Time-of-Flight Secondary Ion Mass Spectrometry Analysis of

Apr 19, 2002 - Time-of-Flight Secondary Ion Mass Spectrometry Analysis of Conformational Changes in Adsorbed Protein Films. Nan Xia,Collin J. ..... Th...
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Time-of-Flight Secondary Ion Mass Spectrometry Analysis of Conformational Changes in Adsorbed Protein Films Nan Xia,† Collin J. May,‡ Sally L. McArthur,§ and David G. Castner*,†,§ National ESCA and Surface Analysis Center for Biomedical Problems, Departments of Chemical Engineering and Bioengineering, University of Washington, Box 351750, Seattle, Washington 98195-1750; and Department of Biomedical Engineering, Yale University, New Haven, Connecticut 06520 Received January 7, 2002 The characterization of adsorbed protein films with ultrahigh vacuum (UHV) surface analysis techniques requires dehydration of the samples, which can cause significant alterations in protein conformation. Trehalose coating was used in this study to inhibit these conformational changes from occurring when preparing samples for analysis in UHV. Surface plasmon resonance (SPR) analysis showed that air-dried films of trehalose-stabilized antiferritin and anti-IgM both retained a significant proportion of their hydrated antigen binding activity. In contrast, air-drying without trehalose protection resulted in the adsorbed protein films losing most of their antigen binding activity. Structural differences between trehalosestabilized and unstabilized protein films were then analyzed with static time-of-flight secondary ion mass spectrometry (ToF-SIMS). By application of principle component analysis (PCA) to the static ToF-SIMS spectra, the biological activity difference observed in SPR was correlated to changes in protein conformation. Trehalose-protected proteins retained a greater degree of their original conformation than the unprotected proteins. This suggests coating adsorbed protein films with trehalose prior to air-drying and introduction into UHV allows ToF-SIMS to analyze adsorbed proteins in a state that is more representative of their actual structure in an aqueous environment.

1. Introduction When a synthetic material comes into contact with a biological system, one of the first events to occur is the adsorption of proteins at the solid-liquid interface. Protein adsorption is involved in a wide range of phenomena and applications. The fouling of contact lenses,1,2 the initiation of blood coagulation and platelet consumption by bloodcontacting devices,3,4 the microorganism fouling of marine equipment,5 and the blockage of filtration membranes in bioseparation processes6 are examples of unfavorable aspects of protein adsorption. However, the extensive uses of a variety of proteins in food emulsion, in stabilization,7,8 and in the fabrication of biosensors9-13 are positive * Corresponding author (use the Department of Chemical Engineering, University of Washington address). Telephone: (206)5438094. Fax: (206)543-3778. E-mail: [email protected]. † National ESCA and Surface Analysis Center for Biomedical Problems, Department of Chemical Engineering, University of Washington. ‡ Yale University. § National ESCA and Surface Analysis Center for Biomedical Problems, Department of Bioengineering, University of Washington. (1) Begley, C. G.; Waggoner, P. J. J. Am. Optom. Assoc. 1991, 62, 208. (2) Ruben, M.; Guillon, M.; Ed.; Contact Lens Practice; Chapman and Hall Medical: London, 1994; 1083-1098. (3) Albelda, S. M.; Buck, C. A. FASEB J. 1990, 4, 2868. (4) Gluszko, P.; Rucinski, B. J.; Musia, J.; Wenger, R. K.; Schmaier, A. H.; Colman, R. W.; Edmunds, L. H. Jr.; Niewiarowski, S. Am. J. Physiol. 1987, 252, H615. (5) Costerton, J. W.; Cheng, K.-J.; Geesey, G. G.; Ladd, T. I.; Nickel, J. C.; Dasgupta, M.; Marrie, T. J. Annu. Rev. Microbiol. 1987, 41, 435. (6) Birk, H. W.; Kistner, A.; Wizemann, V.; Schutterle, G. Artif. Organs 1995, 19, 411. (7) Bos, M. A.; van Vliet, T. Adv. Colloid Interface Sci. 2001, 91, 437. (8) Rodriguez Patino, J. M.; Navarro Garcia, J. M.; Rodriguez Nino, M. R. Colloids Surf. B 2001, 21, 207. (9) Nagaoka, S.; Mikami, M.; Shimizu, Y. Biomaterials 1990, 11, 414. (10) Sasso, S. V.; Pierce, R. J.; Walla, R.; Yacynych, A. M. Anal. Chem. 1990, 62, 1111.

