Affinity capture of specific DNA-binding proteins for mass spectrometric identification

Affinity Capture of Specific DNA-Binding Proteinsfor Mass Spectrometric Identification

Mariana Yaneva and Paul Tempst*

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, and Weill Graduate School of Medical Sciences,Cornell University, New York, New York 10021

We describe a general approach for affinity microcaptureof site-specific, nucleic acid-binding proteins. The majordifficulties to developing this method into a widely ap-plicable protocol derived from the need for a massiveenrichment and the inadvertent, extensive binding ofnonspecific proteins to the bait. On the basis of a detailedanalysis, we propose (i) a one-step fractionation of crudeextracts on P11 phosphocellulose, followed by (ii) adiscrete series of positive/negative selections on wild-typeand site-mutated ligand DNA in a magnetic microparticu-late format, with cobalt magnets, concatamerized andbiotinylated ligands, selective salt conditions, and im-proved competitor DNAs. We also present rules fordetermining the precise number and order of selections.The approach and protocol allowed isolation of four, low-abundance transcription factors and repressors from 2× 109 cultured leukemia cells. Captured proteins were10-20 000-fold enriched from the nuclear extract, in aform and amounts that permitted facile MALDI-TOF andTOF/TOF MS-based protein identification. This is 1-2orders of magnitude better than many previous efforts andin a fraction of the time (∼1 factor/week). The methodcan be applied to any protein that binds DNA, includingthose with modest to low affinity, and bridges functional-biochemical studies on replication, transcriptional regula-tion, and DNA repair with the analytical power of massspectrometry-based proteomics.

Targeted proteomics denotes the examination of subsets ofthe proteome. We seek to apply this concept to the biochemicalanalysis of genome function, integrity, and dynamics. Transcrip-tion, replication, recombination, and DNA repair all involve theaction of sequence-specific, DNA-binding proteins. Those interactwith additional proteins, some also binding DNA directly andothers tethered to it through the partner. In this way, largefunctional complexes are formed and similarly anchored to specificDNA sites.1-3 Biochemical analysis requires purification, identifica-tion, reconstitution, and functional characterization of the com-

ponents. Targeted proteomics can contribute to this by generatinga catalog of physical interactions that may help define thefunctional modules. Many of the auxiliary proteins, enzymes, andcomplexes have important, more general functions and are presentin the nucleus in small to moderate amounts. By contrast, thosebinding at select sites, such as transcription factors (TFs) tospecific promoters, represent only 0.01-0.001% of the total cellularprotein.4

Historically, purification of TFs by conventional chromato-graphic methods has been very difficult, often requiring nuclearextract from hundreds to several thousand liters of cultured cellsto achieve a 10 000-100 000-fold enrichment and still recoverenough protein for chemical and functional analysis.4-6 Theinherent capacity of these proteins to bind nucleic acids has longbeen exploited as a means to purification, initially by couplingtotal calf thymus DNA to solid supports7 and later by immobilizingselected DNA fragments or synthetic oligonucleotides for sequence-specific affinity chromatography.8,9 Attachment is either donethrough chemical means, usually by CNBr activation,4,5 or byutilizing the biotin- streptavidin system.8,9 Still, sequence-specificDNA affinity chromatography has rarely, if ever, succeeded inpurifying human TFs to homogeneity directly from crude extract,a shortcoming related to (i) the massive enrichment factor thatis required and (ii) the presence of large numbers of abundant,nonspecific DNA- and RNA-binding proteins in the nucleus.Therefore, multicolumn chromatography is still very much thenorm with an affinity step at the end, usually in the presence ofnonspecific competitor DNAs such as Escherichia coli DNA orpoly(dI-dC).7,10 Most protocols remain laborious and very time-consuming and generally end with large-volume preparations,impeding downstream mass spectrometric analysis. Subsequentconcentration of the sample by evaporation or precipitationfrequently leads to a large or total loss of the proteins.

Although the use of mass spectrometry (MS) has made proteinidentification several orders of magnitude faster and more sensi-

* To whom correspondence should be addressed at Memorial Sloan-KetteringCancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-8923. E-mail: [email protected].(1) Carey, M.; Smale, S. T. Transcriptional Regulation in Eukaryotes; CSHL

Press: Cold Spring, Harbor, NY, 2000.(2) Malik, S.; Roeder, R. G. Trends Biochem. Sci. 2000, 25, 277-283.(3) Naar, A. M.; Lemon, B. D.; Tjian, R. Annu. Rev. Biochem. 2001, 70, 475-

501.

(4) Kerrigan, L. A.; Kadonaga, J. T. In Current Protocols in Molecular Biology;Asubel, F. M., et al., Eds.; John Wiley and Sons: New York; 1993; Unit12.10, pp 1-11.

(5) Kadonaga J. T. Methods Enzymol. 1991, 208, 10-23.(6) Andrews, N. C.; Erdjument-Bromage, H.; Davidson, M. B.; Tempst, P.; Orkin,

S. H. Nature 1993, 362, 722-728.(7) Alberts, B.; Herrick, G. Methods Enzymol. 1971, 21, 198-217.(8) Kasher, M. S.; Pintel, D.; Ward, D. C. Mol. Cell. Biol. 1986, 6, 3117-3127.(9) Blanks, R.; McLaughlin, L. W. Nucleic Acids Res. 1988, 16, 10283-10299.

(10) Gadgil, H.; Jurado L. A.; Jarrett, H. W. Anal. Biochem. 2001, 290, 147-178.

Anal. Chem. 2003, 75, 6437-6448

10.1021/ac034698l CCC: $25.00 © 2003 American Chemical Society Analytical Chemistry, Vol. 75, No. 23, December 1, 2003 6437Published on Web 11/01/2003

tive,11,12 the classical purification protocols have been slowly ornot at all adapted, resulting in virtually zero overall time and effortsavings. Instead, recent methods and technology research in theproteomics field has been largely directed at mass spectrometrichardware, software, quantitational issues, and mixed analysis, inthe hope of overcoming current limitations in sample preparationand homogeneity.11,12 While laudable, this approach can be costlyand in some cases prohibitively sophisticated for all but a fewexpert laboratories. Real micro-biochemical methods are thereforeneeded to match the analytical ones and to provide a bridgebetween classical molecular biology/biochemistry and massspectrometry-based protein identification and proteomics.

The ideal method starts with the smallest possible amount ofcells or tissue and follows a fast and efficient protocol to prepareprotein(s) in amounts, form, and final volume fully compatible withpopularly used MALDI-TOF-based peptide mass fingerprinting oran alternative identification technique. This has been tried by one-step capture from crude extract with direct MS analysis on acommercial SELDI-TOF system.13,14 However, these efforts didnot progress beyond detecting a protein with an apparent molec-ular mass somewhere in the range of what was expected for theanalyte;14 positive identification was either not made or, in caseof an abundant bacterial protein, purified off-chip for standard massfingerprinting.13 A more pragmatic alternative is off-line captureon magnetic particles coated with double-stranded DNA thatcontains specific binding sequences for the factor(s) of interest.In its early configuration, the technique was used for purificationof yeast TFs without, however, making any attempts to identifycaptured proteins.15 This method also exists in a one-step,microassay format with detection by Western blot,16 rather thanMS. More recent applications with successful identification stillrequired the inclusion of four chromatographic steps before finalcapture and MS readout.17 There is scant evidence in thepublished literature that one-step capture with MS-based identi-fication would, in fact, be feasible at all as a routine method.Nordhoff and colleagues identified RXRR and PPARγ in this waybut only after ectopic expression in yeast or after inducedexpression in cultured mouse fibroblasts.18 Finally, Masternak etal. discovered a novel RFX-associated protein after a single roundof DNA affinity purification yielded about 500 ng (∼15 pmol) formass spectrometric analysis,19 a uncommonly large amount bytoday’s standards. All in all, there is currently no robust, generallyapplicable method for affinity capture of specific DNA-bindingproteins.

