Selective Binding of RNase B Glycoforms by Polydopamine

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Anal. Chem. 2009, 81, 5413–5420

Selective Binding of RNase B Glycoforms by Polydopamine-Immobilized Concanavalin A Todd A. Morris,* Alexander W. Peterson, and Michael J. Tarlov National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899 Glycoanalysis is important in the manufacture and quality control of protein therapeutics. An emerging method for glycoanalysis is the use of lectin arrays. Critical to the performance of these arrays is the immobilization of lectin molecules. Polydopamine has recently been shown to adsorb to a wide variety of surfaces. In this study, polydopamine (pDA) was used to modify gold, indium, and iridium surfaces and promote the adhesion of the r-mannose-specific lectin concanavalin A (Con A). The activity of the surface-bound lectin was demonstrated with the r-mannose-presenting glycoprotein ribonuclease B (RNase B). Surface plasmon resonance spectroscopy (SPRS) was used to demonstrate the selective affinity of RNase B for Con A. Surface-MALDI-TOF MS experiments revealed that the affinity of polydopamine-immobilized Con A for the glycoforms of RNase B is significantly affected by slight variations in oligosaccharide structure and composition. Specifically, surface-bound Con A binds certain Man7, Man8, and Man9 RNase B glycoforms more strongly than Man5 and Man6 glycoforms. Glycosylation is one of the most common post-translational modifications of proteins and occurs in the endoplasmic reticulum and Golgi complex of eukaryotic cells.1,2 This multistep process results in the net addition of sugar moieties to the peptide backbone and is accomplished by a series of glycosyltransferase and glycosidase enzymes. The activity of these enzymes is cell line-specific and can be influenced by several environmental factors.3 As a result, glycoproteins can exhibit considerable heterogeneity in their glycosylation. This characteristic has important implications for the manufacturing of glycosylated protein therapeutics. Glycosylation impacts a wide range of properties that are directly linked to the safety and efficacy of glycoprotein therapeutics.4,5 As a consequence, characterization of the oligosaccharide profile of glycoproteins is an essential element in quality control procedures used in manufacturing these biopharmaceuticals.2 Detailed glycoanalysis of protein therapeutics is typically performed with mass spectrometric6-9 and chromato* Author to whom correspondence should be addressed. Fax: 301-975-2643. E-mail: [email protected]. (1) Helenius, A.; Aebi, M. Science 2001, 291, 2364–2369. (2) Walsh, G.; Jefferis, R. Nat. Biotechnol. 2006, 24, 1241–1252. (3) van Kooyk, Y.; Rabinovich, G. A. Nat. Immunol. 2008, 9, 593–601. (4) Chirino, A. J.; Mire-Sluis, A. Nat. Biotechnol. 2004, 22, 1383–1391. (5) Jefferis, R. Biotechnol. Prog. 2005, 21, 11–16. (6) Harvey, D. J.; Wing, D. R.; Kuster, B.; Wilson, I. B. H. J. Am. Soc. Mass Spectrom. 2000, 11, 564–571. 10.1021/ac900715d Not subject to U.S. Copyright. Publ. 2009 Am. Chem. Soc. Published on Web 06/10/2009

