Photoimmobilization of Proteins for Affinity Capture Combined with

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Anal. Chem. 2004, 76, 6643-6650

Photoimmobilization of Proteins for Affinity Capture Combined with MALDI TOF MS Analysis Dariusz J. Janecki, William C. Broshears, and James P. Reilly*

Department of Chemistry, Indiana University, Bloomington, Indiana 47405

Immobilized molecules on solid surfaces can serve many different purposes. Proteins and biopolymers based on solid supports can mimic receptor-drug interactions in high-throughput screening of candidate drugs.1 Immobilized antibodies and binding proteins (such as concanavalin A (Con A) or protein G) are widely used for separation and purification of materials by affinity chromatography and filtration.2,3 Medical diagnostics rely on immobilized proteins since most immunoassays employ immobilized antibodies or enzymes.4-6 The specificity, sensitivity and availability of antibodies has led to a proliferation of antibodybased biosensors.7-11 Affinity-based biosensors can also be employed in environmental monitoring.12 Industrial applications of immobilized biomolecules are also numerous.13 Diverse examples include the use of catalase on a collagen spiral membrane reactor

to sterilize dairy products14 and the clarification of juice using immobilized endopolygalacturonase.15 Protein immobilization can be accomplished in several ways including adsorption onto solid supports,16,17 entrapment into membranes and films,18 and covalent binding to carriers such as polymeric beads.2,19,20 The characteristics of the support are very important since they exert considerable influence on the performance of covalently attached proteins. Due to either their hydrophilic properties, ease of use, or low cost, the most commonly used support materials include agarose, dextran, cellulose, polystyrene, and polyacrylate.21,22 Covalent binding can be performed in many ways;2 solid supports often possess hydroxy, amino, amide, and carboxy groups that must be activated before they are able to directly react with proteins. Matrixes such as agarose or dextran (Sepharose TM, Sephadex TM) are modified using cyanogen bromide or the less toxic 1,1′-carbonylimidazole.23 After the hydroxyl groups of the matrixes are activated, several types of coupling reactions can be used but most involve proteins’ amino, thiol, or carboxyl groups.2 These reactions are often performed in extreme conditions (e.g., very high pH with CNBr activation) and may result in denaturation of the protein. One more disadvantage of coupling through functional groups is that the latter may be essential for protein activity and thus immobilization can lower or eliminate this activity. Agarose and dextran beads with immobilized binding proteins are commercially available from several manufacturers and are commonly employed as packing materials for affinity chromatography columns. Removal of nonspecifically bound material is facilitated by the hydrophilic nature of these hydrogels.24 On the other hand, it has been reported that their flexible structure may bury ligands in the interior of cross-linked matrixes.25 Ligand accessibility is then lessened and affinity for it is diminished.

* To whom correspondence should be addressed: (phone) 812-855-1980; (e-mail) [email protected]. (1) Waller, A.; Simons, P.; Prossnitz, E. R.; Edwards, B. S.; Sklar, L. A. Comb. Chem. High Throughput Screening 2003, 6, 389-397. (2) In Protein Immobilization, Fundamentals and Applications; Tylor, R. F., Ed.; Marcel Dekker: New York, 1991. (3) Sii, D.; Sadana, A. J. Biotechnol. 1991, 19, 83-98. (4) Lu, B.; Smyth, M. R.; O’Kennedy, R. Analyst 1996, 121, 29R-32R. (5) Butler, J. E. Struct. Antigens 1992, 1, 209-259. (6) Gupta, S.; Suresh, M. J. Biochem. Biophys. Methods 2002, 51, 203-216. (7) North, J. R. Trends Biotechnol. 1985, 3, 180-186. (8) Heineman, W. R.; Halsall, H. B. Anal. Chem. 1985, 57, 1321A-1331A. (9) Place, J. F.; Sutherland, R. M.; Dahne, C. Biosensors 1985, 1, 321-353. (10) Dent, A. H.; Aslam, M. Bioconjugation 1998, 1-49. (11) Collings, A. F.; Caruso, F. Rep. Prog. Phys. 1997, 60, 1397-1445. (12) Marco, M. P.; Barcelo, D. Tech. Instrum. Anal. Chem. 2000, 21, 10751105. (13) Chibata, I.; Tosa, T.; Sato, T. Bioprocess Technol. 1991, 14, 339-362.

(14) Chu, H. D.; Leeder, J. G.; Gilbert, S. G. J. Food Sci. 1975, 40, 641. (15) Pfferi, P. G.; Tramontini, M.; Malacarne, A. Biotechnol. Bioeng. 1989, 33, 1258. (16) Woodward, J.; Wieseman, A. Enzyme Microb. Technol. 1982, 4, 73. (17) Nelson, J. M.; Griffin, E. G. J. Am. Chem. Soc. 1916, 38, 1109-1115. (18) Dinelli, D.; Marconi, W.; Morisi, F. Methods Enzymol. 1976, 44, 227-243. (19) Bahulekar, R. V.; Prabhune, A. A.; SivaRaman, H.; Ponrathnam, S. Polymer 1993, 34, 163-166. (20) Kitano, H.; Hasegawa, M.; Kaku, T.; Ise, N. J. Appl. Polym. Sci. 1990, 39, 241-248. (21) Narayanan, S. R.; Crane, L. J. Trends Biotechnol. 1990, 8, 12-16. (22) Navratil, M.; Sturdik, E. Chem. Listy (Prague) 2000, 94, 380-388. (23) Fodor, S. P.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767-773. (24) Cao, X.; Shoichet, M. S. J. Biomater. Sci., Polym. Ed. 2002, 13, 623-636. (25) Amatschek, K.; Necina, R.; Hahn, R.; Schallaun, E.; Schwinn, H.; Josic, D.; Jungbauer, A. J. High-Resolut. Chromatrogr. 2000, 23, 47-58.

