS sample buVer and separated by SDS–PAGE,and bands were excised, trypsin digested, and subjected toLC–MS/MS. The remainder of the material was elutedwith 8 M urea, reduced, alkylated, diluted to 2 M urea,trypsin digested, desalted by C18 HPLC (1 mmi.d.£ 5 mm), and subjected to two-dimensional LC–MS/MS (SCX and C18). MS was performed on a quadrupoleTOF instrument. To control for nonspeciWc binding, thebiotinylated complementary RNA template was in vitrotranscribed. Quadruplicate biological experiments wereperformed, and only proteins occurring in two of fourreactions were further considered. A total of 22 proteinsspeciWcally interacting with the IRES RNA template andnot the antisense control were identiWed. The relativeabundance of these proteins was estimated by percentageprotein coverage, that is, the number of observed trypticpeptides divided by the number of theoretical tryptic pep-tides within the mass range analyzed.

Similarly, protein interactions with nucleic acid apta-mers have been demonstrated by aYnity approaches. Inthis application, both protein-speciWc and scrambledaptamers were covalently attached to a fused silica slideto create DNA-coated spots. The oligonucleotide-coatedslides were incubated with proteins, rinsed with buVer toremove nonspeciWcally bound proteins, and allowed to

dry. The subsequent addition of the MALDI matrix andsinapinic acid allowed for the aptamers to unfold andrelease the proteins after crystallization. Ultimately, theslides were mounted directly to a MALDI plate and ana-lyzed by TOF MS for thrombin capture versus nonspe-ciWc protein binding. The aptamer spots generally yieldedsignals 10 times greater than signals detected for thescrambled oligonucleotide spots, proving successful aYn-ity capture of thrombin and prothrombin by the protein-speciWc aptamers. Furthermore, following both an addi-tional rinse and reconstitution process of the aptamer, theslide surface becomes ready for another experiment, pro-viding a DNA aYnity capture matrix that is reusable [75].

Conclusions

Scalable methods for aYnity selection using domains,peptides, and nucleic acids have been described and reWnedrecently. These methods have the potential to measure alarge number of interactions within a relatively small num-ber of experiments. The tendency of these large-scale pro-teomic analyses to generate new testable hypotheses isperhaps their strongest attribute. On the other hand, inter-actions are measured on a global scale, where structuralinformation related to cellular localization often is lost.Thus, one measures the repertoire of possible interactionsrather than the interactions that actually occur within thecell at any given moment or in response to a given biologi-cal stimulus. The constraints of protein compartmentaliza-tion within the cell, such as Xuorescent colocalization, needto be considered in follow-up studies.

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