graphic methods.10 Although these are highly developed and effective methods for determining the product’s glycoprofile, they require highly trained operators and costly equipment and are time-consuming. An alternative strategy for glycosylation screening involves the use of lectin arrays.11-13 In this approach, lectins, which are sugarbinding proteins of nonimmunologic origin, are immobilized to a solid substrate in a specific array pattern. Lectins are generally specific for a small number of saccharide motifs (e.g., mannose, galactose, glucose). Therefore, when a glycoprotein is applied to a properly designed lectin array, the resulting pattern can be used as a fingerprint of the protein’s glycoprofile. In contrast to the more established analytical methods, lectin arrays potentially offer lower cost and near real-time glycoform reporting. In addition to screening glycoprotein therapeutics, glycoprofiling by lectin arrays has been reported to be useful for discriminating between healthy and malignant cells.11 In both cases, lectins are employed for their ability to report gross changes in the glycosylation patterns of the samples (e.g., changes in the terminal saccharide residue). More nuanced differences in a glycoprofile, such as structural isomerism, are more difficult to monitor by lectin arrays. As a first step in the construction of lectin arrays, the substrate typically needs to be modified with a linker molecule to mediate the protein-substrate interaction. This molecular adhesion layer is important because protein adsorption to bare, unmodified surfaces may result in their denaturation and loss of biological activity.14 A common approach is to form an amide linkage between a N-hydroxylsuccinimidyl (NHS) ester from a linker molecule to a ε-NH2 of the protein’s lysine groups.15 Other surface chemistries include the maleimide group for sulfur (cysteine) attachment,16 streptavidin/avidin coupling,17 and the relatively novel “click” chemistry approach using either con(7) Kawasaki, N.; Ohta, M.; Hyuga, S.; Hashimoto, O.; Hayakawa, T. Anal. Biochem. 1999, 269, 297–303. (8) Lim, A.; Reed-Bogan, A.; Harmon, B. J. Anal. Biochem. 2008, 375, 163– 172. (9) Dell, A.; Morris, H. R. Science 2001, 291, 2351–2356. (10) Kamoda, S.; Kakehi, K. Electrophoresis 2006, 27, 2495–2504. (11) Chen, S.; Zheng, T.; Shortreed, M. R.; Alexander, C.; Smith, L. M. Anal. Chem. 2007, 79, 5698–5702. (12) Kuno, A.; Uchiyama, N.; Koseki-Kuno, S.; Ebe, Y.; Takashima, S.; Yamada, M.; Hirabayashi, J. Nat. Methods 2005, 2, 851–856. (13) Hirabayashi, J. J. Biochem. 2008, 144, 139–147. (14) Mrksich, M. Chem. Soc. Rev. 2000, 29, 267–273. (15) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777–790. (16) Mallik, R.; Wa, C.; Hage, D. S. Anal. Chem. 2007, 79, 1411–1424. (17) Davies, J.; Roberts, C. J.; Dawkes, A. C.; Sefton, J.; Edwards, J. C.; Glasbey, T. O.; Haymes, A. G.; Davies, M. C.; Jackson, D. E.; Lomas, M.; Shakesheff, K. M.; Tendler, S. J. B.; Wilkins, M. J.; Williams, P. M. Langmuir 1994, 10, 2654–2661.

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jugated or bioengineered alkyne/azide functionalities.18-20 Polymers have also been used to control the adsorption of proteins on surfaces. For example, Corn et al. have demonstrated that poly(L-lysine) can be electrostatically21 or covalently22 bound to a carboxylic acid terminated self-assembled monolayer. The abundance of primary amine groups for conjugation reactions is one reason poly(L-lysine) is a suitable choice for certain protein adsorption experiments. However, the cationic properties of poly(Llysine)-modified substrates at neutral pH may limit adsorption of basic proteins due to repulsive electrostatic forces.21 The surface chemistries mentioned above for immobilizing proteins are often substrate specific, which can be a limitation because the choice of a particular substrate can constrain the analytical methods used to interrogate surface structure or binding events. In contrast, it was recently reported that polydopamine can adsorb to virtually any surface and serve as an adhesion layer to immobilize biological molecules and mercapto-functionalized self-assembled monolayers to the surface.23 In addition, the fabrication and application of polydopamine as an adhesion layer are straightforward and relatively quick. In the presence of dissolved O2 and under alkaline conditions, aqueous solutions of dopamine will spontaneously oxidize in about 10 min to polydopamine (pDA), a brown-colored melanin-like polymer.24 A suitable adhesion layer will form on a clean substrate after a less than 30 min immersion in the freshly prepared polydopamine solution. In contrast, most self-assembled monolayerbased adhesion layers require the clean substrate to be immersed in the adsorbate solution for at least several hours. This paper describes the use of a variety of polydopaminemodified substrates for the immobilization of lectins for glycoanalysis. Surface plasmon resonance spectroscopy (SPRS) and surface matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (surface-MALDI-TOF MS) are employed to demonstrate the reversible, specific interaction between the polydopamine-immobilized lectin concanavalin A (Con A) and the glycoprotein ribonuclease B (RNase B). In addition, the oligomannose specificity of Con A is reported with a discussion of its significance. MATERIALS AND METHODS Concanavalin A was purchased from Vector Laboratories25 and used without further purification. Dopamine hydrochloride (98%), ribonuclease A, ribonuclease B (>80%), indium foil (99.99%), potassium bromide, sodium chloride (99.9%), calcium chloride (18) Deiters, A.; Cropp, A.; Mukherji, M.; Chin, J. W.; Anderson, J. C.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125, 11782–11783. (19) Devaraj, N. K.; Collman, J. P. QSAR Comb. Sci. 2007, 26, 1253–1260. (20) Gauchet, C.; Labadie, G. R.; Poulter, C. D. J. Am. Chem. Soc. 2006, 128, 9274–9275. (21) Frey, B. L.; Jordan, C. E.; Kornguth, S.; Corn, R. M. Anal. Chem. 1995, 67, 4452–4457. (22) Frey, B. L.; Corn, R. M. Anal. Chem. 1996, 68, 3187–3193. (23) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Science 2007, 318, 426–430. (24) Herlinger, E.; Jameson, R. F.; Linert, W. J. Chem. Soc. Perkin Trans. 2 1995, 259–263. (25) The full description of the procedures used in this paper requires the identification of certain commercial products and their suppliers. The inclusion of such information should in no way be construed as indicating that such products or suppliers are endorsed by NIST or are recommended by NIST or that they are necessarily the best materials, instruments, software, or suppliers for the purposes described.