Here, we report a fast, simple, and highly efficient method forprotein capture on specific DNA-magnetic beads and identifica-tion by MALDI-TOF MS. It involves a single-step fractionation ofcrude extracts on phosphocellulose and a discrete series ofpositive/negative selections and ends with purified protein,concentrated in a small volume, that allows successful MS analysis.After appropriate optimizations, this method can be applied to anyfactor, of human or other origin, that binds DNA including thosewith modest to low affinity.

EXPERIMENTAL SECTIONAbbreviations: DNA, deoxyribonucleic acid; ds, double-

stranded; EMSA, electrophoretic mobility shift assay; MALDI-TOF, matrix-assisted desorption/ionization time-of-flight; MS,mass spectrometry; mut, mutated; NB4, human myeloid leukemiacell line; NE, nuclear extract; P11, phosphocellulose; PCR, poly-merase chain reaction; PMF, peptide mass fingerprinting; ppm,parts per million; SDS, sodium dodecyl sulfate; ss, single-standed;TF, transcription factor; wt, wild type.

Materials. Tween-20 was purchased from Fisher Scientific(Pittsburgh, PA); NP-40 was from Sigma (St. Louis, MO); acrylamide, bis(acryl amide), and Coomassie Blue R250 were fromBioRad (Hercules, CA); PVDF Immobilon-P membranes werefrom Millipore (Bedford, MA). Poly(dI:dC) was purchased fromAmersham Pharmacia (Piscataway, NJ). Oligo(dI:dC), 30 bp inlength, was custom synthesized by Integrated DNA Technologies(IDT, Coralville, IA). Regular and 5′-biotinylated (with 6-carbonlinker), single-stranded oligonucleotides were either custom-synthesized by IDT or purchased in double-stranded form fromSanta Cruz Biotechnology (Santa Cruz, CA). Antibodies to PU.1,Fos, Jun, and RARR were also from Santa Cruz. Rabbit anti-GABPRpolyclonal antibodies were custom-raised by Pocono Rabbit Farm(Canadis, PA) against KHL-coupled, synthetic peptides (Yanevaet al., unpublished).

Cell Culture. Human promyelocytic leukemia NB4 cells weregrown at 37 °C and 5% CO2 in RPMI-1670 medium (Life Technolo-gies, Rockville, MD) supplemented with 10% fetal calf serum(Sigma), nonessential amino acids, and penicillin and streptomycinat 5 µg/mL each. Cell cultures were passaged twice a week tomaintain the cell density between 0.5 and 1.5 × 106 cells/mL.

Preparation of Nuclear Extracts (NE) and Chromatogra-phy on Phosphocellulose (P11). All procedures were per-formed at 4 °C. Nuclear extracts from NB4 cells were preparedas described previously20 and were then fractionated on a phos-phocellulose P11 (Whatman, Clifton, NJ) column equilibrated withbuffer D (20 mM HEPES pH 7.9, 0.2 mM EDTA, 0.5 mM DTT,0.01% NP-40, 0.2 mM PMSF, and 10% glycerol) containing 75 mMNaCl. As a rule, approximately 3 mg of NE is loaded onto 1 mLof P11 resin. Bound proteins were eluted either with a lineargradient of 0.075-0.85 M NaCl or stepwise with 0.1, 0.3, 0.5, and0.85 M NaCl, in the same buffer. Gradient or stepwise, the entireelution is done with 20 column volumes and at a flow rate of 0.4mL/min. Fractions containing the respective DNA-binding activi-ties, as monitored by EMSA (see below), were pooled, dialyzedovernight at 4 °C against 50 volumes of buffer D containing 0.1M NaCl, and then bound directly to DNA-magnetic beads,

(11) Yates, J. R., 3rd. Trends Genet. 2000, 16, 5-8.(12) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207.(13) Forde, C. E.; McCutchen-Maloney, S. L. Mass Spectrom Rev. 2002, 21, 419-

39.(14) Bane, T. K.; LeBlanc, J. F.; Lee, T. D.; Riggs, A. D. Nucleic Acids Res. 2002,

30, 69.(15) Gabrielsen, O. S.; Huet, J. Methods Enzymol. 1993, 218, 508-530.(16) Kumar, V. N.; Bernstein, L. R. Anal. Biochem. 2001, 299, 203-210.(17) Schweppe, R. E.; Melton, A. A.; Brodsky, K. S.; Aveline, L. D.; Resing, K.

A.; Ahn, N. G.; Gutierrez-Hartman, A. J. Biol. Chem. 2003, 278, 16863-16872.

(18) Nordhoff, E.; Krogsdam, A. M.; Jørgensen, H. F.; Kallipolitis, B. H.; Clark,B. F. C.; Peter Roepstorff, P.; Kristiansen, K. Nat. Biotechnol. 1999, 17,884-888.

(19) Masternak, K.; Barras, E.; Zufferey, M.; Conrad B.; Cortals, G.; Aebersold,R.; Sanches, J. C.; Hochstrasser, D. F.; Mach, B.; Reith, W. Nat. Genet. 1998,20, 273-277.

(20) Dignam, J. D.; Martin, P. L.; Shastry, B. S.; Roeder, R. G. Methods Enzymol.1983, 101, 582-598.

6438 Analytical Chemistry, Vol. 75, No. 23, December 1, 2003

constructed with concatamerized oligonucleotides (see below),and equilibrated with the same buffer.

Concatamerization of DNA Oligonucleotides. Multimers ofDNA-binding sites were generated by a self-priming PCR method21

using two complementary, direct repeats of single-strandedoligonucleotides with either wild-type or mutant versions ofspecific binding sites (Table 1), as required. Only the forward, ssoligonucleotides were biotinylated at the 5′ end, using a 6-carbonspacer arm (IDT). PCRs (50-µL volume) contained 460 ng of eacholigonucleotide, 8 µM of dNTPs (Roche Molecular Biochemicals,Indianapolis, IN), and 2 units of “Vent” polymerase (New EnglandBioLabs, Beverly, MA) in 10 mM KCl, 10 mM (NH4)2SO4, 3.5 mMMgSO4, 0.1% Triton X-100, and 20 mM Tris-HCl pH 8.8 (at 25 °C).Cycling conditions were optimized for each pair of oligonucle-otides. For GABP-binding concatamers, the PCR conditions wereas follows: 95 °C for 2 min followed by 14 cycles at 95 °C for 1min, 55 °C for 1 min, and 72 °C for 3 min. Conditions for PURR:95 °C for 2 min followed by 9 cycles at 95 °C for 1 min, 55 °C for1 min, and 72 °C for 3 min. Conditions for AP.1 and PU.1: 92 °Cfor 2 min followed by 14 cycles at 95 °C for 1 min, 55 °C for 1min, and 72 °C for 3 min. Conditions for RARR: 95 °C for 2 minfollowed by 9 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72°C for 5 min. PCR products were purified using QiaQwick kit(Qiagen, Valencia, CA) and analyzed by electrophoresis in agarosegel/Tris-borate-EDTA buffer. Each PCR yielded about 3-5 µgof DNA, ranging between 200 bp and 5-10 kb in length.