applications. Thus, it is clear that characterization of the adsorbed protein layer, including its composition, conformation, and orientation, could greatly aid biomedical device development and many other protein-based technologies. Various methods have been developed to characterize protein films, including radiolabeling,14,15 ellipsometry,16,17 Fourier transform infrared spectroscopy (FTIR),17-21 X-ray photoelectron spectroscopy (XPS),22-24 and atomic force microscopy (AFM).25-29 Among these techniques is the recent application of static time-of-flight secondary ion mass spectrometry (ToF-SIMS). This surface analytical (11) Brinkman, E.; van der Does, L.; Bantjes, A. Biomaterials 1991, 12, 63. (12) Coughlan, M. P.; Alcock, S. J. Biosens. Bioelect. 1991, 6, 87. (13) Wang, J.; Wu, L.-H.; Martinez, S.; Sanchez, J. Anal. Chem. 1991, 63, 398. (14) Broughton, A.; Strong, J. E. Clin. Chem. 1976, 22, 726. (15) Pestka, S.; Lin, L.; Wu, W.; Izotova, L. Protein Expr. Purif. 1999, 17, 203. (16) Elwing, H. Biomaterials 1998, 19, 397. (17) Tengvall, P.; Lundstrom, I.; Liedberg, B. Biomaterials 1998, 19, 407. (18) Haris, P. I.; Chapman, D. Biopolymers 1995, 37, 251. (19) Jackson, M.; Mantsch, H. H. Crit. Rev. Biochem. Mol. Biol. 1995, 30, 95. (20) Chittur, K. K. Biomaterials 1998, 19, 357. (21) Vigano, C.; Manciu, L.; Buyse, F.; Goormaghtigh, E.; Ruysschaert, J. M. Biopolymers 2000, 55, 373. (22) Blomberg, E.; Claesson, P. M.; Froberg, J. C. Biomaterials 1998, 19, 371. (23) Weser, U.; Jung, G.; Ottnad, M.; Bohnenkamp, W. FEBS Lett. 1972, 25, 346. (24) Paynter, R. W.; Ratner, B. D.; Horbett, T. A.; Thomas, H. R. J. Colloid Interface Sci. 1984, 101, 233. (25) Balashev, K.; Jensen, T. R.; Kjaer, K.; Bjornholm, T. Biochimie 2001, 83, 387. (26) Mu¨ller, D. J.; Fotiadis, D.; Engel, A. FEBS Lett. 1998, 430, 105. (27) Hansma, H. G.; Kim, K. J.; Laney, D. E. J. Struct. Biol. 1997, 119, 99. (28) Galli, C.; Collaud Coen, M.; Hauert, R.; Katanaev, V. L.; Wymann, M. P.; Gro¨ning, P.; Schlapbach, L. Surf. Sci. 2001, 474, L180. (29) Hansma, H. G.; Pietrasanta, L. I.; Auerbach, I. D.; Sorenson, C.; Golan, R.; Holden, P. A. J. Biomater. Sci. Polym. Ed. 2000, 11, 675.

10.1021/la020022u CCC: $22.00 © 2002 American Chemical Society Published on Web 04/19/2002