Affinity capture surfaces can be prepared in a number of ways. A method of obtaining such surfaces through UVactivated immobilization of binding proteins using a benzophenone derivative is reported. Photoimmobilized protein G was used to selectively capture and preconcentrate bovine IgG from a mixture with BSA, and the affinity of photoattached concanavalin A toward ovalbumin was compared with that of commercially available concanavalin A on agarose beads. The results of the capture after tryptic digestion were analyzed by MALDI TOF MS. Immobilized trypsin was also prepared through photoimmobilization and later used to digest hemoglobin. Immobilized enzyme digestion resulted in more partial cleavages than solution-phase digestion. More methionine and tryptophan oxidation was also observed. Photoimmobilization was shown to be a quick and easy way of immobilizing ligands on surfaces.

10.1021/ac049212v CCC: $27.50 Published on Web 10/15/2004

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An alternative way of attaching proteins to a surface involves the use of photoactive substrates as linkers between a solid support and a protein. Chemical cross-linking reagents typically possess succinimidyl ester, maleimide, or iodoacetamide reactive groups.2 In contrast, photoreactive cross-linking reagents contain groups that require light activation before reacting with another molecule. Photoactivatable cross-linking reagents have been known for over 30 years, but their usage has recently increased due to the development of more efficient photophores.26,27 Most commonly used photophores belong to three groups: tetrafluorophenyl azides, trifluoromethylphenyl diaziranes, and benzophenone.27 The photolysis products of the fluorinated aryl azides are aryl nitrenes that undergo characteristic nitrene reactions such as C-H bond insertion.28 Likewise, photoactivation of diazirine photophores generates highly reactive carbenes that can undergo intermolecular C-H bond insertion.29,30 The benzophenone photophore has been found to be superior to these photophores because it can be chemically bound to an amine-containing surface and subsequently activated in a reversible manner via an excitation-relaxation cycle. Although stable in common protic solvents,26 upon photoactivation, it reacts with C-H bonds. The reliability and efficiency of these different types of photolabels were compared recently in several studies.31,32 Mass spectrometry is frequently used to detect proteins captured and separated using affinity methods. With the development of MALDI33 and electrospray34 as ionization techniques, a new era in protein research began. Advances such as delayed extraction for MALDI MS35 and nanospray for ESI MS36 have improved the accuracy and sensitivity of these techniques. Due to its speed and sensitivity, MALDI TOF MS is often the first choice for protein identification by peptide mass fingerprinting.37,38 The combination of affinity capture techniques with MALDI mass spectrometric detection was first described by Hutchens and Yip.39 They immobilized single-stranded DNA with high affinity for lactoferrin on agarose beads, used it to capture the protein from a urine sample, and analyzed products by MALDI MS. Subsequently Brockman and Orlando40,41 immobilized a lysozyme antibody on a gold-plated probe surface via thiol groups and successfully captured and detected lysozyme from human tears. Likewise Liang et al. immobilized antibodies on a probe with a nitrocellulose film.42 The strong interaction of immunoglobulin G (IgG) and nitrocellulose fixed the IgG to the surface, which (26) Dorman, G.; Prestwich, G. D. Biochemistry 1994, 33, 5661-5673. (27) Dorman, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18, 64-77. (28) Keana, J. F. W.; Cai, S. X. J. Org. Chem. 1990, 55, 3640-3647. (29) Kauer, J. C.; Erickson-Viitanen, S.; Wolfe, H. R., Jr.; DeGrado, W. F. J. Biol. Chem. 1986, 261, 10695-10700. (30) Shih, L. B.; Bayley, H. Anal. Biochem. 1985, 24, 132-141. (31) Fleming, S. A. Tetrahedron 1995, 51, 12479-12520. (32) Weber, P. J.; Beck-Sickinger, A. G. J. Pept. Res. 1997, 49, 375-383. (33) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (34) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (35) King, T. B.; Colby, S. M.; Reilly, J. P. Int. J. Mass Spectrom. Ion Processes 1995, 145, L1-L7. (36) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (37) Cottrell, J. S. Pept. Res. 1994, 7, 115-124. (38) Pappin, D. J. C.; Hojrup, P.; Bleasby, A. J. Curr. Biol. 1993, 3, 327-332. (39) Hutchens, T. W.; Yip, T. T. Rapid Commun. Mass Spectrom. 1995, 10, 1688. (40) Brockman, A. H.; Orlando, R. Anal. Chem. 1995, 67, 4581-4585. (41) Brockman, A. H.; Orlando, R. Rapid Commun, Mass Spectrom. 1996, 10, 1688-1692. (42) Liang, X.; Lubman, D. M.; Rossi, D. T.; Nordblom, G. D.; Barksdale, C. M. Anal. Chem. 1998, 70, 498-503.