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dihydrate (98%), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, 99.5%), D-(+)-galactose (99%), methyl-R-D-mannopyranoside (99%), sodium carbonate, sodium bicarbonate, and manganese chloride tetrahydrate (99.99%) were purchased from SigmaAldrich and used as received. Thick gold films (∼150 nm) were prepared by vapor deposition on Cr-primed (2 nm) Si (100) wafers (Silicon Quest International). The base pressure in the vacuum chamber for deposition was typically ∼3 × 10-5 Pa (∼2 × 10-7 Torr). Thin gold films (∼55 nm) were prepared by magnetron-sputtering on Cr-primed (∼1 nm) BK7 glass (Corning). Iridium films (∼200 nm) were prepared by sputter deposition onto Ti-primed (2 nm) Si wafers. Dopamine hydrochloride was dissolved in a 10 mmol/L carbonate/bicarbonate buffer (pH ) 8.5) at a concentration of 1 mg/mL. After 10 min of incubation time, substrates were immersed in the solution for 30 min. The samples were retrieved, rinsed thoroughly with nanopure water (18 MΩ cm) to remove unbound polydopamine, and dried with nitrogen. Protein solutions were prepared to the desired concentrations by dissolving in HEPES buffered saline, HBS (pH ) 7.4, 150 mmol/L NaCl, 0.1 mol/L HEPES, 1 mmol/L Mn2+, 1 mmol/L Ca2+), and were used immediately. All protein solutions were prepared at a nominal concentration of 1.0 mg/mL unless otherwise specified. Reflection-absorption infrared spectroscopy (RAIRS) was performed on a Bio-Rad (FTS-7000) Fourier transform spectrometer equipped with a wire-grid polarizer (p-polarized), a variableangle reflectance accessory, and a liquid nitrogen-cooled mercury cadmium telluride (MCT) detector. Spectra were typically acquired at an incident angle of 80°, a resolution of 2 cm-1, and summed over at least 100 scans. Dry house compressed air was used to purge the sample compartment of ambient carbon dioxide and water vapor. Transmission IR samples were prepared by mixing 1 mg of the protein into 125 mg of dry KBr and pressing into a translucent pellet. All spectra have been corrected for baseline drift. Surface plasmon resonance spectroscopy (SPRS) experiments were performed on a home-built angle-scanned instrument. Unlike a traditional rotating prism assembly, this optical setup focuses and collects a wedge of light (angle width of ∼10°), allowing for a simultaneous collection of full SPR angle curves.26 White light from a 150 W halogen lamp (Thorlabs, OSL1-EC) was consecutively passed through a spatial filter, a 850 nm (10 nm fwhm) bandpass filter, and a rotatable linear polarizer (Newport). The polarized light was focused with a hemicylindrical lens and directed toward a mounted hemicylindrical BK7 glass prism/gold coated glass slide assembly. The gold-coated BK7 slides were optically coupled to the prism with index matching fluid (n ) 1.515) (Cargille). The reflected wedge of light was focused to a line with a horizontally aligned hemicylindrical lens and detected with a 3000 pixel linear CCD camera. (Thorlabs). A computercontrolled syringe pump (Cole-Parmer) was filled with buffer and used to push solution through Teflon tubing (dead volume ≈30 µL) into a Teflon flow cell fixed to the gold film/prism assembly. Sample solutions entered into the flow system via a FPLC assembly with a sample injection loop of 250 µL allowing for (26) Homola, J., Ed. Surface Plasmon Resonance Based Sensors; Springer: New York, 2006.