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technique uses a focused primary ion beam to bombard the sample, resulting in atoms, clusters, and molecules being sputtered from the sample surface. A small fraction of the sputtered particles are positively and negatively charged secondary ions. These secondary ions provide information about the surface elemental and chemical composition of the material being analyzed. Some recent ToF-SIMS studies have shown the potential of this technique for analyzing adsorbed protein films due to its combination of chemical specificity and surface sensitivity.30-34 In particular, the sampling depth of static ToF-SIMS is approximately 10 Å, which is smaller than the dimensions of most proteins. Therefore, static ToFSIMS should be sensitive to the conformation and orientation of adsorbed proteins. This has been discussed and addressed in two previous publications.31,33 However, two major limitations were realized in both studies. First, the data analysis was limited to the selection of a few peaks in the mass spectra. Second, the samples were airdried prior to analysis, which probably resulted in the protein conformation changing from that in aqueous solution. Thus, the ToF-SIMS results from these studies are difficult to relate directly to the original, hydrated conformation of the adsorbed proteins. Previous investigators have identified the characteristic mass peaks generated by the fragmentation of amino acids.30,32,35-37 Since these peaks can be present in the ToF-SIMS spectra of any protein film, it is necessary to analyze the relative intensities of these peaks for characterizing adsorbed protein films. This requires multivariate analysis to reduce the complexity of the ToFSIMS spectra from adsorbed protein films. For example, by combining ToF-SIMS with principal component analysis (PCA), Wagner et al. were able to classify proteins by their type and relative surface concentration.32 If the conformation of adsorbed proteins can be retained when the samples are air-dried and introduced into ultrahigh vacuum (UHV), then the combination of ToF-SIMS with PCA should be a powerful method for identifying conformation differences in adsorbed protein films. This study involves the development of a method for preserving the structure of the adsorbed proteins for static ToF-SIMS analysis in UHV. A cold stage technique is one possibility, and it has been applied to SIMS with some success.38 However, special hardware is required and the sample preparation can be time-consuming. This has limited the widespread use of cold stage methods. Thus, it is desirable to develop simpler preservation techniques for preparing adsorbed protein samples for ToF-SIMS analysis. Trehalose (a disaccharide composed of two [1,1]linked R,R units of glucopyranose) is known for its ability to inhibit protein unfolding at high temperatures and during water removal.39-43 It has been proposed that (30) Lhoest, J.-B.; Wagner, M. S.; Tidwell, C. D.; Castner, D. G. J. Biomed. Mater. Res. 2001, 57, 432. (31) Lhoest, J.-B.; Detrait, E.; van den Bosch de Aguilar; Bertrand, P. J. Biomed. Mater. Res. 1998, 41, 95. (32) Wagner, M. S.; Castner, D. G. Langmuir 2001, 17, 4649. (33) Tidwell, C. D.; Castner, D. G.; Golledge, S. L.; Ratner, B. D.; Meyer, K.; Hagenhogg, B.; Benninghoven, A. Surf. Interface Anal. 2001, 31, 724. (34) Ferrari, S.; Ratner, B. D. Surf. Interface Anal. 2000, 29, 837. (35) Mantus, D. S.; Ratner, B. D.; Carlson, B. A.; Moulder, J. F. Anal. Chem. 1993, 65, 1431. (36) Bartiaux, S. Undergraduate Thesis, Unite de Chimie des Interface, Faculte des Sciences Agronomiques, Universite Catholique de Louvain, Louvain-la-Nerve, Belgium, 1995. (37) Bouchonnet, S.; Denhez, J.-P.; Hoppilliard, Y.; Mauriac, C. Anal. Chem. 1992, 64, 743. (38) Derue, C.; Gibouin, D.; Lefebvre, F.; Rasser, B.; Robin, A.; LeSceller, L.; Verdus, M. C.; Demarty, M.; Thellier, M.; Ripoll, C. J. Trace Microprobe Tech. 1999, 17, 451.