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allowed them to selectively capture a 25-amino acid peptide from a plasma suspension. Numerous medical diagnostic applications of immobilized biomolecules involve glycoconjugates, and every day brings more insight into their role in biorecognition. Plant lectins such as Con A were described in 1919 by Sumner,43 and their carbohydrate recognition44 led to the establishment of the blood group system. A recent review by Rudiger45 provides interesting insights into plant lectin research. The most abundant and frequently used lectin is Con A. Its specific affinity for glucosides and mannosides can be used to separate and concentrate glycoproteins from complex mixtures.46 Bundy and Fenselau showed how lectins and carbohydrates directly immobilized on a membrane surface can be used to capture microorganisms by forming complexes with carbohydrates present on their surface.47,48 In a similar way, commercially available streptavidin-coated glass slides can be derivatized with biotinylated lectin and used for microorganism capture and direct MALDI MS analysis.49 Various other applications have made both activated glass and microbeads readily available. Another large field of application of immobilized biomolecules consists of enzymes attached to solid matrixes.2,19,20 Proteases are used frequently in MALDI MS experiments and immobilized proteases may have many advantages over their soluble counterparts. These include the following: (a) resistance to autolysis so that activity is maintained for longer periods; (b) minimal contamination of a protein digest with enzyme fragments; (c) efficiency with small volumes of proteins; (d) reproducibility of digests.50,51 During the coupling reaction, the enzymes should be stabilized and if necessary additional steps taken in order to protect the active site. Buffer and pH conditions need to be selected such that the enzyme does not readily inactivate. Trypsin immobilized on a gold-coated MALDI probe52 has been employed to rapidly digest hemoglobin in whole human blood.53 Photoinduced coupling using photophores may provide a way of immobilizing enzymes under alternative mild or desirable conditions. In this paper, we combine covalent immobilization of proteins using a photoreactive linker to create an affinity capture surface and use this to capture other proteins. As noted above, the benzophenone photophore that we employ is covalently bound to an amine-containing surface and then photoactivated to capture other molecules that contain C-H bonds, especially peptides and proteins. We have investigated the extent to which biomolecules photolinked to a surface retain their activity (e.g., enzymatic) and affinity for other proteins (protein G affinity for immunoglobulin G and Con A for ovalbumin). We also explore the capability of using photoimmobilized molecules to preconcentrate or separate biological samples for subsequent MALDI MS analysis. (43) Sumner, J. B. J. Biol. Chem. 1919, 37, 137-142. (44) Sumner, J. B.; Howell, S. F. J. Bacteriol. 1936, 32, 227-237. (45) Rudiger, H.; Gabius, H. J. Glycoconjugate J. 2001, 18, 589-613. (46) Mattiasson, B.; Borrebaeck, C. FEBS Lett. 1978, 85, 119-123. (47) Bundy, J.; Fenselau, C. Anal. Chem. 1999, 71, 1460-1463. (48) Bundy, J. L.; Fenselau, C. Anal. Chem. 2001, 73, 751-757. (49) Afonso, C.; Fenselau, C. Anal. Chem. 2003, 75, 694-697. (50) Bornscheuer, U. T. Angew. Chem., Int. Ed. 2003, 42, 3336-3337. (51) Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand Techniques; Academic Press: San Diego, CA, 1992. (52) Nelson, R. W. Mass Spectrom. Rev. 1997, 16, 353-373. (53) Houston, C. T.; Reilly, J. P. Anal. Chem. 1999, 71, 3397-3404.

MATERIALS AND METHODS Chemicals. 4-Benzoylbenzoic acid, succinimidyl ester (BBSE) was purchased from Molecular Probes (Eugene, OR). HypoGel 400 NH2 beads were obtained from Rapp Polymere GmbH (Tu¨bingen, Germany). Bovine trypsin (TPCK treated), horse heart myoglobin, horse cytochrome c, recombinant protein G (Streptococcus sp.), bovine IgG, Con A, and chicken egg albumin (ovalbumin) were all purchased from Sigma (St. Louis, MO). Agarose beads labeled with Con A were obtained from Sigma (C6904, according to the manufacturer, 15 µL of gel slurry has a maximum binding capacity of 80 µg of yeast mannose). Porcine trypsin modified by methylation was also obtained from Sigma as “proteomics grade”. Preparation of Photoactive Beads. HypoGel NH2 is a hydrophilic gel-type resin consisting of poly(ethylene glycol)s (PEGs) grafted on to a low cross-linked (1% DVB) polystyrene substrate. The reactive primary amine centers are located at the termini of the glycol spacers. These long spacer “arms” and the hydrophilic properties of the resin were the main reasons why this material was chosen. According to the manufacturer, the beads have 0.69 mmol of active sites/g.54 Assuming the beads to be spherical with a mean particle size of 130 µm (manufacturer’s information), the area of a bead was calculated to be 5.31 ×10-8 m2. A 1-mg sample of beads was weighed out on a balance, and the number of beads in the sample was found to be 800. This is consistent with manufacturer’s data and allows us to estimate bead area to be 4.25 × 10-2 m2/g. Combining this with the stated density of 0.69 mmol/g implies a surface density of 9.78 × 1021 sites/m2 or 9.78 × 103 sites/nm2. Although these high numbers suggest that beads are not smooth spheres but have additional surface area, the high density of amine sites allows for a potentially high density of affinity ligand. To prepare photoactive beads, ∼4 mg of HypoGel NH2 beads was first shaken with 200 µL of 20 mM phosphate buffer pH 7.8 in a 1.6-mL tube and after 15 min 150 µL of the buffer was discarded. This cleaning step removed some PEG that may have leaked from the beads and it also kept them wet. Later, 70 µL of 2.5 mM BBSE solution in dimethylformamide (DMF) was added every 15 min (4 times), and the tubes were gently shaken. After all portions of BBSE were added, the beads were shaken for 30 min more, washed with DMF and water, and then dried in a Speedvac. If not used immediately, they were kept desiccated in a refrigerator. Photoimmobilization. On the order of 10-15 µg of a protein to be linked to the beads was added to 4 mg of photoactivated beads in 15 µL of water. The mixture was then irradiated with either a mercury UV lamp (Fluorolume 645L; American Optical Co., Rochester, NY) or a Nd:YAG laser (third harmonic 355 nm, 10 Hz, 2.3 mJ/pulse, Quanta Ray, Mountain View, CA) for 5 or 10 min. After irradiation, the beads were thoroughly washed to remove nonspecifically adsorbed proteins. Trypsin Digestion. In solution, digests were prepared using 200 pmol of a protein in 20 mM NH4HCO3 buffer incubated with porcine or bovine trypsin (50:1 w/w ratio) for 16 h at 37 °C. Digestion was stopped by adding 10 µL of 10% trifluoroacetic acid (TFA) (total digest volume 100 µL). Immobilized proteins were tryptically digested by adding 100 mM ammonium bicarbonate buffer to the beads and 15 pmol of trypsin (either porcine or bovine) and incubating the resultant (54) http://www.rapp-polymere.com/preise/hypo_d.htm, accessed May 2004.

