Accelerated Articles Anal. Chem. 1994,66, 2609-2613
Direct Analysis of Affinity-Bound Analytes by MALDI/TOF MS Damon I. Papac,t John Hayes,* and Kenneth B. Tomer'pt Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.0. Box 12233, Research Triangle Park, North Carolina 27709, and VG Anal'ical, Floats Road, W'hensha we, Manchester M23 9LE, UK Identification of ligands separated with affinity chromatography has been facilitated by direct analysis of the bound ligand using matrix-assisted laser desorption time-of-flightmass spectrometry. The mass spectral detection of analytes separated by immunoaffinity chromatography and immobilized metal ion affinity chromatography is shown. For example, cytochrome c is used as an affinity support to purify the anti-cytochrome c monoclonal antibody from ascites, and the mass spectrum of the anti-cytochrome c monoclonal antibody was obtained by direct analysis of an aliquot of the column bed. Direct analyses of metal binding proteins and phosphopeptides bound to immobilized metal ion affinity columns are also demonstrated. The method is characterized by minimal sample manipulation and high sensitivity with low picomoles and high femtomoles of analytes being readily observed.
Immobilized affinity chromatography (IAC) has become a widely used tool for the detection and isolation of biomolecu1es.l It differs from conventional chromatography in that it exploits specific biological/ biochemical interactions, such as those of an antibody and an antigen or an enzyme and a substrate, rather than differential solubilities. Because of the high specificity associated with affinity binding, IAC is typically used to determine known substances. In general, either half of a biological interaction can be used in the stationary phase as an immobilized ligand. It is now possible to purchase a number of affinity columns or stationary phases to which specific ligands can be bound. The separation and purification techniques based upon these methodologies are of increasing importance in biotechnology. National Institute of Environmental Health Sciences. VG Analytical. (1) Hundbook ofAflinniry Chromurogruphy;Kline, T.,Ed.; Marcel Dekker: New York, 1993; Vol. 63. f
0003-2700/94/0366-2609$04.50/0 0 1994 American Chemical Society
Immobilized metal ion affinity chromatography (IMAC) is another type of affinity chromatography where the separation is based on the interaction of a ligand and a metal ion.' IMAC exploits a peptide's/protein's ability to form coordination complexes with the immobilized metal ions and is, therefore, less specific than IAC. Affinity techniques have also been adapted for use with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI/TOF MS)2by Hutchens and Yip3and by Nakanishi et aL4 In the technique of Hutchens and Yip, which they call surface-enhanced affinity capture (SEAC), the probe surface was modified by attachment of the affinitycapture device. They showed the application of this technique to the detection of lactoferrin in untreated urine. Nakanishi et al. directly analyzed immunoprecipitates by MALDI/TOF MS for transferrin. They observed that the technique allowed rapid identification of the transferrin without prior purification. A potential limitation of this approach is that other proteins can coprecipitate and complicate the spectra. In addition, ions due to the antibody are also observed and may interfere with the observation of the analyte. Youngquist et al. have used MALDI/TOF MS as the analysis step in the screening of support-bound combinatorial peptide libraries. In this study, the beads were exposed to a monoclonal antibody and the beads containing active peptides were identified with a secondary antibody conjugated to alkaline phosphatase, followed by a reaction which stains the active beads. The stained beads identified by eye were treated with cyanogen bromide to release the active peptides, which (2) Karas, M.; Bahr, U.; Giessmann, U. MassSpectrom. Rev. 1991,10,335-358. (3) Hutchens, T. W.; Yip, T.-T. Rapid Commun. Muss Spectrom. 1993, 7, 576-580.
(4) Nakanishi. T.;Okamoto, N.; Tanaka, K.; Shimizu, A. Eiol. Muss Specrrom. 1994, 23, 230-233.