precise control of injection volumes and times. At the start of an experiment, a reflected s-polarized light angle scan was taken by manually rotating the linear polarizer 90°. During the experiment, real-time reflected p-polarized light scans were collected and divided by the s-polarized light scan. Only p-polarized light interacts with the surface plasmons while s-polarized light remains proportional to the incident light. Dividing p- by s-polarized light provides a normalized value for reflectivity and also normalizes for spatial inhomogeneity in incident light. Flow rates were typically between 5 and 25 µL/min, as described for each experiment. All instrument control, data collection, and data fitting were performed using custom and stock code written in MATLAB, MATLAB Instrument Control toolbox, and MATLAB Optimization toolbox (Mathworks, Natick, MA). The SPR reflectivity curves were analyzed by fitting the data to a four-layer Fresnel model27 using published optical constants28,29 for the prism, the gold layer, adsorbed protein, and aqueous solution that correspond to the experiments described below. The thickness of the adsorbed protein layer was determined using a protein layer refractive index of 1.45. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) data were collected on an Applied Biosystems instrument in the positive ion and linear modes using a 337 nm nitrogen laser for irradiating samples. The spectra are the result of at least 6000 laser pulses and have been smoothed. The matrix solution for all MALDI and surface-MALDI experiments was prepared by dissolving sinapinic acid (Fisher) in 0.1% TFA/50% acetonitrile at a concentration of 20 mg/mL. For MALDI experiments, 0.3 µL of matrix was added to 0.3 µL of sample, mixed on the MALDI target plate, and allowed to air-dry before analysis. For surface-MALDI experiments, the indium foils were modified with pDA and proteins as required and reversibly attached to the MALDI plate via double-sided tape (Scotch). Before analysis, 0.5 µL of the matrix solution was added to the modified indium foil and allowed to air-dry. RESULTS AND DISCUSSION Immobilization of Concanavalin A to PolydopamineModified Surfaces. In situ adsorption of concanavalin A to polydopamine-modified gold was followed by surface plasmon resonance spectroscopy (SPRS). Before introduction of concanavalin A (Con A), the thin Au film was coated with a polydopamine film by floating the gold-coated glass slide face down in an alkaline aqueous dopamine solution for 30 min, followed by rinsing with water and drying. Figure 1a shows the SPR curves of the bare gold substrate before and after the ex situ adsorption of polydopamine. As a result of polydopamine deposition, the surface plasmon resonance angle (i.e., angle of minimum reflectivity) increased and the resonance of the curve dampened (i.e., reflectivity at the surface plasmon resonance angle increased). Figure 1b shows the change in surface plasmon resonance angle (∆θsp) during a concanavalin A solution exposure to a polydopamine (pDA)-modified gold surface. HBS buffer was allowed to flow for several minutes to establish a (27) Yamamoto, M. Rev. Polarogr. 2002, 48, 209–237. (28) Palik, E. D., Ed. Handbook of Optical Constants of Solids; Academic Press, Inc.: Orlando, FL, 1985. (29) Palik, E. D., Ed. Handbook of Optical Constants of Solids II; Academic Press, Inc.: San Diego, CA, 1991.

Figure 1. (a) SPR curves of a bare Au substrate before and after a 30 min ex situ deposition of polydopamine. The curves were acquired in water. (b) SPR response as a result of exposure of a Con A solution to a pDA/Au substrate. The down arrow indicates the beginning of the Con A exposure. The up arrow indicates return to HBS buffer flow.

baseline measure of the SPR response before protein exposure. After 6 min, a solution of Con A was introduced at a flow rate of 5 µL/min. As a consequence of protein adsorption, the surface plasmon angle increased and reached a plateau about 20 min later, indicating the cessation of net protein adsorption. Buffer flow was resumed after about 30 min. The small decrease in the surface plasmon angle that was observed soon after the return to buffer may be due to the slight difference in refractive index between the protein in the buffer solution and the protein-free buffer solution. After several minutes of buffer flow, a new flat baseline was established, and a net increase of 0.8° in the surface plasmon angle was determined. A thickness of (12.3 ± 0.4) nm was calculated for the modeled protein layer. Performing the identical experiment without the pDA layer (i.e., exposure of Con A to a bare Au substrate) resulted in a calculated protein layer thickness of about 2.6 nm (data not shown). This latter thickness is inconsistent with any known physical dimensions of an intact Con A molecule and is assumed to result from denaturation of the lectin upon adsorption to the gold surface.14 Analytical Chemistry, Vol. 81, No. 13, July 1, 2009

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Table 1. IR Assignments for Major Features of Bound and Unbound Con A

Figure 2. (a) RAIRS spectrum of pDA/Ir after a 30 min exposure to 1 mg/mL Con A solution. (b) Difference spectrum obtained by subtracting the RAIRS spectrum of pDA/Ir from the spectrum in part a. (c) Transmission IR spectrum of 1% Con A in KBr.