mixture for 16 h at 37 °C. Tubes were shaken during digestion using a top-to-bottom rotor. Digestion was stopped by adding 5 µL of concentrated TFA (total digest volume 45 µL). After 5 min of sonication, the solution was removed from above the beads and moved to another tube where it was kept until analysis by MALDI TOF MS. Samples of bovine IgG and ovalbumin were denatured for 20 min at 90 °C before digestion. MALDI TOF Mass Spectrometry. Mass spectra were acquired on a Bruker Reflex III (Billerica, MA) mass spectrometer in reflector mode. A solution of R-cyano-4-hydroxycinnamic acid (10 g/L) in acetonitrile/water (0.1% TFA) (1:1 v/v) was used as matrix. The analyte solution was mixed with matrix solution in a 1:5 ratio before applying 0.65 µL of the resulting mixture to the probe. RESULTS AND DISCUSSION Washing Off the Adsorbed Proteins. Since the goal of the experiments was to photoimmobilize biomolecules, considerable effort was expended to ensure that our proteins were not simply adsorbed to the beads. For each experiment, a stringent washing protocol was developed as described in each section below. To develop these protocols, control samples of photoactivated beads were prepared and mixed with protein but not irradiated with UV. Conditions were sought such that after tryptic digestion of material left on a washed surface no mass spectral peaks were observed. Determination of Optimal Bead/Protein Ratio. To determine appropriate amounts of photoactivated beads and protein to be employed, we investigated the photoactivated capture of cytochrome c. HypoGel NH2 beads were first modified with the photoactive linker BBSE. Different amounts of BBSE and beads were tried in an attempt to maximize the amount of captured protein. For each experiment, 4 mg of HypoGel NH2 beads were reacted with 0.88, 0.44, 0.22, and 0.088 mg of BBSE. Based on calculations presented above, these corresponded-to-molar ratios of BBSE to NH2 sites of 1:1, 1:2, 1:4, and 1:10. A 15-µL aliquot of 1 g/L cytochrome c solution was added to each sample of activated beads and all irradiated with the UV lamp for 10 min. After intensive washing of the beads to remove protein that was not covalently bound, the immobilized protein was digested with trypsin and peptides analyzed by MALDI TOF MS. Bradykinin was spiked into the MALDI spots to serve as a rough internal standard (20 fmol on a MALDI spot). For each sample, 12 spectra were recorded and averaged. With a high amount (1:1 ratio) of photolinker on the beads, we noticed an increased signal for some tryptic peptides but sequence coverage was reduced (Supporting Information, Figure 1) possibly due to multiple attachment of cytochrome c to the beads. We concluded that a 1:4 ratio of BBSE to primary amine sites results in good sequence coverage for captured protein and the best signal in MALDI. All later experiments were performed using beads derivatized with this ratio. Since the photolinker reacts with at most 1/4 of the active primary amine sites, the remaining basic amine groups must create a slightly positive surface at neutral pH. The interaction of this surface with negatively charged areas of proteins during coupling may help to establish preferred binding orientations. Photoattachment of Model Proteins. Using the optimal conditions for BBSE/beads determined above, two model proteins were photoimmobilized and mass spectra obtained following tryptic digestion of immobilized protein. These spectra were Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

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Figure 1. MALDI TOF spectra of trypsin digestion of (A) cytochrome c photoimmobilized on HypoGel beads, (B) cytochrome c in solution, (C) myoglobin photoimmobilized on HypoGel beads, and (D) myoglobin in solution. (*) denotes tryptic peptides of cytochrome c or myoglobin; (T) trypsin autolysis fragment.

compared with those obtained after solution-phase digestion. The model proteins were cytochrome c and myoglobin. A 4-mg aliquot of photoactivated beads was mixed with 15 µL of a 1 g/L solution of each protein and irradiated with UV light. After irradiation, the beads were washed 10 times with 200-µL portions of ACN/water (0.1% TFA) (1:1 v/v) solution in order to remove nonspecifically adsorbed protein. The beads were then dried and immobilized proteins digested with bovine trypsin as described in Materials and Methods. No MALDI MS signals from digested proteins were observed when UV irradiation did not take place. Figure 1 displays a comparison of MALDI TOF spectra from tryptic digests of cytochrome c and myoglobin either photoattached to HypoGel beads (Figure 1A and C) or dissolved in solution (Figure 1B and D). Two striking observations can be made about cytochrome c spectra: the distributions of tryptic fragments differ in the two cases, and the signal-to-noise ratio of the spectrum obtained with immobilized cytochrome c is significantly worse. It is not surprising that the digest of cytochrome c photolinked to the surface leads to fewer tryptic fragments because some of the peptides are most likely bound to the beads. One of the most intense peaks in the solution-phase digestion spectrum at 1633.82 Da, the heme-containing peptide (a porphyrin molecule attached to cysteines Cys14 and Cys17), is absent in the photoattached cytochrome c spectrum. Another missing heme-containing peptide appears at 1875.8 Da in Figure 1B. Some of the peptide fragments not observed in the immobilized protein spectrum arise from a large region of cytochrome c sequence between Lys54 and Lys86. Four closely spaced glutamic acid residues (Glu61, Glu62, Glu66, Glu69) between Lys54 and Lys71 make this part of cytochrome c negatively charged at neutral pH. Figure 2A shows a 3D structure of the cytochrome c molecule55 (structure 1AKK from Protein Data Bank; www.pdb.org). It is likely that these negative charges are attracted toward the positively charged beads. If these parts of cytochrome c were immobilized on the beads, after tryptic digestion, these peptides might not be released to the solution phase. Other peaks missing from the immobilized (55) Banci, L.; Bertini, I.; Gray, H. B.; Luchinat, C.; Reddig, T.; Rosato, A.; Turano, P. Biochemistry 1997, 36, 9867-9877.