Preparation of Double-Stranded (ds) DNA-MagneticBeads. The 5′-biotinylated ds oligonucleotides or concatamerizedDNA, (DNA)n, were attached to M280 magnetic beads coated withstreptavidin (Dynal Biotech, Oslo, Norway), using a Kilobase-BINDER kit according to the manufacturer’s instructions. Theefficiency of concatamer binding to those beads depends on thelength of DNA. According to the manufacturer’s specifcations,binding of 1000-bp DNA is on the order of 2-5 µg/mg of beads;lower and higher molecular weight (DNA)n bind in up to ∼12µg/mg. For quantitations, small aliquots of ds oligonucleotides

or (DNA)n were end-labeled with T4 polynucleotide kinase (NewEngland Biolabs) and γ-32P-ATP (PerkinElmer Life Sciences,Boston, MA) and then used as a tracer to monitor final attachment.This varied from 3 to 9 µg of concatamers/mg of beads (detailsin Table 2). To prepare ds oligonucleotides, complementary ssoligonucleotides were mixed in a 1:1 molar ratio (50 µM each) in10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, and 100 mM KCl, heatedat 88 °C for 3 min, and then gradually cooled (10 min at 65 °C; 1min at 55 °C; 10 min at 37 °C; 5 min at room temperature) andstored at -20 °C. About 200 pmol of ds oligonucleotides wasattached/mg of beads.

Protein Binding to DNA-Magnetic Beads. All procedureswere carried out at 4 °C. The beads with attached concatamericDNA were first washed in DNA-binding solution (20 mM HEPES,pH 7.9, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.01% NP-40,10% glycerol). The binding buffer also contained oligo(dI:dC) andpoly(dI:dC) at 0.1 mg/mL each, except where indicated. Ifnecessary, the protein fractions after P11 chromatography weredialyzed against 50 volumes of the binding buffer, mixed with theDNA-beads, and incubated for at least 3 h at 4 °C with rotationon LabQuake shaker (Labindustries, Berkeley, CA). For collectionof the beads from large volumes of nuclear extracts or columnfractions, high-powered cobalt magnetic disks (catalog no. CR30352-75, Edmund Scientific, Tonawanda, NY) were placed underneath15-mL or 50-mL Falcon tubes during centrifugation in GS-6KRrotor (Beckman, Fullerton, CA) for 5 min at 700g. For smallervolumes, in Eppendorf tubes, magnetic disks were hand-held. Afterprotein binding, beads were washed with binding buffer (≈1 mL/mg of beads) and three times in the presence of E. coli ds and ssDNA, at 0.1 mg/mL each, as a nonspecific competitor. Single-stranded E. coli DNA was prepared by heating ds DNA in boilingwater for 20 min and quick chilling on ice. Finally, the beads wereeluted with 50 µL of binding buffer, containing 0.5 M NaCl, for15 min on ice.

After elution, the beads were suspended directly in 50 µL ofLaemmli sample loading buffer, and after being heated for 3 minat 95 °C, the eluates were subjected to analysis by SDS gelelectrophoresis and Western blotting, at room temperature. Gelswere always stained with Coomassie Blue R250. Silver stainingof the sodium dodecyl sulfate gels prior to mass spectrometricanalysis is not advisable as silver binds to traces of competitorDNA left in the final preparations.

Electrophoretic Mobility Shift Assay (EMSA). EMSA wasperformed essentially as described previously,22 with minormodifications. In brief, prebinding of nuclear extract (5 to 10 µg/mL), or respective protein fraction, to the poly(dI-dC) was carriedout at 25 °C for 10 min in buffer containing 4% glycerol, 1 mMMgCl2, 0.5 mM EDTA, 0.5 mM DTT, 25 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.05 mg/mL poly(dI-dC)‚poly(dI-dC). For thecompetition experiments 5-100-fold molar excess of unlabeledwild-type or mutated oligonucleotides was included in the incuba-tion mixture. Probe (3.5 fmol, ∼2 × 104 cpm) was then added tothe above reaction, which was mixed and incubated at 25 °C for20 min. One microliter of 10× gel loading buffer, containing 250mM Tris-HCl (pH 7.5), 0.2% of bromophenol blue, 0.2% xylenecyanol, and 40% glycerol, was added to the reaction and thenloaded onto a 6% native gel (which was prerun for 90 min at 100

(21) Hemat, F.; McEntee, K. Biochem. Biophys. Res. Commun. 1994, 205, 475-481 (22) Ma, Y.; Qin, S.; Tempst, P. J. Biol. Chem. 1998, 273, 8727-8740.

Table 1. Sequences of the Wild-Type (wt) and Mutant(mut) Double-Stranded DNA Oligonucleotides Used forConcatamerizations, Immobilization on MagneticBeads, and Capture of GABP (Yaneva et al.,Unpublished), PU.1,22 Ap1,27 PURr (Kippenberger etal., Unpublished), and RARr37 with Mutations Boxed

Analytical Chemistry, Vol. 75, No. 23, December 1, 2003 6439

V) in 0.5× nondenaturing Tris-borate-EDTA buffer. Electro-phoresis was performed at 25 °C and 100 V for about 3.5 h. Thegel was then transferred onto Whatman paper, vacuum-dried, andexposed to Hyperfilm (Amersham Pharmacia) for the desiredperiod of time at -80 °C and with an intensifier screen.

Western Blotting. Protein solutions were separated by elec-trophoresis in 4-15% gradient polyacrylamide sodium dodecylsulfate gels and the proteins transferred onto a PVDF membranein Tris-glycine-methanol buffer. The respective antibodies wereadded to the membranes at concentrations of 1 µg/mL in PBS/0.05% Tween-20, for 2 h at room temperature. Anti-mouse or anti-rabbit-horseradish peroxidase-conjugated antibodies (Santa Cruz)and Enhanced chemiluminescence kit (Pierce; Rockford, IL) wereused for visualization of the immune complexes.

Mass Spectrometry (MS). Gel-resolved proteins were di-gested with trypsin, the mixtures fractionated on a Poros 50 R2RP micro-tip, and resulting peptide pools analyzed by matrix-assisted laser-desorption/ionization reflectron time-of-flight (MALDI-reTOF) MS using a Bruker UltraFlex TOF/TOF instrument(Bruker Daltonics; Bremen, Germany), as described.23,24 Selectedexperimental masses (m/z) were then taken to search a nonre-dundant protein database (NR; ∼1.4 × 106 entries; National Centerfor Biotechnology Information; Bethesda, MD), utilizing thePeptideSearch (Matthias Mann, Southern Denmark University,Odense, Denmark) algorithm. A molecular weight range twicethe predicted weight was covered, with a mass accuracy restrictionbetter than 40 ppm and maximum one missed cleavage siteallowed per peptide. Mass spectrometric sequencing of selectedpeptides was done by MALDI-TOF/TOF (MS/MS) analysis onthe same prepared samples, using the UltraFlex instrument in“LIFT” mode. Fragment ion spectra were then taken to searchthe NR database using the MASCOT MS/MS Ion Search program(Matrix Science Ltd.; London, U.K.).25 Any identification thusobtained was verified by comparing the computer-generatedfragment ion series of the predicted tryptic peptide with theexperimental MS/MS data.

In general, positive identifications were made on the basis ofa Mascot MS/MS score g74 (p < 0.05) for a single peptide, or ascore g40 for each of two or more peptides (combined score g80).Alternatively, a Mascot MS/MS score g40 for a single peptide

was combined with a peptide mass fingerprinting (PMF) resultthat yielded g15% sequence coverage.