The immobilization of Con A on a pDA-modified surface was further characterized by infrared spectroscopy as shown in Figure 2. Figure 2a is a representative RAIRS spectrum of Con A adsorbed to pDA-modified Ir. The features in this spectrum are characteristic of both the immobilized Con A and the adsorbed pDA. To deconvolute the data, the IR spectrum of pDA/In (data not shown) was subtracted from Figure 2a and the result is Figure 2b. For comparison purposes, Figure 2c is the KBr pellet transmission IR of Con A. The spectra in parts b and c of Figure 2 are qualitatively similar and both include the major vibrational features characteristic for proteins.30 Assignment of these features are 5416

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Con A/pDA IR peaks (cm-1)

Con A IR peaks (cm-1)

assignment

3311 1670 1543

3313 1654 1535

ν(NH) amide A ν(CdO) amide I ν(C-N) amide II

provided in Table 1. For the Con A/pDA films, the amide I and II vibrational features are shifted 8-16 cm-1 higher relative to the transmission spectrum of Con A. We do not believe that this small shift indicates denaturation of Con A adsorbed on the pDA surface. Instead we attribute this shift to an optical effect that is associated with the spectra being obtained from a thin film at a glancing angle of incidence. Allara et al.31 have noted that vibrational features obtained in the reflection mode from thin film samples tend to be shifted to higher frequencies relative to those obtained by transmission. Therefore, the IR data suggest that Con A adsorbs to the pDA surface largely intact and does not displace the bound pDA. The nature of the interaction between the bound Con A and pDA is not clear at this time. In contrast to poly(L-lysine), another amine containing polymeric film that has been used extensively for attaching proteins to surfaces, the isoelectric point of the protein was observed to have no effect on its adsorption efficiency to a polydopamine-coated surface (data not shown). Furthermore, there was no spectroscopic evidence that the integrity of the lectin film on the pDA was ever significantly affected by water rinses, incubation in protein solutions, or moderate aging. The observed stability could be attributed to Schiff base coupling between the quinone/catechol groups of pDA and the ε-NH2 of the lysine residues of Con A.23 We have plans to further investigate this hypothesis. Biological Activity of Con A Immobilized to pDA-Modified Substrates. Ribonuclease B (RNase B) was chosen as a model glycoprotein to demonstrate that the biological activity of Con A was retained upon adsorption to polydopamine-modified substrates. RNase B is a ∼15-15.5 kDa protein which possesses a single N-linked oligosaccharide with a variable composition of (Man)x-(GlcNAc)2-Asn where Man is R-D-mannose, GlcNAc is N-acetylglucosamine, and x ) 5-9.32,33 Con A selectively binds R-mannose-terminated glycans, and the favorable interaction between RNase B and Con A has been reported in solution34 and in the adsorbed state.12 Surface plasmon resonance spectroscopy was used to investigate the reversible, specific interactions between RNase B and pDA-immobilized Con A. As before, after ex situ adsorption of a pDA film on the gold slide, Con A was introduced in situ and allowed to reach saturation coverage on the surface. To render the surface resistant to nonspecific adsorption events, consecutive solutions of ribonuclease A (RNase A) were introduced to the Con (30) Revell, D. J.; Knight, J. R.; Blyth, D. J.; Haines, A. H.; Russell, D. A. Langmuir 1998, 14, 4517–4524. (31) Allara, D. L.; Baca, A.; Pryde, C. A. Macromolecules 1978, 11, 1215–1220. (32) Fu, D.; Chen, L.; O’Neill, R. A. Carbohydr. Res. 1994, 261, 173–186. (33) Tarelli, E.; Byers, H. L.; Wilson, M.; Roberts, G.; Homer, K. A.; Beighton, D. Anal. Biochem. 2000, 282, 165–172. (34) Sparbier, K.; Koch, S.; Kessler, I.; Wenzel, T.; Kostrzewa, M. J. Biomol. Techniques 2005, 16, 407–413.

Figure 3. SPR sensorgram illustrating the effect of inhibitory and noninhibitory sugars on the dissociation of RNase B from pDAimmobilized Con A. Abbreviations: Me-R-D-Man ) methyl-R-D-mannopyranoside, Gal ) galactose. See text for details.