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Figure 2. Cytochrome c (A) 3D structure 1AKK from Protein Data Bank (www.pdb.org) (B) Sequence coverage: dotted lines show coverage for in solution digest and boxes for digest of immobilized cytochrome c.

protein spectrum at 2081.0 and 2209.1 Da also account for peptides from this region of cytochrome c ([G56-K72] and [G56-K73], respectively). Heme is another negatively charged entity (two carboxylic groups) that may be drawn to face the beads. Figure 2B compares the sequence coverage for cytochrome c digested in solution and immobilized to the HypoGel beads. Only 40.4% of the sequence is covered for immobilized cytochrome c compared with 76.0% for the solution-phase digest. Absent from the sequence coverage in immobilized cytochrome c tryptic digest are peptides reported above from negatively charged areas of cytochrome c. Immobilization of cytochrome c on a negatively charged resin was recently reported by Regnier,56 who concluded that adsorption of cytochrome c to negatively charged resin is due to adsorption at multiple residues. In our study, we found that cytochrome c interacts strongly with positively charged beads due to its negatively charged areas. When comparing tryptic digests of immobilized and solutionphase myoglobin once again we observe lower S/N ratio for the immobilized protein spectrum (Figure 1C), but in this case, the two peptide fragment distributions are very similar. The sequence coverage for myoglobin in solution digest is 73.2% and only slightly less (70.0%) for the photoattached protein (Figure 1C). Myoglobin, unlike cytochrome c, does not have large negatively charged areas that would preferentially adsorb to a positively charged surface. In both experiments described above, the MALDI spectra of tryptic peptides from immobilized proteins show lower signal then spectra for in-solution digests. Although the exact amount of protein that was immobilized is not known, an upper limit for this can be calculated. The 1 µg of cytochrome c equals 1.2 nmol, and 15 µg of myoglobin is 0.88 nmol. Assuming that each sample was completely photoimmobilized and digested in 45-µL total volume and that MALDI spots were prepared by mixing the digest with matrix solution with 1:5 ratio and then depositing 0.65 µL of this (56) Xu, W.; Zhou, H.; Regnier, F. E. Anal. Chem. 2003, 75, 1931-1940.

mixture onto the probe, 2.8 and 2.1 pmol of cytochrome c and myoglobin, respectively, would have been deposited onto the probe. In contrast, solution-phase digests of 250 fmol of cytochrome c (Figure 1B) and 720 fmol of myoglobin (Figure 1D) yielded stronger MALDI signals. The above calculations suggest that only a fraction of the available protein is immobilized through photoattachment or that the digestion of the immobilized protein is very inefficient. In addition, the HypoGel beads contain PEG spacers that produce PEG ions in MALDI mass spectra. Their appearance may decrease the ionizability of tryptic peptides. C18 RP microcolumns has been proven very effective for desalting and preconcentration of tryptic digests before MALDI TOF analysis. Larsen57 described a way to clean off PEG before MALDI analysis using C18 microextraction columns or pipet tips with activated graphite, but our experience with these approaches was not successful. In general, the intensity of peptide peaks in spectra increased when samples were cleaned up with microcolumns but PEG peaks spaced by 44 Da were still present in all of our spectra of immobilized protein digests in the mass range 1000-1300 Da. (Additional data are presented as Supporting Information, Figure 2). Photoimmobilization of Trypsin on the Beads. To determine whether the activity of an enzyme is retained after photoimmobilization, we photoattached bovine trypsin to the beads and then investigated the ability of this enzyme to digest human hemoglobin. HypoGel beads with benzophenone photolinker were prepared as described above. A 4-mg aliquot of the beads activated by reaction with BBSE were mixed with a 15 µL (1 g/L) solution of bovine trypsin and irradiated with a UV lamp or YAG laser. To remove adsorbed (not photolinked) trypsin, the beads were moved to a fresh tube and washed thoroughly with phosphate buffer (PBS) (pH 7.2, 20% ACN, 0.1% Triton X-100, 0.8M NaCl). Considerable time was spent to find the best washing solution to remove adsorbed trypsin. Typically, 10 washes of buffer solution such as Tris or phosphate combined with vigorous shaking of beads and solution in a tube were enough to remove proteins from the beads but that was not enough to completely eliminate the proteolytic activity of adsorbed trypsin. When hemoglobin was added to such “cleaned” beads, hemoglobin tryptic fragments were observed in MALDI mass spectra, indicating that some trypsin was still adsorbed. To remove adsorbed trypsin from HypoGel beads, as many as 15 washes with 200 µL of phosphate buffer containing also 20% ACN, 0.1% Triton X-100, and 0.8 M NaCl had to be used followed by two washes with 200 µL of water. To the washed beads, 10 µg of human hemoglobin in 20 mM ammonium bicarbonate buffer was added and the resultant mixture was digested overnight at 37 °C. In this experiment, 32.3 nmol of hemoglobin interacted with the beads and 0.12% of the digest solution was deposited on the MALDI spot. The MALDI TOF spectrum of the hemoglobin digest achieved with photoimmobilized trypsin is shown in Figure 3A. The distribution of peptides appears to be somewhat different compared to the solution-phase experiment (Figure 3B). Digestion fragments of hemoglobin obtained with immobilized trypsin have more missed cleavage sites (peaks in the mass range above 2000 Da). The reason for more missed cleavage peptides is that the digest rate is reduced when the contact between enzyme and protein is constrained. This results in fewer sites being cleaved. (57) Larsen, M. R.; Cordwell, S. J.; Roepstorff, P. Proteomics 2002, 2, 12771287.