RESULTS AND DISCUSSIONWe sought to develop a protocol for the microisolation and

handling of unidentified transcription factors (TFs), characterizedonly by the binding to a specific DNA-recognition site, all the wayfrom crude nuclear extracts to a downstream, facile massspectrometric identification. The ideal method should allowprepararation of TFs from the minimum amount of tissue ornumber of cultured cells, in the fewest possible steps, and yielda small (i.e. e30-50 µL), concentrated sample for gel electro-phoretic fractionation and visualization. The entire procedureshould only require tools and reagents commonly available in theaverage molecular biology or biochemistry laboratory. Twoenabling concepts can be considered up front. Affinity capturemay in principle allow a single-step isolation by taking advantageof the highly specific factor-nucleic acid interactions. Samplevolume and handling losses may be kept to a minimum byimmobilizing the protein(s) of interest throughout the purificationprocess onto the smallest amount of solid-phase support, operatedin batch-mode for simplicity reasons. Ligand (e.g. ds DNA)bearing low-micrometer (e3 µm) diameter, magnetic particlespotentially satisfy both requirements. Magnetic microparticles alsohave the advantage of facile collection from relatively largevolumes, with resultant analyte concentration, and of minimallosses during washings and transfers. To accommodate largervolumes, we selected strong, 6-mm-diameter, cobalt magneticdisks (see Experimental Section) that can be used in either manualpull-downs or in combination with centrifugation.

Nonspecific Nucleic Acid-Binding Proteins. At the outsetof this study, we typically generated magnetic particles ligandedwith specific ds DNA sequences by annealing two syntheticoligonucleotides, one of which was biotinylated at the 5′-end, andthat were then bound to commercially available, streptavidin-coated beads. In a first, comparative experiment, three differentparticles were prepared, each with a specific DNA sequence (21-bp long) containing the known binding site for one of threecommon TFs, namely AP.1, NFκB, and Sp1 (Figure 1A).26-28

These beads were separately incubated with nuclear extract fromhuman leukemia NB4 cells under standard binding conditions(NaCl and Mg2+ concentrations; poly(dI:dC) competitor) com-monly used in EMSA.1,22 After extensive washing, bound proteins

(23) Erdjument-Bromage, H.; Lui, M.; Lacomis, L.; Grewal, A.; Annan, R. S.;McNulty, D.; Carr, S. A.; Tempst, P. J. Chromatogr., A 1998, 826, 167-181.

(24) Winkler, G. S.; Lacomis, L.; Philip, J.; Erdjument-Bromage, H.; Svejstrup, J.Q.; Tempst, P. Methods 2002, 26, 260-269.

(25) Perkins, D. N.; Pappin, D. J. C.; Creasy, D. M.; Cottrell, J. S. Electrophoresis1999, 20, 3551-3567.

(26) Kadonaga, J. T.; Tjian, R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 5889-5893.

(27) Lee, W.; Mitchell, P.; Tjian, R. Cell 1987, 49, 741-752.(28) Lenardo, M. J.; Baltimore, D. Cell 1989, 58, 227-229.

Table 2. Affinity Capture of GABP, PU.1, AP.1, and PURr on DNA-Specific Magnetic Beads for Identification by MSa

captured protein cell culture (L) no. of cells (109) P11 fraction (M) P11 protein (mg) capture scheme (+)DNA/mg of beads (µg)

GABP 0.9 1.4 0.1 3.5 + - + 5 (×2)RARR 4.5 6.8 0.3 22 + - - - + 6 (×2)AP.1 1.1 1.7 0.5 5 - + 9PURR 1.5 2.3 0.5 7 - - + 3PU.1 1.9 2.9 0.85 7 - - - + 8

a RARR was identified by Western blotting. Human NB4 myeloid leukemia cell cultures were grown to 1.5 × 106 cells/mL; NE protein yieldswere on average 1 mg/108 NB4 cells. Amounts of ds DNA attached to beads were calculated as described under Experimental Section.


were eluted with Laemmli sample buffer and analyzed by SDSgel electrophoresis and Coomassie Blue staining. The profiles ineach lane looked nearly identical, regardless of the difference inDNA sequences attached to the beads (Figure 1B), suggestive ofa high degree of nonspecific binding. In support of this view,several major bands from the gel were excised and identified byMS as mostly nonspecific RNA or DNA-binding proteins, somewith preference for binding to free DNA ends (as indicated onthe right in Figure 1B).29 It showed that, under conditions wherespecific bindings were observed in EMSA (data not shown), mostof the DNA-binding sites on the beads were occupied nonspecifi-cally. Poly(dI:dC) competitor at a concentration of 0.1 mg/mL,already high, was clearly insufficient to prevent these unwantedinteractions in a chromatographic setting. Raising the concentra-tion any further leads to viscosity-induced anomalies and potentialloss of specific protein.

Concatamerized DNA-Binding Sites and Oligo(dI:dC)Competitor. From the premise that several major, nonspecificallybinding proteins (e.g. DNAPK, Ku autoantigen, PARP) have highaffinities for DNA breaks or ends, we reasoned that the resultantbackground could be reduced by using (i) beads with DNA affinityligands of a lower ends-to-binding site ratio and, conversely, (ii)competitor DNA of a higher ends-to-weight ratio. The latter option

was implemented by including 30-bp long, custom synthesizedoligo(dI:dC) in the binding reaction, in addition to the standardpolymeric competitor.30 A 0.1 mg/mL oligo(dI:dC) concentrationwas empirically found to be the highest that did not increaseviscosity and did not visibly interfere with TF-DNA binding inEMSA (data not shown). As for the first option, long (g1000 bp)double stranded concatamers of 5′-biotinylated, specific oligo-nucleotides were produced in a self-priming PCR, effectivelyincreasing ligand density on the beads without added DNA ends.It should be noted that PCR-based concatamerization reactionsare sequence specific and must be carefully optimized first duringpilot experiments.

In binding experiments with AP.1-DNA beads (see Figure1A), the effect of oligo(dI:dC) as competitor was evaluated andprotein binding to single DNA sites vs concatamers compared.As anticipated, the nonspecific protein bands were reduced in thepresence of oligodI:dC (Figure 2A; lanes 2 and 3) and did notvisibly increase when concatamerized DNA instead of oligonucle-otide was used as affinity ligand on the beads (Figure 2A, lane4). Western blot analysis of the bound proteins demonstrated thatboth subunits of AP.1 (i.e. Jun and Fos) were successfully capturedand that their levels were significantly higher on beads withconcatamerized binding sites (Figure 2B, lane 4). However, theamount of AP.1 was insufficient for identification as severalcontaminating, more abundant proteins still precluded unbiasedMS detection (data not shown). We concluded that, despite theimprovements, direct capture from crude nuclear extracts wouldnot be successful under the current conditions, not even of arelatively abundant TF such as AP.1. At least one enrichment stepwill be needed before capture on specific DNA-beads.

(29) Yaneva, M.; Kowalewski, T.; Lieber, M. R. EMBO J. 1997, 16, 5098-5112. (30) West, R.; Yaneva, M.; Lieber, M. Mol. Cell. Biol. 1998, 18, 5908-5920.

Figure 1. Capture of nuclear proteins on specific oligonucleotides.(A) Sequences of double-stranded DNA oligonucleotides, with specificbinding sites for AP1,27 NFkB,28 and Sp1,41 immobilized on magneticparticles. Factor binding sites are shown in bold. (B) Electrophoreticprofile of the proteins captured on magnetic beads with DNA specificfor AP.1 (lane 2), NFkB (lane 3), and Sp1 (lane 4). A 1 mg amountof DNA-beads, containing approximately 100 pmol of oligonucle-otides, was incubated with 36 mg of NB4 nuclear extract in thepresence of 0.1 mg/mL poly(dI:dC) as competitor. After wash andelution, proteins were separated on a 10% SDS gel and stained withCoomassie Blue. Lane 1: molecular weight markers. Selectedproteins were identified by MALDI-TOF MS as indicated on the right.