A/pDA/Au system until no net adsorption of RNase A was observed (i.e., return to pre-RNase A injection baseline soon after buffer flow resumed). RNase A was chosen to block nonspecific adsorption sites because it is the nonglycosylated form of RNase B.12 Figure 3 displays SPRS data showing the effects of an inhibitory sugar and a noninhibitory sugar on the dissociation rate of RNase B from pDA-immobilized Con A. All injections of protein or sugar solutions were at a flow rate of 20 µL/min for 800 s in duration, followed by a 200 or 400 s flow of buffer. After a 10 min flow of buffer to establish the baseline response, the substrate was exposed to a solution of RNase B followed by a 50 mmol/L methyl-R-D-mannoside solution, a well-known35,36 inhibitory sugar for Con A-glycan interactions. The substrate was then exposed again to a solution of RNase B followed by consecutive injections of 50 mmol/L galactose and 50 mmol/L methyl-R-D-mannoside solutions. Finally, another solution of RNase B was introduced followed by buffer for the remainder of the experiment. The increase in the SPRS response after introduction of the sugar solutions was caused by the different refractive indices of the buffer and sugar solutions. Following the first RNase B solution injection, introduction of the methyl-R-D-mannoside solution was able to effectively promote the dissociation of RNase B-Con A as evidenced by the return to baseline of the SPRS data. In contrast, in a similar experiment where galactose, a noninhibitory sugar of Con A-glycan interactions, is introduced, a slow dissociation process qualitatively similar to that for pure buffer is observed. An additional injection of methyl-R-D-mannopyranoside was required to completely regenerate the lectin for additional RNase B binding experiments in a reasonable amount of time. In addition to the above-mentioned regeneration ability of the inhibitory sugars, these experiments demonstrate the specificity of the RNase B-Con A interactions.37 If the RNase B were nonspecifically bound, the type of sugar would be expected to have little effect on its interaction with the pDA-modified substrate. Since the sugar solutions behave as expected, we conclude that RNase B is specifically bound to the sugar-binding sites of pDAimmobilized Con A. (35) Goldstein, I. J.; Hollerman, C. E.; Smith, E. E. Biochemistry 1965, 4, 876– 883. (36) Gallego, R. G.; Haseley, S. R.; van Miegem, V. F. L.; Vliegenthart, J. F. G.; Kamerling, J. P. Glycobiology 2004, 14, 373–386. (37) Pilobello, K.; Krishnamoorthy, L.; Slawek, D.; Mahal, L. K. ChemBioChem 2005, 6, 985–989.

Selectivity of Con A/pDA toward RNase B Glycoforms. The selectivity of Con A/pDA toward the various RNase B glycoforms was investigated with surface-MALDI-TOF MS. SurfaceMALDI-TOF MS is a MALDI-TOF MS variant in which the compositions and identities of molecules first adsorbed either specifically or nonspecifically to a surface are analyzed.38,39 Conventional MALDI-TOF MS, in contrast, is typically used to analyze molecules originating from solution. Surface-MALDI-TOF MS has been applied in the characterization of the chemical reactions of self-assembled monolayers,40 the study of protein fouling on contact lenses,38 and the capture and identification of bacteria.41,42 This technique has also been referred to in the literature as surface-enhanced laser desorption/ionization (SELDI)43,44 and probe affinity MS.45 When combined with a selectively binding surface, surface-MALDI-TOF MS may be used in conjunction with conventional MALDI-TOF MS or other techniques46 to simplify the determination of the composition of a complex mixture. An example of this application, presented in this section of the paper, is the use of surface-MALDI-TOF MS and lectin-modified substrates for the glycoanalysis of RNase B. Commercially available RNase B is a mixture of glycoforms that differ in the number and branching pattern of the mannose sugars on the nonreducing end of the saccharide moiety. Table 2 shows the structures, composition, and reported affinities of Con A for the nine glycoforms of RNase B.32,47 A representative MALDI-TOF mass spectrum of RNase B is shown in Figure 4. Peaks at m/z values of 14 898, 15 062, 15 226, 15 385, and 15 544 are identified as the pseudo parent ions ([M + H]+) of the Man5, Man6, Man7, Man8, and Man9 glycoforms and their structural isomers, respectively.33 Minor peaks attributable to impurities of unknown composition are also evident. Adjacent oligomannose peaks are separated by an average of (161 ± 3) units, in good agreement with glycosidic addition of a mannose moiety. Because peaks are not baseline resolved in this experiment, even relative quantification is difficult; however, the glycoprofile pattern of Figure 5 agrees well with reported values and published MALDI-TOF MS spectra.7,32,33 Figure 5a is a surface-MALDI TOF mass spectrum of Con A immobilized on pDA-modified indium foil after a 30 min exposure to RNase B. Although the same five glycoform peaks previously observed in Figure 4 are present, the glycoprofile has changed. In comparison to the MALDI data, the intensity of glycoforms with higher mannose content (i.e., Man7-9) has increased relative to that of Man5, the glycoform with the lowest mannose content. The relatively higher intensity of the higher mannose content glycoforms can be attributed to the different relative affinities of (38) Griesser, H. J.; Kingshott, P.; McArthur, S. L.; McLean, K. M.; Kinsel, G. R.; Timmons, R. B. Biomaterials 2004, 25, 4861–4875. (39) McLean, K. M.; McArthur, S. L.; Chatelier, R. C.; Kingshott, P.; Griesser, H. J. Colloids Surf., B 2000, 17, 23–35. (40) Su, J.; Mrksich, M. Langmuir 2003, 19, 4867–4870. (41) Bundy, J.; Fenselau, C. Anal. Chem. 1999, 71, 1460–1463. (42) Bundy, J. L.; Fenselau, C. Anal. Chem. 2001, 73, 751–757. (43) Hutchens, T. W.; Yip, T.-T. Rapid Commun. Mass Spectrom. 1993, 7, 576– 580. (44) Tang, N.; Tornatore, P.; Weinberger, S. R. Mass Spectrom. Rev. 2004, 23, 34–44. (45) Brockman, A. H.; Orlando, R. Anal. Chem. 1995, 67, 4581–4585. (46) Nedelkov, D.; Tubbs, K. A.; Nelson, R. W. Electrophoresis 2006, 27, 3671– 3675. (47) Mega, T.; Oku, H.; Hase, S. J. Biochem. 1992, 111, 396–400.