Figure 3. Digest of human hemoglobin by bovine trypsin: (A) photoimmobilized on beads; (B) in solution.

A similar missed cleavage phenomenon is often observed in polyacrylamide gel digests58 because trypsin mobility in the gel is limited. Likewise Houston and Reilly noted that on-probe digestion of hemoglobin with immobilized trypsin produces more partial cleavage products then solution-phase digestion.53 Several peaks appearing in Figure 3 display spacings of 16 or 32 Da. Some of these can be attributed to oxidation of methionine to methionine sulfoxide. Other peaks correspond to peptides containing oxidized tryptophan;59 e.g., in the experiments when photoimmobilized trypsin digested hemoglobin, the peptide LLVVYPWTQR (m/z 1274.7) was observed also as singly (+16 Da) or doubly (+32 Da) oxidized (inset Figure 3). Methionine oxidation during chemical treatment has been described in the literature, and it is sometimes incorporated into peptide mass fingerprinting (PMF) search algorithms.60 Tryptophan oxidation has also been previously reported in anchor chip experiments61 where it was attributed to spot preparation on microcrystalline CHCA surfaces. The surface may also be responsible for oxidation of peptides during in-gel digestion. The oxidation of methionine and tryptophan in our experiments is likely caused by our resin. The high number of oxidized forms of peptides decreases the sensitivity of the method. Sequence coverage for the immobilized trypsin digest of hemoglobin is 49.6% (70/141) for the R chain and 70.5% (103/ 146) for the β chain. These are lower than what was reported by Houston for solution-phase hemoglobin digest (64.5% (91/141) R chain and 84.9% (124/146) β chain),53 but in our experiment, small peptides (below 1000 Da) were difficult to observe due to PEG signal interference. Partial digestion does allow for some small peptides to be detected due to their incorporation into larger fragments (e.g., R[56-60] m/z 398.2 Da detected as R[41-60] 2213.11Da and R[41-61] 2341.20 Da). Some other peptides not (58) Thiede, B.; Lamer, S.; Mattow, J.; Siejak, F.; Dimmler, C.; Rudel, T.; Jungblut, P. R. Rapid Commun. Mass Spectrom. 2000, 14, 496-502. (59) Bienvenut, W. V.; Deon, C.; Pasquarello, C.; Campbell, J. M.; Sanchez, J. C.; Vestal, M. L.; Hochstrasser, D. F. Proteomics 2002, 2, 868-876. (60) http://www.matrixscience.com/cgi/search_form.pl?FORMVER)2& SEARCH)PMF, accessed May 2004. (61) Gobom, J.; Schuerenberg, M.; Mueller, M.; Theiss, D.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2001, 73, 434-438.

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Table 1. Tryptic Peptides Observed from Digestion of Human Hemoglobin expl [M + H]+ masses

sequence β [9-17] β[1-8] R[32-40] R[91-99] (or R[32-40]a) R[1-11] β[31-40] β[31-40]b β[31-40]c β[18-30] β[121-132] β[133-146] R[17-31] β[67-82] R[1-16] R[1-16]b R[41-56] R[12-31] β[41-59] β[41-59]a R[41-60] β[9-30] β[9-30]b β[9-30]c β[41-61] β[41-61]a R[41-61] R[17-40] R[17-40]a R[17-40]d β[1-30] β[1-30]b β[1-30]c c

calc [M + H]+ masses

solutionphase digest

immobilized trypsin digest

no. of missed cleavages

932.52 952.51 1071.55 1087.62

932.51 952.50 1071.58 1087.66

952.50 1071.56 1087.60

0 0 0 1

1171.64 1274.72 1290.70 1306.68 1314.64 1378.63 1449.72 1529.73 1669.91 1684.90 1700.91 1833.88 2043.01 2058.96 2074.93 2213.11 2228.21 2244.20 2260.17 2286.21 2302.10 2341.20 2582.33 2598.25 2614.33 3161.66 3177.63 3193.61

1 0 0 0 0 0 1 0 0 2 2 0 1 0 0 1 1 1 1 1 1 2 1 1 1 2 2 2

1171.67 1274.72 1290.72 1306.72 1314.66 1378.70 1449.79 1529.73 1669.89 1684.94 1700.94 1833.89 2043.00 2058.95 2074.94 2213.09 2228.17 2244.17 2260.17 2286.11 2302.11 2341.18 2582.27 2598.26 2614.26 3161.66 3177.66 3193.66

1274.72 1314.65 1378.65 1449.83 1529.74 1669.91 1833.86 2058.97

a Methionine partially oxidized. b Tryptophan partially oxidized. Tryptophan completly oxidized. d Methionine completly oxidized.