Figure 2. Effect of oligo(dI:dC) competitor and DNA concatamer-ization on the capture of AP.1. (A) Nuclear extracts were incubatedwith monomeric, AP.1-specific DNA oligonucleotides bound to mag-netic beads in the absence (lane 2) or the presence of a 100-foldmolar excess of oligo(dI:dC) (lanes 3 and 4); alternatively, beadscontained concatamerized oligonucleotides (lane 4). All incubationmixtures contained 0.1 mg/mL poly(dI:dC) as well. The proteins elutedfrom the beads were analyzed on a 10% SDS gel and stained withCoomassie Blue. Lane 1: molecular weight markers. (B) Westernblot of the proteins analyzed in (A) using a mixture of anti-Fos andanti-Jun antibodies at 1 µg/mL each. Lane 1: molecular weightmarkers.


Chromatography on P11 Phosphocellulose. Ion exchang-ers (DEAE-cellulose, BioRex70) or Heparin-Sepharose, Phenyl-Sepharose, and gel-filtration columns (Sephacryl S300) have beenused as a prefractionation step for affinity purification of nucleicacid-binding proteins.5,10 However, we chose to fractionate nuclearextracts on phosphocellulose P11 as it provided a unique combi-nation of ion-exchange and affinity properties. P11 chromatogra-phy is a simple, popular procedure that has been used as a firststep in many proven TF purification schemes and which allowsenrichment of multiple factors, cofactors, and corepressors inseparate fractions.31-33 In this way, we could conceivably alsoperform parallel affinity captures of several different factors, eachfrom a separate P11 fraction but all derived from a single batchof nuclear extract.

NB4 nuclear extracts (75 mg of total protein in 40 mL of bufferD) were applied to a P11 column (22-mL bed volume) andextensively washed, and bound proteins were then eluted witheither a linear gradient (Figure 3A) or stepwise increase of NaClconcentration from 0.1 to 0.85 M, always in a 200-mL total elutionvolume. As monitored by specific EMSA, we detected the presenceof GABP in the 0.1 M fraction, RARR in 0.3 M fraction, AP.1 andPUR in the 0.5 M fraction, and PU.1 activity in the 0.85 M fraction(Figure 3B and data not shown). The choice of these particularfactors was directed at covering the entire P11 elution range andalso determined by prior observations in our laboratory whilepurifying several factors (later identified as PU.1, GABP, and PUR)involved in the regulation of the myeloid defensin promoter or byscrutinizing published TF purification schemes.27,34,35 Note that,initially, every other 1-mL column fraction was assayed for eachof the five factors. On the basis of the distribution of the variousbinding activities, fractions were arbitrarily combined into fourpools, as indicated in Figure 3A. For reasons of simplicity andtime efficiency, all subsequent P11 columns were batchwise elutedwith 0.1, 0.3, 0.5, and 0.85 M salt (5 × column volumes each) foruse in further protocol development.

As the model DNA-binding factors were present in differentpools of significantly varying ionic strenth, we investigated theeffects of salt concentration on the in vitro binding to their cognatesequences as a prelude to the choice and optimization ofsubsequent purification steps. Also, low Mg2+ concentrationswould reduce the probability for degradation of DNA concatamers,attached to the beads, by endogeneous nucleases. We noticed thatall five chosen proteins bound equally well to their cognate DNAin the presence or absence of Mg2+ (data not shown). Conse-quently, all further DNA captures were performed in the absenceof magnesium. As for the salt effects, selectively illustrated forGABP and PU.1 in Figure 4, we observed a striking correlationbetween the salt concentration at which a given protein elutedfrom the P11 column and the stability of the DNA-proteincomplex in the presence of salt as measured by EMSA. Proteinseluting at e300 mM NaCl formed less stable complexes with DNAthan proteins that eluted at g500 mM. For example, the formation

of GABP-DNA complex was adversely affected by the presenceof 500 mM as compared to 50 mM KCl, whereas high salt had nosuch destabilizing effect on the PU.1-DNA complex (Figure 4).In some cases (e.g. AP.1 and PUR), the presence of high saltcaused a slight change in the mobility of the DNA-proteincomplexes (data not shown), but the DNA-binding specificity waspreserved as confirmed in subsequent capturing experiments.

Effect of High/Low Salt on DNA-Affinity Capture. In effect,these findings allowed us, as a rule, to perform specific DNA-affinity capture on beads in the presence of 0.5 M NaCl of anyprotein that has previously been eluted from a P11 column at high(g500 mM) salt. We speculated that, under such high saltconditions, the number of nonspecific, low-affinity binding con-taminants might be significantly reduced, a characteristic thatshould definitely be taken advantage of whenever possible. Onthe other hand, capture of proteins that bind weakly to a P11column (eluting at e300 mM NaCl), such as GABP, require adifferent strategy. To this end, the 0.1 M salt cut from the P11column (Figure 3A; Figure 3B,C, lanes 2) was incubated withconcatamerized GABP-DNA beads under conditions as optimizedup to that point, washed, eluted with 0.5 M NaCl, and analyzedby standard gel electrophoresis (Figure 3C, lane 3) and by EMSAwith a GABP-specific probe (Figure 3B, lane 3). While the bindingactivity had clearly been enriched by this affinity step, more thana dozen protein bands were still visible on the Coomassie-stainedgel, the majority of which were identified to be nonspecific nucleicacid binding proteins or various housekeeping proteins. No GABPcould be detected by MS-based methods, making further purifica-tion necessary. Similarly, “high salt” (i.e. in the presence of 0.5M NaCl) affinity capture of proteins from the P11 0.85 M salt cuton PU.1-DNA beads also failed to produce sufficiently homoge-neous PU.1 factor detectable by standard peptide MALDI-TOFmass fingerprinting. It appeared, therefore, that direct DNA-affinitycapture from P11 fractions may be generally inadequate to purifyspecific TFs for facile mass spectrometric identification. It isimperative that any additional purification steps should also becarried out in a microcapture format.

Negative Selection Enhances DNA-Affinity Capture Speci-ficity. As nonspecific protein binding appeared to be the majorobstacle for successful TF affinity capture, the problem could bereduced to the adequate removal of such interfering proteins.Conceptually, the suitable medium could be an immobilized DNAsequence that binds many or all of the nonspecific proteins butnot the specific one. We envisioned this could be readily ac-complished by using DNA ligands consisting of minimally modi-fied TF binding sites, for instance, by one or more point mutations,just enough to abolish specific factor binding in vitro. The mutatedDNA sequences were derived from pilot competition EMSAexperiments performed in the presence of an excess of unlabeled,mutant DNA probes. Point mutations that fully abolished competi-tion were then selected. Preclearing of protein solutions with beadsloaded with such DNA will be further referred to as “negativeselection”. Sequences for negative selections for the capture ofGABPs, PU.1, and PURs were established in our laboratory(Yaneva et al., unpublished; Kippenberger et al., unpublished),as listed in Table 1; mutant DNAs for capture of AP.1 and RARRwere taken from the published literature.27,36,37

(31) Dignam, J. D.; Lebovitz, R. M.; Roeder, R. G. Nucleic Acids Res. 1983, 11,1475-1489.