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were subsequently identified by MALDI/TOF MS analy~is.~ IMAC has been used off-line in conjunction with fast atom bombardment (FAB) mass spectrometry for the analysis of phosphopeptides.6 Nuwaysir and Stults have demonstrated on-line coupling of IMAC with electrospray ionization (ESI) MS at the 10-pmol level and off-line with MALDI/TOF MS with sensitivities better than 30 p m ~ l . ~ We have been exploring the use of affinity chromatographic techniques for two applications,one involving epitope mapping, and the other, identification of metal binding proteins. The final analyses in these projects has been performed by MALDI/TOF MS. On the basis of the work of Hutchens and Yip,3 we thought that it should be possible to probe the affinity-bound analyte directly from the affinity media rather than to isolate the analyte from the media. This approach has proven to be sensitive, very facile, and rapid.
EXPERIMENTAL SECTION Chemicals. Phosphokemptide was prepared by dissolving 2.4 mg of kemptide (Sigma, St. Louis, MO) in 750 pL of a 50 mM potassium phosphate (pH 6.8), 18 mM MgC12 and 10 mM adenosine triphosphate solution, Protein kinase-A catalytic subunit (250 units; Sigma) dissolved in 80 pL of a dithiothreitol solution (6 mg/mL) was added to the kemptide solution, and the resulting solution was incubated at 37 "C for 3.5 h.7 The anti-cytochrome c monoclonal antibody E8 (mAb E8) was a generous gift from Dr. Yvonne Paterson, University of Pennsylvania. Horse heart cytochrome c was purchased from Fluka (Ronkonkoma, NY), and neurotensin, human lactoferrin, human apotransferrin, and bovine serum albumin were purchased from Sigma. All water used was obtained from a Milli Ro/Milli-Q system (Millipore Corp., Bedford, MA). IAC. Column Preparation. The cytochrome c IAC was prepared by placing 1 mL of Affi-Gel 10 (mean bead size, 188 pm; Bio-Rad, Hercules, CA) into a 10-mL polypropylene column. The gel was washed four times with 1.OmL of water. The column was then loaded with a solution of cytochrome c (19 mg) in 10 mM HEPES (pH 8.0,2 mL) and incubated at 4 OC for 4 h while being rotated at 2 rpm. The column was drained after coupling and washed three times with 1 mL of 10 mM HEPES (pH 8.0). The remaining active sites were blocked with 150 pL of 1 M ethanolamine hydrochloride (pH 8.0) in 1 mL of 10 mM HEPES buffer (pH 8.0) at 4 OC for 1 h while rotating at 2 rpm. The column was subsequently drained and washed three times with 1.OmL of 10 mM sodium phosphate (pH 7.0) containing 150 mM sodium chloride. A 250-pL aliquot of beads was placed into a compact reaction column (frit size, 35 pm; United States Biochemical,Cleveland, OH) and washed three times with 0.5 mL of 10 mM sodium phosphate (pH 7.0) containing 150 mM sodium chloride. The mAb E8 column was prepared as follows. A compact reaction column was packed with 150pL of aldehyde activated agarose (mean bead size, 105 pm; Aminolink gel, Pierce Chemical, Rockford, IL). The anti-cytochrome c monoclonal ( 5 ) Youngquist, R. S.;Fuentes, G. R.;Lacey, M. P.; Keough, T. Rapid Commun. Mass Spectrom. 1994, 8, 17-8 1. (6) Michel, H.; Hunt, D. F.;Shabanowitz, J.; Bennet, J. J. B i d . Chem. 1988,263, 1123-1130. (7) Nuwaysir, L.M.:Stults, J. T. J. Am. SOC.MassSpectrom. 1993,4,662-669.