observed are known to precipitate during tryptic digestion because of their hydrophobicity. They need to be derivatized in order to be observed.62,63 All observed peptides, along with their calculated masses, are listed in Table 1. The wavelength used to activate benzophenone is ∼300 nm, which should not have great influence on the attached enzyme, but standard mercury lamps also provide light below 300 nm (184.9, 253.6 nm).64 Several authors described the possibility of destabilizing proteins with UV light,65-68 causing inactivation or loss of aromatics. In our experiment, we used both the Nd:YAG (third harmonic line at 355 nm) laser and mercury UV lamp to activate benzophenone and found no difference in the activity of immobilized trypsin (Supporting Information, Figure 3). (62) Wada, Y.; Matsuo, T.; Sakurai, T. Mass Spectrom. Rev. 1989, 8, 379-434. (63) Ross, G. A.; Lorkin, P.; Perrett, D. J. Chromatogr., A 1993, 636, 69-79. (64) Winge, R. K. Inductively coupled plasma-atomic emission spectroscopy: an atlas of spectral information; Elsevier: New York, 1985. (65) Drzazga, Z.; Michnik, A.; Bartoszek, M.; Beck, E. J. Therm. Anal. Calorim. 2001, 65, 575-582. (66) Kovaleva, T. A.; Kozhokina, O. M.; Trofimova, O. D. Radiatsionnaia Biologiia, Radioecologiia 2000, 40, 23-27. (67) Miles, C. A.; Sionkowska, A.; Hulin, S. L.; Sims, T. J.; Avery, N. C.; Bailey, A. J. J. Biol. Chem. 2000, 275, 33014-33020. (68) Durchschlag, K.; Hefferle, T.; Zipper, P. Radiat. Phys. Chem. 2003, 67, 479486.

6648 Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

The hemoglobin digest obtained by using immobilized trypsin produces a spectrum with lower signal-to-noise ratio than the solution digest. This is most likely a consequence of less trypsin being available to digest the hemoglobin. Calculations shown above for cytochrome c and myoglobin immobilization suggested that only a small amount of protein may be attached to beads. The enzymatic activity of trypsin can be also lowered due to immobilization as has been sometimes noticed for other enzymes.69 The suppressed ionization of peptides due to the presence of PEG (peaks in ∼1000-Da range) is also evident in the spectrum obtained for the immobilized enzyme digest (Figure 3A). The presence of oxidized species further lowers the overall signal-tonoise ratio. Capture of IgG with Photoimmobilized Protein G. Methods of purifying proteins from complex mixtures are fundamental to immunochemistry. A specific antibody bound to a matrix allows the capture and detection of low-abundance proteins. The most commonly used protein for binding monoclonal and polyclonal IgGs is protein G isolated from streptococci bacterium.70,71 Immunoglobulin G often has to be captured and preconcentrated from solutions containing serum albumin protein. In our experiments, we used a recombinant form of protein G that lacks the albumin binding region of the native molecule, leaving two IgG binding sites that can bind all subclasses of bovine IgG.72,73 Experiments were conducted to selectively capture bovine IgG from a mixture containing bovine serum albumin (BSA). To photoimmobilize protein G to the beads, the photoactivated beads were prepared by reacting 4 mg of HypoGel NH2 beads with BBSE as described in Materials and Methods. A 10-µg aliquot of recombinant protein G in 15 µL of water was added to photoactivated beads, and the resultant mixture was irradiated with the UV lamp for 5 min. Unattached protein G was washed off with immunoprecipitation (IP) buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl). Beads with photoimmobilized protein G were then incubated for 1 h at 4 °C with 10 µg (∼68 pmol) of bovine IgG and 10 µg (∼150 pmol) of BSA in 400 µL of IP buffer and water mixture (1:1 v/v). The beads were washed with IP buffer and heated to 90 °C for 20 min to denature the bound proteins. The latter were digested overnight with trypsin, and digests were analyzed using MALDI TOF MS. Spectra of digests of captured IgG and solution-phase IgG are compared in Figure 4. The spectrum of selectively captured IgG (Figure 4A) shows only peaks for IgG (labeled *) and protein G (labeled G) tryptic peptides. In total, eight peaks can be attributed to bovine IgG (either light chain or IgG2a heavy chain) and six peaks to protein G. Just as important, no signal is observed from BSA, which is not supposed to be captured by protein G. When no photoimmobilization of protein G was performed, all proteins were washed off the beads and the spectrum displayed neither IgG nor BSA tryptic peptide peaks (Figure 4B). The experiment proves that the affinity of protein G toward IgG is preserved after photoimmobilization and that it is possible to selectively capture bovine IgG from a mixture that contains (69) Zoungrana, T.; Findenegg, G. H.; Norde, W. J. Colloid Interface Sci. 1997, 190, 437-448. (70) Bjorck, L.; Kronvall, G. J. Immunol. 1984, 133, 969-974. (71) Akerstrom, B.; Brodin, T.; Reis, K.; Bjorck, L. J. Immunol. 1985, 135, 25892592. (72) Kochan, J. E.; Wu, Y. J.; Etzel, M. R. Ind. Eng. Chem. Res. 1996, 30, 11501155. (73) Kacskovics, I.; Butler, J. E. Mol. Immunol. 1996, 33, 189-195.

Figure 4. MALDI TOF spectra of porcine trypsin digest of (A) bovine IgG selectively captured by protein G photoattached to the beads, (B) control experimentsphotoactive beads mixed with protein G but not irradiated with UV light, and (C) solution-phase digest of bovine IgG. (*) denotes IgG peptides, (G) protein G, and (T) trypsin autolysis peaks.