(32) Ge, H.; Roeder, R. G. Cell 1994, 78, 513-523.(33) Roeder, R. G. Trends Biochem. Sci. 1996, 21, 327-335.(34) Yeh, W. C.; Hou, J.; McKnight, S. L. Methods Enzymol. 1996, 274, 101-

112.(35) Haas, S.; Thatikunta, P.; Steplewski, A.; Johnson, E. M.; Khalili, K.; Amini,

S. J. Cell Biol. 1995, 130, 1171-1179.


The utility of this approach is best illustrated by the subsequentsuccessful efforts on AP.1 and PU.1 isolation. Starting with the0.5M P11 fraction, AP.1 was captured on “positive” (i.e. wild type)DNA-beads in the presence of 500 mM NaCl, after one round ofnegative selection. Each of the four visible Coomasie-stained bands(Figure 5A; lane 7) was identified by MS (MALDI-TOF and TOF/

TOF) as either Fos or Jun proteins (i.e. a “AP.1” heterodimer) orbreakdown products thereof (Figure 5C,D); experimental detailsare given in the figure legends. Western blot analysis with mixedanti-Fos and -Jun antibodies on a fraction of the final sampleindicated that nearly all of the protein of interest was recovered(Figure 5B). Similarly, PU.1 protein was captured from the 0.85M P11 fraction but not until a more extensive removal ofnonspecific proteins was done by three consecutive rounds ofnegative selection (Figure 6A,B). MS-assisted identification wasvery straightforward at that point (Figure 6C). The additional

(36) Perez, A.; Kastner, P.; Sethi, S.; Lutz, Y.; Reibel, C.; Chambon, P. EMBO J.1993, 12, 3171-3182.

(37) Leid, M.; Kastner, P.; Lyons, R.; Nakshatri, H.; Saunders, M.; Zacharevsky,T.; Chen, J.-Y.; Staub, A.; Garnier, J. M.; Mader, S.; Chambon, P. Cell 1992,68, 377-395.

Figure 3. Partial purification of GABP on phosphocellulose P11 and DNA-affinity beads. (A) Elution profile of P11 column chromatography.Nuclear extract (75 mg) was applied to a P11 column (22-mL bed volume) and eluted with a 200-mL linear gradient of NaCl as described in theExperimental Section. The fractions in the “0.1 M” pool contained GABP DNA-binding activity. These fractions were combined and applied toGABP-DNA affinity beads. Other fractions were all pooled as indicated (0.3 M; 0.5 M; 0.85 M; flow through ) FT). (B) DNA-binding activitiesof GABP-containing fractions. EMSA using a GABP-specific, ds oligonucleotide was performed as described in the Experimental Section. Key:lane 1, free DNA probe; lane 2, NB4 nuclear extract; lane 3, pooled “0.1 M” fractions from P11 column; lane 4, proteins eluted from the DNA-affinity beads. The two arrows on the right indicate the positions of the DNA/GABPR (bottom) and DNA/GABPR+â (top) complexes (Yaneva etal., unpublished). (C) Electrophoretic profile of eluted proteins. Proteins were analyzed by electrophoresis in 4-15% SDS gels and stained withCoomassie Blue. Lanes 1-3 correspond to lanes 2-4 of panel B. The proteins in lane 3 identified by MS are indicated at right.


negative selections were required in this case because of theapparent, relatively low PU.1 concentration in NB4 cell nuclearextract and P11 fraction. As visualized by Western blotting, PU.1enrichment throughout the entire procedure was quite dramatic(Figure 6B; lane 9). PUR proteins were also captured under thesame conditions from the 0.5M P11 fraction using two rounds ofnegative selection. Both subunits (see Figure 7, lane 3) were thenpositively identified by MS (data not shown).

“Positive/Negative/Positive” Affinity Selections. The cap-ture of lower affinity binders, such as GABP and RARR, was doneusing a different sequence of positive/ negative selections. Theseproteins could be readily eluted from wild-type DNA-magneticbeads in 0.5 M salt, which was then removed by dialysis, andfollowed by one or more rounds of negative selection, asnecessary. The advantage of a front-end, positive selection is thesizable reduction of protein mass, all the while retaining the TFof interest, which enables subsequent rounds of capture on amicroscale (i.e. Eppendorf tubes). A final round of GABP purifica-tion was then also carried out by positive capture, and the proteinswere eluted from the beads in 0.5 M NaCl for gel electrophoresis(Figure 7A, lane 2) and identification by MS (data not shown).

RARR, easily detected by EMSA and Western blotting in the0.3 M NaCl fraction of P11, was taken through three rounds ofnegative selection before capture on “positive” DNA-beads.Again, Western blot analysis clearly demonstrated that the proteinwas not bound to the mutant but only to the wild-type DNA-beads, from which it could be readily eluted in 0.5 M salt (Figure7B). However, RARR was not found by MALDI-TOF MS analysisin the final preparation (Figure 7A,B; compare lanes A4 and B10).We concluded that for successful capture of RARR, additionalpurifications, or further scaling up of the present procedure, wouldbe necessary because the protein level seemed to be unusuallylow. By contrast, Nordhoff et al. reported MS identification of adifferent nuclear receptor, RXRR, isolated from crude extracts of

hormone-induced mouse 3T3-F442A cells.18 This discrepancy maybe related to genetic differences between mouse fibroblasts andhuman myeloid leukemia NB4 cells. Indeed, two forms of RARRexist in NB4 cells, namely the free protein and an oncogenic fusionPML-RARR.36 To our knowledge, neither wild-type nor fusionprotein have ever been purified.

It also should be noted that conventional biochemical proce-dures have rarely allowed purification of specific TFs to homo-geneity in such a few steps and from such small number of cells.10

The four out of five factors, totaling six proteins out of seven, thatwere positively identified here by MS after capture on DNA-magnetic beads derived from only 1.5 to 3 × 109 human cells(Table 2). Taken together, these results suggest a wider ap-plicability of the procedure, with RARR and PML-RARR beingamong the exceptions.

General Rules for DNA-Affinity Capture of Site-SpecificProteins. On the basis of the results reported here, we proposethe following empirical rules for future identifications of TFs byMS, as illustrated in Figure 8. After initial fractionation of nuclearextract on a P11 column, it is critical to determine whether theprotein of interest will form a stable complex with its target DNAin the presence of at least 500 mM salt. This is generally the casefor all proteins that elute from P11 at or higher than 500 mM salt(e.g. AP.1, PURR, PU.1, and others). Binding affinities may alsobe known from prior molecular characterization of the promoterof interest. Affinity and salt tolerance, often directly linked, arethe primary determinants of the order in which positive andnegative selections are then carried out: “negative/positive” forhigh-affinity, high-salt binders; “positive/negative/positive” for low-affinity, low-salt binding TFs. Addition of a positive step prior tothe negative selections serves dual purpose: (i) the protein massis drastically reduced; (ii) protein eluates are very concentrated,enabling further microcapture format. This is not possible for high-affinity binders as approximately 1 M salt is required for elution,in most cases resulting in some degree of denaturation whichcomplicates both assaying and subsequent affinity captures.Consequently, the first positive selection must be omitted. Thiscan be compensated, in part, by the option to include 0.5 M saltthroughout the entire selection process, thereby also increasingcapturing stringency, albeit by a different mechanism. Bothoptions appear to be mutually exclusive.

Regardless of the selection sequence, the number of negativeselections on mutant DNA-magnetic beads always depends onthe abundance of the TF in the respective fraction. Low-abundantspecies require more nuclear extract and proportionally morerounds of negative selection than the higher abundant ones, forexample, one round for GABP and AP.1, two for PURR, three forPU.1, but more than three for RARR (Figure 8). All capturingschemes end with a positive selection, including extensive washingof the beads with E. coli competitor ds and ss DNA. Final elutionis done with 0.5 M NaCl for low-affinity binders or by boiling inLaemmli gel-loading buffer for the others, followed by gelelectrophoresis and identification by the mass spectrometricmethod of choice. The purification scheme generates proteins inamounts and form that are readily compatible with MALDI TOF-based peptide mass fingerprinting.