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antibody E8 from ammonium sulfate precipitated ascites fluid was attached to the agarose gel via a secondary amine using the Aminolink immobilization kit No. 1 from Pierce. Following the blocking of the unreacted aldehyde groups with 1 M Tris-HC1, (pH 7.4), the compact reaction column was washed with 10 mM sodium phosphate (pH 7.0) containing 150 mM sodium chloride. mAb E8 Attachment. mAb E8 mouse ascites fluid (0.35 mL) was diluted with 10 mM sodium phosphate (pH 7.0) containing 150 mM sodium chloride (0.35 mL). The diluted ascites fluid was loaded onto the immobilized cytochrome c affinity column, and antibody binding was allowed to occur for 1 h at room temperature while rotating at 2 rpm. The column was washed three times with 0.5 mL of water, and a 1-pL aliquot of beads was removed for MALDI/TOFanalysis. The column was next washed three times with 0.5 mL of 10 mM sodium phosphate (pH 7.0) containing 500 mM NaC1, followed by threewashes with0.5 mLofwater. A 1-pL aliquot of the beads was removed for MALDI/TOF analysis. The antibody was eluted from the column with 0.5 mL of 2 M acetic acid containing 500 mM sodium chloride into 1.O mL of 1 M sodium phosphate buffer (pH 7.9) prior to immobilization. The sodium phosphate buffer neutralized the acetic acid and prevented denaturation. The column was washed three times with 0.5 mL of water, and a 1-pL aliquot of beads was removed for MALDI/TOF analysis. Cytochrome c Attachment. Horse heart cytochrome c (25 pg) was dissolved in 500 pL of 10 mM sodium phosphate (pH 7.0) containing 150 mM sodium chloride, and the solution was added to the compact reaction column containing the immobilized E8 monoclonal antibody. Binding of the antigen was accomplished at room temperature while the column was gently rotated at 2 rpm for 2 h. The unbound cytochrome c was then removed by washing the column three times with 0.5 mL of water. Following washing, a 1-pL aliquot of beads was removed for analysis by MALDI/TOF. IMAC. Metal Binding Proteins. An IMAC column was prepared by placing 150 pL of HiTrap Chelating sepharose (mean bead size, 34 pm; Pharmacia Biotech Inc., Piscataway, NJ) into a compact reaction column, which was then washed three times with 0.5 mL of water. The column was charged with 0.1 N CuC12 and washed three times with 0.5 mL of water, three times with 0.5 mL of a 100 mM sodium acetate solution (pH 3.5) containing 0.5 M NaCl, and three times with 0.5 mL of a 200 mM sodium phosphate solution (pH 7.0) containing 3 M urea and 500 mM NaCl. The column was then loaded with a solution of horse heart cytochrome c, human lactoferrin, and human apotransferrin (50 pg each) in 200 pL of 200 mM sodium phosphate (pH 7.0) containing 3 M urea and 500 mM NaC1. The column was flushed three times with 0.5 mL of a 200 mM sodium phosphate solution (pH 7.0) containing 3 M urea and 500 mM NaCl and three times with water (0.5 mL). A 0.5-pL aliquot of beads was removed for MALDI/TOF analysis. The column was further washed threetimes with0.5 mLof a 200mM sodiumphosphate solution (pH 4.5) containing 3 M urea and 500 mM NaCl and three times with 0.5 mL of water. A 0.5-pL aliquot of beads was removed for MALDT/TOF analysis. The column was then washed three times with 0.5 mL of a 200 mM sodium phosphate solution (pH 3.5) containing 3 M urea and 500