another protein using photoimmobilized protein G. The capture of IgG was obtained from 25 µg/mL solution (∼0.17 µM; 68 pmol of IgG total amount) implying that preconcentration of IgG is possible with immobilized protein G. Comparison of the spectrum for IgG solution-phase digest (Figure 4C) with that for captured IgG indicates a small decrease of signal-to-noise ratio. Once again a signal for PEG in the mass range around 1000 Da is evident. PEG is interfering with ionization of peptides and is undoubtedly contributing to this small decrease in signal. Capturing Ovalbumin with Photoimmobilized Concanavalin A. The ability of Con A photoattached to HypoGel beads to selectively capture chicken egg albumin (ovalbumin) from a mixture of proteins was explored next. Commercial beads with Con A immobilized on agarose are also available, and we compared their performance with that of Con A photoimmobilized on HypoGel beads. A 4-mg aliquot of HypoGel beads was activated by reaction with BBSE. Con A was dissolved in water, and 15 µL of 1 g/L solution was added to the beads activated with BBSE. The mixture was irradiated for 10 min with 355-nm laser light, and then the beads were pretreated with wash solution (1 M NaCl, 5 mM MgCl2, 5 mM MnCl2, and 5 mM CaCl2) and equilibrated with Tris buffer (pH 7.15, 0.5 M NaCl). HypoGel beads with attached Con A were mixed later with a solution containing 450 pmol each of ovalbumin, cytochrome c, and myoglobin in 200 µL of Tris buffer and slowly shaken for 2h at 4 °C. After this binding step, the beads were thoroughly washed with equilibrating buffer and digested with porcine trypsin as described in Materials and Methods. The MALDI TOF mass spectrum of the digest is presented in Figure 5A. Only peaks for ovalbumin (labeled *) and Con A (labeled C) are present in the spectrum showing that immobilized Con A selectively captured only glycoprotein. To see whether nonspecific adsorption on positively charged beads played a role in the capture, we mixed the same amount of beads but without photolinker with the mixture of proteins and subjected it to the same procedures as photoactivated beads. The MALDI spectrum for the digest is shown in Figure 5B. The only signal observed

Figure 5. Selective capture of ovalbumin from mixture with myoglobin and cytochromec by Con A on beads: (A) Con A photoimmobilized, (B) control experimentsphotoactive beads mixed with Con A but not irradiated with UV light, and (C) commercially available Con A on agarose beads. (*) ovalbumin fragments; (c) Con A fragments; (T) trypsin autolysis fragments.

was for one trypsin autolysis fragment (labeled T), proving that the washing protocol was stringent enough to remove adsorbed proteins. An attempt to compare commercially available agarose beads labeled with Con A (Sigma C6904) with 4 mg of the beads prepared by photoimmobilization of Con A was made as follows. A 15-µL aliquot of the agarose/Con A bead slurry was prewashed and equilibrated with Tris buffer (pH 7.15, 0.5 M NaCl). A 200µL aliquot of protein mixture (ovalbumin, cytochrome c, myoglobin; 450 pmol each) in Tris buffer was added to the beads and the resultant mixture was shaken for 2 h at 4 °C. After this binding step, agarose beads were washed in the same way as HypoGel beads with Con A. Finally, captured proteins were digested and MALDI mass spectra recorded. Figure 5C displays the result. The overwhelming signal in the spectrum comes from Con A peptides (labeled C), and in order to show ovalbumin signal (labeled*), the intensity scale was broken at 850 intensity units. The Con A peptide peak at 2103.05 Da has an intensity over 9500 counts while the 1687.83-Da peak for ovalbumin (the strongest ovalbumin peak in solution-phase digests) has an intensity of only 130 counts. Peptides from the other two proteins are not present in both spectra. Although the extent of labeling of the commercial agarose/Con A beads is very high, many of these labels are possibly inaccessible to the ligate due to the structure of the polysaccharide matrix. Also, the method of immobilizing Con A on agarose involves p-nitrophenyl chloroformate activation and reaction with amino groups of Con A to form urethane bonds. Bond formation with lysines of Con A active sites may lower its binding capacity. Although the amount of Con A immobilized on HypoGel beads is probably smaller, the affinity ligand (Con A) is open toward the analyte (due to the rigid structure of the beads and ligand attachment on their surface) and we may obtain the same selectivity and better capture using photoattached Con A. Once again the overall observed signal-to-noise ratio in the spectrum of digested ovalbumin captured with photoimmobilized Con A is probably diminished by the production of PEG ions. Peaks spaced by 44 Da are clearly seen in the spectrum (Figure 5A). In the future, other materials that reduce leakage of PEG Analytical Chemistry, Vol. 76, No. 22, November 15, 2004

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Table 2. Summary of the Results immobilized protein cytochrome c myoglobin trypsin protein G concanavalin A

sequence coverage (%) activity

digest of hemoglobin

on beads

in solution

40.4 70.0 49.6 R chain 70.5 β chain

76.0 73.2 64.5 R chain 84.9 β chain53

capture IgG from mixture with BSA capture ovalbumin from mixture with cytochrome c and myoglobin

will be utilized in the experiments. This should improve both the signal-to-noise ratio and the sensitivity of the experiment. CONCLUSIONS The immobilization of macromolecules for affinity capture using photoactivation as presented in this paper provides a quick and easy way of preparing beads with immobilized ligands. This method can be used to bind virtually any peptide or protein since they contain numerous C-H bonds that can be reacted with the photophore. Chemical modifications of solid supports and protein coupling reactions are often performed in extreme pH conditions or using highly toxic reagents. During these reactions, protein functional groups are modified and this can lead to lowered activity. Photoactivation, on the other hand, can be performed in water or buffer solution providing mild conditions for biomolecule attachment. We showed that irradiation with UV light and immobilization on the surface does not destroy the activity of several proteins. Use of beads with a photolinker can be an

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alternative way to prepare affinity surfaces for quick screening of protein-protein interactions. This preparation of an affinity surface is also economical. The traditional methods to immobilize antibodies or binding proteins often denature or modify these molecules. After being washed off, they are unusable. In contrast, after photoimmobilization on beads, the protein that is not attached during the process can be removed and used for preparation of another portion of beads. The results of the experiments described in this paper are summarized in Table 2. The main disadvantage of the method of photoimmobilizing macromolecules described in this paper is currently the low yield of the attachment. Another type of photophore might yield superior results. The presence of leaked PEG suppresses ionization in MALDI MS and complicates part of each spectrum. The problem of PEG leakage may be reduced in the future by using another type of beads in which the PEG is attached through a urethane linkage. ACKNOWLEDGMENT This work was supported by the National Science Foundation Grant CHE 0094579. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review May 28, 2004. Accepted August 30, 2004. AC049212V