On a more molecular biological note, the precise DNAsequence to which a protein of interest binds both in vivo and in

Figure 4. Effect of salt on the stability of DNA-protein complexes.DNA binding was measured in EMSA using crude nuclear extractsand DNA oligonucleotides specific for GABP (lanes 1-3) or for PU.1(lanes 4-6). Proteins and DNA were incubated in the presence ofeither 50 mM (lanes 2 and 5) or 500 mM KCl (lanes 3 and 6) prior toelectrophoresis. Lanes 1 and 4: free DNA probes. Faster migratingDNA-protein complexes in lanes 5 and 6 were typically observeddue to partial proteolysis of PU.1 protein in crude extracts.22


Figure 5. Affinity capture of AP.1 on specific DNA-magnetic beads. (A) Protein analysis. Proteins from the P11 “0.5 M” fraction (lane 2) wereincubated with 1 mg of beads derivatized with (mutAP1-DNA)n, i.e., negative selection; unbound (lane 3, “NB” ) not bound) and bound (lane4, ‘beads’; boiled in Laemmli buffer) fractions are shown, after analysis on a 4-15% SDS gel and staining with Coomassie Blue. Unboundprotein was then incubated with 1 mg of (wtAP1-DNA)n beads; the unbound (lane 5, wt-NB), wash (lane 6), and terminally bound (lane 7,beads) are indicated. All bindings, washes, and elutions were performed as described in Experimental Section, in the presence of 500 mM KCl.The arrows on the right, labeled A-D, denote bands that were identified by MS; band A is Jun (C), band B is Fos (D), and bands C and D arebreakdown products of Jun and Fos. (C) is an MS/MS spectrum of a peptide selected from band A for further identification by sequencing, and(D) is a peptide from band B also selected for MS/MS. (B) Western blot analysis. An SDS gel with 1-5% aliquots of the same protein fractionsas shown in (A) was transferred to a PVDF membrane and incubated with a mixture of anti-Fos and anti-Jun antibodies (see ExperimentalSection). (C) MALDI-TOF/TOF (MS/MS) identification of a Jun tryptic peptide. Protein band A (A; lane 7) was digested with trypsin; peptideswere processed over a RP-microtip and analyzed by MALDI-TOF MS (not shown), and the data were used for peptide mass fingerprinting(PMF).24 Jun was independently identified by TOF/TOF (MS/MS) analysis of a peptide observed as a peak at m/z ) 1746.866. Fragment ionspectra were taken for a MASCOT MS/MS ion search of the NR database and retrieved a tryptic peptide sequence, GASTFKEEPQTVPEAR([MH]+ ) 1746.844; ∆ ) 13 ppm) with a Mascot score of 53. b- and y-fragment ions are indicated. (D) MALDI-TOF/TOF (MS/MS) identificationof a Fos tryptic peptide. Protein band B (A; lane 7) was processed as described for (C). In this case, Fos was identified by PMF and also byTOF/TOF analysis of a selected tryptic peptide at m/z ) 1960.966: LQAETEELEEEKSGLQK ([MH]+ ) 1960.972; ∆ ) 3 ppm) with a Mascotscore of 74.


vitro should, of course, always be known in advance. Using highlysensitive and specific assays, it is especially critical to determineminimal mutations in the sequence to completely abolish factor

binding; this information will be used in the negative selectionsas described above. Traditional assays include DNA footprintings,transient expression of a promoter fused to a reporter gene, andEMSA. In addition, EMSA is used to optimize the binding with

Figure 6. Affinity capture of PU.1 on specific DNA-magnetic beads.(A) Protein analysis. Proteins from the P11 “0.85 M” fraction (lane 2)were incubated with 1 mg of beads derivatized with (mutPU1-DNA)n,i.e. negative selection. The bound (lane 3, “mut1-B”; beads boiled inLaemmli buffer) fraction is shown, after analysis on a 4-15% SDSgel and staining with Coomassie Blue. Clearance of the unboundfraction was repeated two more times; bound proteins are shown aftereach cycle (lanes 4 and 5, “mut2-B, 3-B”), as is the unbound fractionafter the third negative selection (lane 6, “mut3-NB”). Unbound proteinwas then incubated with 1 mg of (wtPU1-DNA)n beads; the unbound(lane 7, wt-NB), wash (lane 8), and terminally bound (lane 9, beads)fractions are indicated. All bindings, washes, and elutions wereperformed in the presence of 500 mM KCl. Arrows on the right pointto the bands in lane 9 that were all identified by MS as PU.1 (C). (B)Western blot analysis. An SDS gel with 1-5% aliquots of the sameprotein fractions as shown in (A) was transferred to a PVDFmembrane and incubated with anti-PU.1 antibodies. The arrows onthe right point to the immunoreactive bands that were all identifiedas PU.1 protein by MS. (C) MALDI-TOF MS-based peptide massfingerprinting of PU.1. Protein bands marked with arrows in lane 9(A) were digested with trypsin, peptides were processed over a RP-microtip and analyzed by MALDI-TOF MS, and all three wereidentified by peptide mass fingerprinting as PU.1 (or fragments), with17% sequence coverage and average mass error of 9 ppm for thebest match. CAL indicates the position of internal calibrants. Theprotein was confirmed by TOF/TOF (MS/MS) analysis (not shown)of two peptides, observed at m/z ) 1369.724 and 1497.816, andidentified as tryptic peptides LTYQFSGEVLGR and KLTYQFSGEV-LGR, respectively.

Figure 7. Affinity capture of GABPR, PURR, RARR, AP.1, and PU.1on specific DNA-magnetic beads. (A) Protein analysis. Proteins werecaptured from the following P11 fractions (derived from crude nuclearextract): GABP, “0.1 M”; RARR, “0.3 M”; AP.1, “0.5 M”; PURR, “0.5M”; PU.1, “0.85 M”. The first two pools were initially incubated withwild type (positive) beads. Bound proteins were eluted and desaltedand protein solutions cleared on mutant (negative) beads, one roundfor GABP and three rounds for RARR. The 0.5 and 0.85 M poolswere immediately precleared on negative beads, one round for AP.1,two for PURR, and three for PU.1. All cleared fractions were subjectedto one more round of positive selection; beads were then washedand eluted with salt (GABP; RARR) or boiled in Laemmli sample buffer(others) and analyzed by electrophoresis in a 4-15% SDS gel andstained with Coomassie Blue, as indicated. The circles to the right ofthe gel lanes indicate protein bands that have been identified by MS.Closed circles mark proteins that were confirmed by MS as thosepredicted to bind to specific DNA baits; open circles indicate thosethat were identified as false positives after “RARR-DNA”-mediatedcapture. (B) Western blot analysis of proteins captured on (RARR-DNA)n-beads. An SDS gel with 1-5% aliquots of intermediate andfinal protein fractions of the RARR capture were transferred to a PVDFmembrane and incubated with anti-RARR polyclonal antibodies (1µg/mL): lane 1, crude nuclear extract; lanes 2-8, eluted (EL) andbound (B) proteins during three rounds of negative selection onmutant beads; lanes 9-11, positive selection on wild-type (DNA)n-beads; lane 9, nonbound proteins (NB); lane 10, eluted (EL); lane11, proteins remaining on the beads (B), recovered by boiling inLaemmli sample buffer. Positions of molecular weight markers areindicated on the right; positions of RARR and of the oncogenic fusionprotein PML-RARR are also shown.


respect to salt concentration, divalent metal ions, and competitorDNA requirements.1 As a rule, salt and competitor DNA shouldbe as high as possible. Conversely, divalent cations should beavoided to protect the DNA on the beads from degradation byendonucleases.10 If absolutely necessary (e.g., in the case of Zn-finger proteins), the lowest concentration of metal that stillmaintains specific protein-DNA complex in EMSA should bedetermined.