mM NaCl followed by a 3-fold wash with 0.5 mL of water. A 0.5-pL aliquot of beads was removed for MALDI/TOF analysis. A final wash to remove all metal binding proteins was accomplished with 3 times 0.5 mL of a 200 mM sodium phosphate solution (pH 3.5) containing 3 M urea, 500 mM NaCl, and 200 mM imidazole. Phosphokemptide. An IMAC column was prepared as above except only 50 p L of chelating sepaharose was used. The column was charged with 0.4 mL of 0.1 N FeCl3. Following charging, the column was washed three times with 0.4 mL of water. The phosphokemptide reaction mixture containing maximally 400 pmol of phosphokemptide was spiked with 400 pmol of kemptide. The mixture was loaded onto the column in 50 pL of phosphate buffer solution (pH 7.0), and the column was subsequently washed three times with 0.5 mL of water. A 0.1-pL aliquot of the beads was removed for MALDI/TOF analysis. The column was then washed three times with 0.5 mL of 0.1 N acetic acid containing 500 mM sodium chloride followed by three times 0.5 mL of water. A second 0.1-pL aliquot of beads was removed for MALDI/TOF analysis. The phosphokemptide could be removed from the column by washing with 2% ammonium acetate (pH 9.5). Mass Spectrometry. Mass spectra were acquired on a VG TofSpec (VG Analytical, Wythenshawe, Manchester, UK). This instrument is equipped with an X-Y steerable 337-nm nitrogen laser (focused to a maximum spot size of 150 X 400 pm), and an accelerating voltage of 24 kV was used. A saturated solution of recrystallized a-cyano-4-hydroxycinnamic acid (Aldrich, Milwaukee, WI) in ethanol/water/ formic acid (45:45:10) was used as the MALDI matrix. The standard VG sample targets (1 5 1.5-mm-diameter sample spots/target) were used except that they were roughened with sandpaper prior to use, which may aid in keeping the beads on the target. The samples on the targets could be prepared in either of two ways. In the first method, the samples (0.11.OpL) suspended in deionized water were mixed on the target with a 1-pL aliquot of matrix solution. For the second approach, 10 pL of beads suspended in deionized water was centrifuged. The supernatant was removed, and 20 pL of matrix-containing formic acid was added. The solution was centrifuged, and 0.5 pL of the supernatant was spotted on the target. Crystallization was accelerated by blowing cold air from a hair dryer over the target surface. Approximately 50 scans were averaged to obtain the spectra. Mass calibration was accomplished using an external standard with ions that bracketed the mass range of interest. For high molecular weight species, either bovine serum albumin [MH+ = 66 430.0; (M 2H)2+= 33 215.51 or cytochromec [MH+ = 12 360.1; (M + 2H)2+ = 6180.61 was used while bradykinin (MH+ = 1061.2) and the dimer of a-cyano-4-hydroxycinnamic acid H)+ = 379.41 were used in the kemptide and [(2M phosphokemptide experiments.
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RESULTS AND DISCUSSION The first set of examples of the direct analysis of affinitybound analytes involves antibody/antigen analysis. We have been studying the epitope on cytochrome c for the monoclonal antibody (mAb E8). The antibody was obtained in ascites; therefore, purification was necessary before binding the
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Flgure 1. MALDVTOFMS spectra of agarose beads from a cytochrome c affinity column. (A) After mAb E6 ascites passed over the affinity column and washed wlth water. Peaks designated as E6 are due to the mAb E6. Peaks designated with an asterisk are nonspeclflcally bound proteins from the ascites. (B) After washing column with a 10 mM sodium phosphate/500 mM sodium chloride solution (pH 7.0). The 2+-5+ indicates the respective multiply charged peaks of antlcytochrome c mAb E6. Bovine serum albumin was used as an external calibrant.