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Table 2. Structures, Dissociation Constants, and Composition of RNase B Glycoforms

a

M ) D-mannose, G ) N-acetylglucosamine. b From ref 47. c From ref 32.

the RNase B glycoforms for Con A (cf. Table 2). The affinity of a glycan for Con A is currently thought to depend on several structural features of the glycan. The most important structural motif is that the glycan must possess the nonreducing end trimannose group: ManR1-6(ManR1-3)Man which is enclosed by the dashed box in the Table 2 structure of Man5.11 We note that each of the RNase B glycoforms possesses this motif. 5418

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However, the mannose substitution pattern of this trimannose group also affects RNase B-Con A affinity. It has been reported that affinity increases significantly with addition of an R(1-2) mannose residue on the R(1-6) arm.47 It is worth noting that the four RNase B glycoforms with this structural moiety (i.e., Man7a, Man8a, Man8b, Man9) have measured dissociation constants less than 30 nmol/L, and the five glycoforms lacking

Figure 4. MALDI-TOF mass spectrum of RNase B. Asterisks denote impurities.

Figure 5. (a) Surface-MALDI TOF mass spectrum of Con A/pDA/In after a 30 min exposure to a solution of RNase B. (b) Surface-MALDI TOF mass spectrum of Con A/pDA/In after sequential 30 min exposures to a solution of RNase B and a 1 mmol/L methyl-R-Dmannopyranoside solution.

this structural motif have a dissociation constant of at least 250 nmol/L.48 The addition of an R (1-2) mannose residue on the R (1-3) arm also increases the affinity but to a lesser degree. This structure-affinity relation accounts for the KD differences of the Man7a and Man8a glycoforms, for example.47,48 As a consequence of these structure-affinity relations, the glycoprofile of RNase B as reported by immobilized Con A will depend on the relative concentrations of the RNase B glycoforms in the sample and their relative affinities for Con A. To further investigate this phenomenon, the following experiments were performed. As before, Con A was immobilized to a pDA-modified indium foil and was exposed to a solution of RNase B for 30 min. The sample was then incubated in a 1 mmol/L solution of methyl R-D-mannopyranoside in buffer for 30 min. The

Figure 6. Surface-MALDI TOF mass spectra of Con A/pDA/In after (a) 0.5, (b) 1, (c) 5, and (d) 30 min exposure to a solution of RNase B. For ease in comparison, all spectra are offset and have been normalized to the peak height of the Man5 peak.