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trehalose stabilizes the protein conformation by forming hydrogen bonds to polar residues in the protein.44,45 We have used trehalose to preserve the conformation of adsorbed proteins for ToF-SIMS analysis. The ToF-SIMS spectra of trehalose-preserved and unpreserved protein films were compared with each other. Spectral differences recognized using PCA were correlated to conformational changes of the adsorbed proteins that occurred during drying. Additionally, the biological activity of the adsorbed protein films when dried with and without trehalose protection was examined with surface plasmon resonance (SPR). SPR biosensors have the unique ability to detect the binding of biological molecules to a surface in real time without the use of labels. In this study, SPR was used to compare the antigen binding to adsorbed antibody films without removal from solution, after drying without trehalose protection, and after drying with trehalose protection. 2. Materials and Methods 2.1. Materials. Trehalose (R-D-glucopyranosyl R-D-glucopyranoside, T0167), anti-horse spleen ferritin (AIA, F6136), antihuman IgM antibody (AIA, I0759), horse spleen ferritin (F4503), and human IgM (I8260) were purchased from Sigma Chemical (St. Louis, MO) and were used as received. All water used was purified by treatment in a reverse osmosis unit followed by a Millipore unit (18 mΩ resistivity). 2.2. Sample Preparation for Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Silicon wafers were cut into 1 cm × 1 cm pieces, and cleaned with piranha solution (7:3 (v:v) H2SO4 and H2O2). Caution! Piranha solution reacts violently with organic solvents and should be handled with extreme care. Cleaned wafers were coated by electron beam evaporation at pressures below 1 × 10-6 Torr, first with approximately 100 Å of Ti as an adhesive underlayer and then with approximately 1000 Å of gold. Proteins (antiferritin and anti-IgM) were adsorbed onto clean gold-coated silicon substrates from PBS buffer solutions (0.15 M phosphate-buffered saline, pH 7.4) for 2 h at room temperature. The samples were then rinsed twice in stirred buffer solution and twice in stirred deionized water. Samples without trehalose protection were then dried overnight. Samples to be trehalose protected were immersed in a trehalose solution for 30 min (without air exposure), then spun dry (4000 rpm, 20 s) and set aside for the same time period as the unprotected samples. Prior to ToF-SIMS analysis, the unprotected samples were submerged in the trehalose solution for 30 min and spun dry (4000 rpm, 20 s). This was done to maintain similar levels of trehalose on the two sets of samples. 2.3. ToF-SIMS and Data Analysis. A Model 7200 Physical Electronics instrument (PHI, Eden Prairie, MN) was used for static ToF-SIMS data acquisition. The instrument has an 8 keV Cs+ ion source, a reflectron time-of-flight mass analyzer, chevrontype multichannel plates (MCP), and a time-to-digital converter (TDC). Positive secondary ions mass spectra were acquired over a mass range from m/z ) 0 to 350. The area of analysis for each spectrum was 100 µm × 100 µm. The total ion dose used to acquire each spectrum was less than 2 × 1012 ions/cm2. The mass resolution (m/∆m) of the secondary ion peaks was typically between 5000 and 8000. At least three replicates were prepared with each sample, with three spectra acquired on each replicate. The ion beam was moved to a new spot on the sample for each (39) Crowe, L. M.; Crowe, J. H.; Chapman, D. Science 1984, 233, 701. (40) Carpenter, J. F.; Crowe, L. M.; Crowe, J. H. Biochim. Biophys. Acta 1987, 923, 109. (41) Carpenter, J. F.; Crowe, J. H. Cryobiology 1988, 25, 459. (42) Leslie, S. B.; Israeli, E.; Lighthart, B.; Crowe, J. H.; Crowe, L. M. Appl. Environ. Microbiol. 1995, 61, 3592. (43) Crowe, J. H.; Leslie, S. B.; Crowe, L. M. Cryobiology 1994, 31, 355. (44) Allison, S. D.; Chang, B.; Randolph, T. W.; Carpenter, J. F. Arch. Biochem. Biophys. 1999, 365, 289. (45) Shi, H.; Tsai, W.-B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature (London) 1999, 398, 593.