Prospects for DNA Affinity Capture of Protein Complexes.The studies and general protocol described so far are primarilyaimed at capturing single TFs on single DNA-binding sites. Whatif longer DNA promoter sequences were used as affinity ligands?Provided that separate binding sites for different TFs would mapwithin this sequence, and absence of any steric hindrance, onemight expect that all will bind, possibly together with additionalinteracting proteins such as coactivators or repressors that do notmake direct contact with DNA.1-3 In earlier studies, we were ableto capture GABP and PU.1 simultaneously on an immobilized,concatamerized 120-bp stretch derived from the human defensin1gene promoter (Yaneva et al., manuscript in preparation). How-ever, this could only be achieved from unfractionated nuclearextract as the two TFs are fully resolved on a P11 column (Figure8), in effect precluding this elementary step. Not surprisingly, theresulting preparation was too impure for direct MALDI-TOF-basedidentification, and the presence of both proteins could only beconfirmed by Western blotting. It seems unlikely, therefore, that

use of the protocol described in this report can be extrapolatedto the general capture of multiple factors or complexes forstraightforward MS analysis. Only in those selected cases wheredifferent factors would either cofractionate on P11 (or other resins)and/or stability of the complex would confer chromatographiccomigration can we expect to cocapture proteins on DNA withreasonable efficiency and purity.

Furthermore, gene transcription is regulated by multiplefactors whose assembly is highly cooperative. Formation of thesecomplexes takes place only at particular gene promoters andrequires precise spatial interactions between the binding sites,the bound activators, and the coactivators involved in theirregulation, a process that may be difficult to reproduce in vitro.For example, Drewett et al. were unable to attain quantitativeformation of ternary complexes on immobilized target DNA.38 Twobona fide binding proteins, SRF and Elk-1, were identified by MSbut only after prior overexpression in the cells from which theywere captured; additionally bound proteins were all nonspecific.A more recent attempt to capture the yeast RNA polymerase IIpreinitiation complex on DNA-magnetic beads also demonstratedthe low efficiency of the reconstitution process. Only an estimated10% of the preinitiation complexes reconstituted on immobilizedDNA templates were functional.39 Predominantly nonspecific

(38) Drewett, V.; Molina, H.; Millar, A.; Muller, S.; Von Hesler, F.; Shaw, P. E.Nucleic Acids Res. 2001, 29, 479-487.

(39) Ranish J. A.; Yudkovsky, N.; Hahn, S. Genes Dev. 1999, 13, 49-63.

Figure 8. DNA-specific, affinity microcapture diagram of nucleic factors. The sequence of positive (wt; filled circles) and negative (mut; opencircles) selections performed on 0.1, 0.3, 0.5, and 0.85 M salt elution P11 fractions is shown. The (DNA)n-coated magnetic beads were preparedas described under the Experimental Section and in the text; corresponding, specific DNA sequences (wt; or mut) are listed in Table 1. Theboxes cluster all the steps that were carried out at a similar salt concentration of either 0.05 or 0.5 M, as indicated. Arrows mark the stepswhere, and what, competitor DNA was added to the incubation mixtures; competitor is carried along with unbound fractions but is freshly addedto any bead eluates before the next selection cycle. Elution, when done, was with 0.5 M KCl; tightly bound proteins were recovered from thebeads by boiling in gel loading buffer. The proteins indicated on the bottom of the diagram (with exception of RARR) were identified by MALDI-TOF MS. NE denotes nuclear extract; NB denotes not bound.


proteins were captured on both control and experimental beads,requiring the use of a highly sophisticated, quantitative/compara-tive analysis technique to identify relevant proteins.40 A faithfuland highly efficient reconstitution of the human transcriptionalmachinery that is suitable for MS analysis, especially in the contextof the chromatin fiber, remains one of the unrealized challengesat present.

CONCLUSIONSToday, mass spectrometry accounts for essentially all de novo

protein identifications. For the most part, applications fall into oneof two categories, either of the systems biology variety or standardidentification of proteins isolated by traditional means such asmulticolumn chromatography. These purification schemes can bevery costly and time-consuming, often for the cell culturing alone.Yet, they typically yield only one or a few purified proteins, whichcan be identified within 1 day or so by a proteomics facility or byany laboratory with access to, and elementary expertise in theuse of, a MALDI-TOF mass spectrometer. Biochemists andmolecular biologists could take greater advantage of the muchimproved protein identifying capabilities and throughput if robust,efficient methods were available to scale down and accelerate thepurification process, an effective bridge, as it were, betweentraditional life sciences research and the analytical power ofproteomics. This has been particularly the case in the field ofnucleic acid biochemistry, including the study of DNA-bindingproteins that function in transcriptional regulation, replication, andmaintainance of chromosomal integrity.

Here, we describe an efficient, much accelerated method foraffinity capture of transcription factors on specific DNA-magneticparticles, to yield final peparations in a form and amounts thatare compatible with standard MALDI-TOF MS-based proteinidentification. A major obstacle to developing this approach intoa widely applicable protocol was the inadvertent, extensive bindingof nonspecific proteins to the bait. This problem was addressedat two levels.

First, a single-batch fractionation on P11 serves to increasethe relative concentration of the TF(s) of interest, removes severalof the abundant nonspecific DNA-binding proteins, and mayresolve multiple factors in different fractions which can subse-quently be used for parallel affinity capture. Second, severalcouteracting measures were implemented. Protein fractions are

incubated with beads carrying concatamerized DNA-binding sites,and in the presence of short oligo(dI:dC) competitor, resulting inhigher specific-ligand density and displacement of proteins thatpreferentially bind to DNA nicks and ends. Magnetic beads arecollected from larger volumes on high-powered cobalt magnets.Preclearance with mutant DNA-beads (negative selection) greatlyreduces the background of nonspecific proteins in the finalpreparation. The number of negative selections depends on theabundance and binding kinetics of the respective TFs and isdetermined experimentally. In case of low-affinity binders, negativeselection is preceded by a single positive selection, which resultsin much reduced protein mass and volume. Affinity capture ofhigh-affinity binding TFs, on the other hand, can be performedin high salt, increasing stringency of the procedure that way. Thisapproach and protocol allowed capture of four distinct proteinfactors from comparatively small numbers of cultured humanblood cells, easily permitting standard MALDI TOF-based peptidemass fingerprinting.

A targeted proteomic analysis of this nature should always bepreceded, and later also validated, by a thorough molecularbiological analysis of the system under study. Proteomics is nota standalone science, and the field would benefit greatly if moreprojects were initiated by the traditional, hypothesis-driven re-search groups. The best way to popularize it among biologistsand clinical scientists is by bringing a large portion of proteomicresearch activities into their own laboratories, leaving only thefinal read-out (i.e. protein identifications) to specialized facilities.The approach and protocol that are presented herein should beconsidered in that context. In fact, the benefits go well beyondconvenience and the time and cost savings of having to culturefewer cells, in that we can now initiate related studies in cases oflimited source material, such as specific tumor tissues.

ACKNOWLEDGMENTThe authors are greatly indebted to Arpi Nazarian and Hediye

Erdjument-Bromage for all protein identifications by peptide massfingerprinting and TOF/TOF mass spectrometric analysis. Wethank Margaret McGarvey for technical assistance, Serena Kip-penberger for sharing unpublished data, and Lynne Lacomis forhelp with preparing the figures. This work was supported byDevelopmental Funds from NCI Grant P30 CA08748.

Received for review June 26, 2003. Accepted September24, 2003.

AC034698L

(40) Ranish, J. A.; Yi, E. C.; Leslie, D. M.; Purvine, S. O.; Goodlett, D. R.; Eng,J.; Aebersold, R. Nat. Genet. 2003, 33, 349-355.

(41) Jackson, S. P.; Tjian, R. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 1781-1785


Affinity capture of specific DNA-binding proteins for mass spectrometric identification

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