surface-MALDI-TOF mass spectrum displayed in Figure 5b was obtained after rinsing with water and drying. In comparison to the spectrum in Figure 5a, two noteworthy features are apparent. First, the overall absolute amount of RNase B associated with Con A decreased in agreement with the inhibitory effects of this sugar solution as demonstrated by SPRS (cf. Figure 3). Although normally difficult, a quantitative comparison of MALDI MS data can be made in this case because the intensities of the lectin peaks (data not shown) are similar in both experiments. Second, the amounts of Man7, Man8, and Man9 have increased relative to the amount of Man5. The latter result is consistent with the stronger binding of certain Man7, Man8, and Man9 glycoforms to Con A relative to Man5. Therefore, these glycoforms are expected to dissociate less in competitive binding with methyl R-D-mannopyranoside than the Man5 and Man6 glycoforms which have lower affinities for the lectin. Work by Gallego and coworkers supports these results.36 After PNGase F release and 2-aminobenzamide fluorophore-labeling, RNase B oligosaccharides were introduced to an SPR substrate modified with Con A. Effluents from successively more concentrated methyl R-Dmannopyranoside solution rinsings of the bound RNase B were collected and injected into a HPLC for glycan separation and identification. They concluded that Man7, Man8, and Man9 glycans were more strongly bound to Con A than the Man5 and Man6 glycans because higher inhibitory sugar concentrations were required to effect their dissociation. Exposure of Con A-bound RNase B to an inhibitory sugar solution of low concentration further exacerbated the discrepancy between the solution glycoprofile and the glycoprofile as bound to the lectin. The effects of RNase B exposure time to immobilized Con A were also examined. The results of the surface-MALDI-TOF MS experiment are displayed in Figure 6. The relative amounts of the Man7, Man8, and Man9 RNase B glycoforms were observed to increase with exposure time. Of the higher oligomannose glycoforms, the Man8 peak intensity increased most significantly over the course of the experiment. After 30 s of exposure time, only Man5 and Man6 glycoform peaks were detectable. However, beyond 5 min of exposure time, the Man8 glycoform peak became evident. This result is consistent with the relatively higher affinities (48) Bhattacharyya, L.; Brewer, C. F. Eur. J. Biochem. 1989, 178, 721–726.

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of some of the Man7, Man8, and Man9 glycoforms for Con A. Because the concentrations of Man7, Man8, and Man9 glycoforms are significantly lower than those of Man5 and Man6, short exposure times will favor Man5 and Man6 binding to Con A. At longer exposure times, the higher affinities of Man7, Man8, and Man9 are expected to result in enrichment of these glycoforms at the Con A modified surface. This result is consistent with recent results reported by Sparbier and co-workers where Con A was immobilized on magnetic particles.49 Using MALDI-TOF MS, they showed that although the lectin-functionalized magnetic particles were able to selectively bind RNase B, the glycoform distribution of RNase B after enrichment was significantly different from its actual glycoprofile. In agreement with the results presented here, the glycoprofile of the enriched RNase B sample possessed a higher contribution from the more highly mannose substituted glycoforms. The glycoprofiling discrepancy was solved in Sparbier and co-workers’ investigation by also using 3-aminophenyl boronic acid-functionalized magnetic beads. Boronic acid covalently binds molecules possessing a cis-diol motif (e.g., mannose) without regard to branching or mannose composition heterogeneity. As expected, the glycoprofile of RNase B after enrichment by boronic acid-functionalized magnetic beads accurately reflected its true glycoprofile. CONCLUSIONS First, polydopamine has been shown to be a suitable adhesion layer for the immobilization of lectins. Concanavalin A-RNase B was chosen as the model lectin-glycoprotein system to demonstrate that the biological activity of the lectin is retained upon adsorption to pDA-modified substrates. Since polydopamine adsorbs to practically all substrates, this adhesion layer may be a (49) Sparbier, K.; Wenzel, T.; Kostrzewa, M. J. Chromatogr., B 2006, 840, 29– 36.

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more prudent choice over substrate-specific adhesion layers for protein immobilization assays. With polydopamine as the adhesion layer, the analyst could consider using the substrate that offers the greatest analytical figures of merit (e.g., selectivity, sensitivity) for the ligand-binding assay without regard for the compatibility of the substrate and available adhesion layers. Second, surfaceMALDI has been shown to be a technique suitable for glycoanalysis. The technique is especially advantageous over SPRS if the identity of bound glycan is desirable or needed. Although not demonstrated here, surface-MALDI is ideal for sequentially reading arrays of lectins for evidence of glycoprotein interactions. Third, the glycoprofile of RNase B bound to Con A has been shown to be significantly different than the true glycoprofile in solution. As a practical consequence, lectin arrays composed of Con A will preferentially bind certain mannose-presenting glycoforms over others. It can reasonably be expected that this observation might hold true for other lectin-glycan systems. In this scenario, it is possible that trace amounts of a high-affinity glycan might mask or give false-positives for the lower affinity glycan of interest. Consequently, care must be taken when drawing any quantitative conclusions from lectin array experiments regarding the original sample glycoprofile ACKNOWLEDGMENT T.M. thanks Christopher (Chip) Montgomery for the preparation of the iridium substrates and Wei-Li Liao and Larik Turko for the use of the MALDI-TOF MS instrument. T.M. also acknowledges a NRC-RAP/NIST postdoctoral fellowship for funding.

Received for review April 3, 2009. Accepted May 21, 2009. AC900715D