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spectrum. The mass scale for the spectra was calibrated using the peak sets CH3+, C2H3+, C3H5+, and C7H7+. The differences between the expected and observed masses after calibration were less than 10 ppm. 2.4. Principal Component Analysis (PCA). A complete discussion of PCA for ToF-SIMS analysis of protein films has been written by Wagner et al.32 The peak set for PCA analysis is the same as the one used in that reference, except that the cysteine fragmentation peaks at m/z ) 45 (CHS+) and 59 (C2H3S+) were used instead of the peak at m/z ) 76.46 To make a quantitative comparison between spectra, the peaks were normalized to the total intensity of the PCA peak set to eliminate any systematic differences in total secondary ion yield (absolute intensity) between spectra. The data set was also mean-centered to ensure the differences in samples were due to variations around the means and not the variance of the means.47 Principle component analysis of the normalized ToF-SIMS data was performed using the PLS Toolbox v. 2.0 (Eigenvector Research, Manson, WA) for MATLAB (the MathWorks, Inc., Natick, MA). 2.5. Sample Preparation for Surface Plasmon Resonance (SPR). For SPR measurements, clean glass microscope slides were first coated with 20 Å of Ti and then 500 Å of Au using the same evaporation system as described above. Proteins (antiIgM, antiferritin) were adsorbed onto the gold surfaces either in-situ (i.e., monitored with the SPR biosensor) or by the same method described in section 2.3. 2.6. Surface Plasmon Resonance. The home-built SPR liquid sensing system used in this study has been described and characterized in more detail elsewhere.48 Basically, it uses the planar prism (Kretschmann) configuration. A white light source was passed through the prism and projected onto the backside of a gold-coated substrate at a fixed angle (78° from normal). The gold sensor surface was placed under a Plexiglas flow cell with an 80 µL flow channel. Interactions at or above the gold sensor surface are observed by monitoring the wavelength shift of the SPR minimum. During SPR measurements, the gold substrates were first equilibrated with degassed buffer solution (0.15 M phosphatebuffered saline (PBS) at pH 7.4). Then after a rapid exchange of buffer above the sensor surface with the aqueous protein solutions (0.5 mL at 10 mL/min), the flow was stopped and the protein samples remained in contact with the surface for the desired incubation time. To end the protein adsorption/binding process, PBS buffer was injected at a rate of 10 mL/min into the flow cell to thoroughly rinse the surface.

3. Results and Discussion 3.1. Surface Plasmon Resonance (SPR). Figure 1a presents a typical SPR sensorgram of the bare gold substrate when exposed to sequential injections of antiferritin and ferritin. With the injection of 50 µg/mL antiferritin, a wavelength shift of 14.0 ( 0.9 nm was observed. No additional protein binding was observed from a second antiferritin injection (same concentration), indicating that saturated protein coverage had been achieved. The immunoreactivity of the adsorbed antiferritin layer was then measured. With the injection of 100 µg/mL ferritin, the second increase observed in the SPR curve (10.7 ( 1.1 nm) was due to the specific antibody/ antigen interaction. Further evidence for the specificity of this reaction was the small SPR response of the same antiferritin layer toward the injection of an unrelated protein, IgM (100 µg/mL) (e1.5 nm, results not shown). The addition of a BSA blocking step by exposing the antiferritin layer to a BSA solution (500 µg/mL) had little (46) Dornfeld, K. T.; Wagner, M. S.; Castner, D. G. J. Undergrad. Res. Eng. 2001, in press. (47) Vandeginste, B. G. M.; Massart, D. L.; Buydens, L. M. C.; deJong, S.; Lewi, P. J.; Smeyers-Verbeke, J. Handbook of Chemometrics and Qualimetrics: Part B; Elsevier Science Publishers, B.V.: Amsterdam, 1998. (48) Jung, L. S.; Campbell, C. T.; Chinowsky, T. M.; Mar, M. N.; Yee, S. S. Langmuir 1998, 14, 5636.

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Figure 1. (a) SPR sensorgram for adsorption from 50 µg/mL solution of antiferritin onto a gold-coated glass slide followed by the adsorption from a 100 µg/mL solution of ferritin. (b) SPR sensorgram for adsorption from a 50 µg/mL solution of antiferritin onto a gold-coated glass slide, then dried for 2 h, followed by the adsorption from a 100 µg/mL solution of ferritin. The sequence for fluid injection in this sensorgram is as follows: (a) 50 µg/mL of antiferritin, (b) buffer, (c) DI water, (d) N2 purge, (e) DI water, (f) buffer, (g) 100 µg/mL ferritin, and (h) buffer.