antibody to the affinity support. To accomplish this purification, cytochrome c was first bound to the affinity support (see Experimental Section). The crude antibody solution was passed through the column, and a 1-pL aliquot of the column bed was used to acquire the MALDI/TOF spectrum shown in Figure 1A. The spectrum shows the presence of nonspecifically bound analytes and an analyte with an average molecular weight of 147 077 based on averaging the molecular weights determined from the singly and doubly charged ions. The nonspecifically bound analyte has an M r = 65 867 Da and possibly corresponds to mouse albumin (molecular weight by gel electrophoresis,64 0008). After the column was washed with a 10 mM sodium phosphate (pH 7.0) solution containing 500 mM sodium chloride, only signals for the monoclonal antibody were observed. No signal for cytochrome c could be observed in either part A or B of Figure 1, which shows that an antigen immobilized to the affinity beads is not desorbed during MALDI/TOF MS analysis. In a separate experiment, mAb E8 from ammonium sulfate precipitated ascites was attached to agarose beads and used as an affinity column. A solution of cytochrome c was passed through this column. The column was washed to remove any unbound cytochrome c, and a 1-pL aliquot of the column bed was analyzed (Figure 2). The only species observed is cytochrome c with an observed Mr of 12 448 Da (Mr calc, 12 360 Da). The accuracy of mass measurements of analytes directly desorbed from affinity beads and using an external standard is lower than in typical externally calibrated MALDI/TOF experiments (usually 2X) if the beads are mixed with the matrix on the target as opposed to using the supernatant. Also, use of the matrix containing 10% formic acid as an eluent was observed to be insufficient to release apotransferrin from the column prior to MS analysis. This implies that the sample is being released from the beads during desorption by the laser. We cannot conclusively determine, however, whether or not the IMAC analytes are still bound to the beads through their affinity interaction immediately prior to laser desorption. It should be noted that, if too many beads are applied, most can be lost during the drying process. Thus, with our target, the ideal amount of beads (IMAC) is approximately 0.1 pL.
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Flgure 4. MALDI/TOF MS spectra of sepharose beads from an Fe9+charged IMACcolumn.(A)Sf”acqukedatterthereectknmixture from the protein kinase A catalyzed phosphorylatlonof kemptlde (800 fmol of phosphokemptlde maximum on target) spked with kemptlde (800 fmol maximum on target) was passed over the column. (6) Spectrum acquired after washlng the column with 0.1 M acetic acid containing500 mM sodlumchloride. Bradykininwas usedas an internal calibrant
.
column with a pH 4.5 solution removes thelactoferrin (Figure 3B), while a further pH 3.5 wash removes the cytochrome c (Figure 3C). A final 200 mM imidazole wash removed all analytes from the column (data not shown). Iron-charged IMAC columns are also useful for the separation of phosphorylated peptides from nonphosphorylated peptides, with the phosphorylated peptides being bound to the column.12 Such IMAC columns have been interfaced with electrospray mass spectrometry.’ An Fe(II1) IMAC column was loaded with the products from the protein kinase A catalyzed phosphorylation of kemptide and then washed with water. The MALDI/TOF spectrum shows phosphokemptide (MH+,= 854.3; calc, 852.9) and the spiked kemptide (Figure 4A). The amount of spiked kemptide on-target is 800 fmol, and the maximum amount of phosphokemptide present is also 800 fmol. The column was washed with water followed by 0.1 N acetic acid containing 500 mM sodium chloride to remove nonspecifically bound analytes. A 0.1-pL aliquot of the column bed was removed and directly analyzed. The resulting mass spectrum (Figure 4B) shows ions due to only (1 1) Matsumoto,A.; Ycshima, H.; Takasaki, S.; Kobata, A. J . Bfochem.1982.91,
CONCLUSIONS The combination of affinity chromatography with direct MALDI/TOF MS analysis offers a number of advantages. It combines the high specificity of affinity chromatography with the high sensitivity and high information content of mass spectral analysis in a rapid and facile analysis. Other forseeable applications include reaction monitoring (e.g., following the course of an enzymatic or chemical reaction), following consecutive reactions, identification of sites of phosphorylation, DNA footprinting, and rapid screening for the presence of, for example, metal binding proteins or glycosylated proteins. Along with the SEAC technique of Hutchens and Yip,’ direct analysis of the affinity column bed by MALDI/TOF MS provides simple,but extremely powerful, analytical techniques for bioanalysis. Received for review May 9, 1994. Accepted June 30, 1994.a
143-155. (12) Andcrsson, L.;Porath, J. Anal. Biochem. 1986, 154, 250-254.
Abstract published in Aduance ACS Abstracts, August 1, 1994.
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