effect on the SPR response toward subsequent ferritin injection. Also, the SPR adsorption profile was not altered by a second ferritin injection, excluding the possibility that the reduction in the slope of the SPR curve was caused by the depletion of the supply of ferritin to the surface. The effect of dehydration on the adsorbed antiferritin layer was examined next. As shown by Figure 1b, the sensor surface was adsorbed with antiferritin, rinsed sequentially with buffer and deionized water, dried for 2 h (the sensor surfaces were blown dry for 30 min under a high speed nitrogen stream, and then stored in ambient condition for the remaining time). The large changes observed from points c to f in the sensorgram were caused by switching the fluid over the surface (i.e., buffer to water and water to nitrogen), which have different refractive indices. The drying process was terminated by delivering water then buffer over the dried sensor surface. When ferritin was injected subsequently, the observed SPR response was much lower than that in Figure 1a (less than 20%). No significant difference in SPR wavelength was observed between the water-exposed (or bufferexposed) surfaces of adsorbed antiferritin before and after drying, which excludes the possibility that there was antibody desorption from sensor surface during drying. Therefore, the loss of antigen binding capacity of the adsorbed antiferritin layer was probably caused by protein denaturation during the drying process. The effect of drying was also examined ex situ with a similar loss in the immunoreactivity of adsorbed antiferritin observed. To prevent protein denaturation upon dehydration, trehalose was employed as a stabilizing agent. As shown

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Figure 2. SPR response upon introduction of a 100 µg/mL ferritin solution to antiferritin protein films on gold (adsorbed from a 50 µg/mL solution of antiferritin onto a gold-coated glass slide). The three curves represent antiferritin that was in aqueous, dried (0.1 wt % trehalose protected, 2 h), and dried (unprotected, 2 h) conditions, respectively.

in Figure 2, the adsorbed antiferritin samples dried with trehalose protection (0.1 wt %) did exhibit much higher antigen binding capacity (SPR wavelength shift ∼7.9 nm) than those samples dried without trehalose protection (SPR wavelength shift ∼1.6 nm). Since the protected samples have a trehalose adlayer, it was necessary to examine the affinity between trehalose and the antigen (ferritin). To achieve this goal, the gold-coated substrates were adsorbed with a different antibody, antiIgM, and dried with trehalose protection (0.1 wt %). These samples displayed very low affinity to ferritin (SPR wavelength shift of 0.9 ( 0.3 nm). Moreover, the trehalose-coated antibody samples were always rinsed with a large amount of water and buffer prior to antigen injection into the SPR experiment. This experiment also further shows the specificity of the antigen binding, since significantly more ferritin was bound to trehalose-protected antiferritin compared to trehalose-protected anti-IgM (7.9 nm vs 0.9 nm). Therefore, the SPR response shown in Figure 2 is primarily due to specific antibody/antigen binding. The SPR results demonstrated that the loss in protein functionality upon dehydration could be inhibited by trehalose treatment. Since it is generally accepted that function is related to the structure of a protein, the immunoreactivity differences observed with SPR suggested a difference in conformation between the trehaloseprotected and unprotected adsorbed antibodies. It was expected the protein structure in the trehalose-protected samples would be more analogous to that in aqueous solution, while dehydration without trehalose protection would result in more denaturation of the protein. Our next goal was to characterize the structure difference between the two sets of samples using ToF-SIMS. To facilitate the ToF-SIMS analysis, the protection afforded by different trehalose solution concentrations was evaluated using SPR. In Figure 3, the immunoreactivity of the trehalose-coated, dried antibody samples initially increased rapidly with trehalose concentration up to 0.5 wt % trehalose. Only a small increase in immunoreactivity was observed when the trehalose solution concentration was increased from 0.5 to 5.0 wt %. 3.2. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Because trehalose coating could mask the static ToF-SIMS signals from the adsorbed protein film, it was necessary to determine how much trehalose could be deposited and still allow sufficient protein

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Figure 3. Ferritin binding capacity of antiferritin protein films adsorbed onto gold after drying for 2 h with trehalose protection, as a function of trehalose solution concentration.

fragments to be detected by static ToF-SIMS. Initially, bare gold surfaces coated with trehalose from different solution concentrations were analyzed with ToF-SIMS. For trehalose solution concentrations of 0.5 wt % or higher, the Au+ peak (m/z ) 197) was barely detectable. This suggested that in protein protection experiments, concentrations no higher than 0.5% should be utilized. Since peaks from the Au substrate were virtually eliminated by the trehalose coating from a 5 wt % solution, the ToF-SIMS spectrum from this sample was used as the reference spectrum for pure trehalose. By comparison of the major peaks in the trehalose spectrum to the peak set used for the PCA analysis of the protein films,32 it was observed that peaks at m/z ) 69 (C4H5O+) and m/z ) 71 (C3H3O2+) were present in both trehalose and protein ToF-SIMS spectra. Because the ToF-SIMS spectra of protein films are highly complex and do not have characteristic peaks for each protein, it was essential to evaluate the ToF-SIMS spectra using a multivariate analysis method such as PCA.47 Figure 4 shows the PCA results from the positive ion spectra of unprotected vs protected antiferritin films (0.1 wt % trehalose). The first PC captures approximately 54% of the variance in the data set and clearly separates the two sets of samples in the PCA scores plot (Figure 4a). The cause for this sample separation was determined by examining the PCA loadings plot in Figure 4b. The major variance between protected and unprotected samples was identified as the ratio of hydrophilic to hydrophobic amino acid intensities. The trehalose-protected antiferritin samples had positive PC1 scores, while the unprotected antiferritin samples had negative PC1 scores. In the PC1 loadings plot, the mass peaks with high positive loadings were from hydrophilic (Arg, m/z ) 73, 100, 101; Lys, m/z ) 84; His, m/z ) 110) or polar (Thr, m/z ) 74; Trp, m/z ) 130, 159, 170) amino acids. The amino acids that have side chains with polar OH or NH groups can form hydrogen bonds. Conversely, the protein peaks with the highest negative loadings were from hydrophobic (Val, m/z ) 72; Ile/Leu, m/z ) 86; Phe, m/z ) 120) amino acids with hydrocarbon side chains. Therefore, the unprotected antiferritin samples with negative PC1 scores had higher surface concentrations of hydrophobic amino acids than the trehalose-protected samples. In the native conformation of most proteins, the hydrophobic amino acids are typically located preferentially in the interior of the protein molecule, away from the aqueous biological environment. Conversely, hydrophilic amino acids are typically located preferentially at the exterior of the protein molecule, to interact with the aqueous biological environment. Upon water

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Figure 4. (a) PC1 scores plot from PCA of the positive ion spectra from 0.1 wt % trehalose-protected and unprotected antiferritin films adsorbed from 50 µg/mL solutions. (b) PC1 loadings plot for the scores plot shown in part a. Hydrophilic amino acid fragments are labeled with an [i], hydrophobic amino acid fragments are labeled with an [o], and polar amino acid fragments are labeled with a [p]. The peak at m/z ) 70 labeled with an asterisk (/) contains contributions from several different amino acids.

removal, proteins can unfold or denature to expose hydrophobic domains, which is what was observed for the unprotected antiferritin samples. However, the ToFSIMS results show that the surfaces of trehalose-protected antiferritin samples are enriched with hydrophilic and polar amino acids, analogous to their state in aqueous solution. The extreme surface-sensitive and chemical specificity of the static ToF-SIMS technique allowed the difference in surface amino acid concentrations between the two sets of samples to be easily identified. Although the general trends in Figure 4 were consistent with changes in protein conformation induced by drying, several issues require additional discussion. In Figure 4b, the peak at m/z ) 70 had a high positive loading. Previous studies have found several amino acids, including Pro, Arg, Leu, and Lys, have fragments with similar intensities at this peak, so it is not possible to uniquely attribute this peak to just one amino acid.32,46 Also, unlike the other peaks discussed above, the m/z ) 70 peak was also found to represent the variation among replicates of the same sample type (discussed below). In this study, the proteins were randomly adsorbed onto the gold surface, so the protein films were expected to be heterogeneous. Therefore, it is possible that the m/z ) 70 peak represented the heterogeneity of the surface concentration, conformation, and orientation of the adsorbed proteins. No mass peaks from acidic amino acids had high positive loadings in Figure 4b. Also, peaks from the hydrophilic amino acid Asn (m/z ) 70 and 87) had negative loadings. These observations may be due to the surface charge of

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antiferritin under our experimental conditions or the structure of the protein. To examine these in further detail requires knowledge of the antiferritin structure (amino acid sequence, secondary and tertiary structure, etc.), which is not currently available. Finally, the two trehalose peaks (m/z ) 69 and 71) which overlap with peaks from protein layers had negative loadings in Figure 4b. However, these trehalose peaks were not the exclusive or primary factors for the sample separation observed in Figure 4a. In general, the loading patterns for the amino acid peaks in Figure 4b were readily recognizable and could be correlated to the conformation differences between the trehalose-protected and unprotected protein films. The protective strength of the trehalose coating increased with the concentration of the coating solution (Figure 3). Since the thickness, and therefore the masking effect of trehalose, also increases with solution concentration, an optimum concentration of the trehalose coating solution for protein analysis with static ToF-SIMS needed to be determined. The objective was to minimize the masking effect while maintaining adequate protective strength. The results for trehalose solution concentrations of 0.01 and 0.05 wt % are shown in Figure 5. Separation between the protected and unprotected samples was obtained with PC2. The loading patterns for PC2 (Figure 5c,f) were similar to the PC1 loadings in Figure 4b. The protected samples had high loadings for peaks from basic and polar amino acids, while unprotected samples have high loadings for peaks from hydrophobic amino acids. The samples were not separated by PC1. Thus, PC1 represented sample heterogeneity, both between different samples types and within a given sample type. The peak at m/z ) 70 dominated the PC1 loadings plots (Figure 5b,e), indicating that this peak corresponded to sample heterogeneity. From the results in Figure 5, it was apparent that sample heterogeneity was more important than differences between sample types when 0.01 or 0.05 wt % trehalose solutions were used for coating adsorbed protein films. Thus, these solutions did not provide adequate protection for adsorbed proteins. Comparison of the PCA results for the 0.01 and 0.05 wt % trehalosecoated samples showed an increase in the protection strength with increasing concentration of the trehalose solution. In Figure 5e, loadings of the peaks from hydrophobic amino acids became prominent on the negative side of PC1. Correspondingly, the unprotected samples had predominately negative PC1 scores (9 out of 12 samples), while protected samples had positive PC1 scores (9 out of 14 samples). These tendencies were not observed for the samples treated with 0.01% trehalose in Figure 5a, and the hydrophobic amino acid peaks did not have significant loadings in Figure 5b. The ToF-SIMS observations for the 0.01 and 0.05 wt % trehalose solutions were consistent with the SPR results shown in Figure 3. Figure 6 shows the PCA results for the unprotected vs 0.5% trehalose-protected antiferritin samples. Similar to Figure 4, the first PC clearly separated the two sets of samples (Figure 6a). However, in the PC1 loadings plot (Figure 6b), the trehalose peaks (m/z ) 69 and 71) had the highest absolute loadings. The loadings of the two trehalose peaks corresponded to the protected samples, while the loadings of hydrophobic amino acids peaks corresponded to the unprotected samples. This indicates that the protected samples had thicker trehalose coatings that significantly attenuated the protein signals, while for the unprotected samples the surface enrichment of hydrophobic amino acids could still be detected. These differ-

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Figure 5. (a) PC2 scores plot from PCA of the positive ion spectra from 0.01 wt % trehalose-protected and unprotected antiferritin adsorbed from 50 µg/mL solutions. (b) PC1 loadings plot from PCA of the 0.01 wt % trehalose samples. (c) PC2 loadings plot from PCA of the 0.01 wt % trehalose samples. (d) PC2 scores plot from PCA of the positive ion spectra from 0.05 wt % trehalose-protected and unprotected antiferritin adsorbed from 50 µg/mL solutions. (e) PC1 loadings plot from PCA of the 0.05 wt % trehalose samples. (f) PC2 loadings plot from PCA of the 0.05 wt % trehalose samples. Hydrophilic amino acid fragments are labeled with an [i], hydrophobic amino acid fragments are labeled with an [o], and polar amino acid fragments are labeled with a [p]. The peak at m/z ) 70 labeled with an asterisk (/) contains contributions from several different amino acids.

ences were detected even though the trehalose coating conditions (incubation time, trehalose concentration, spindrying speed, and time) were the same for both the protected and unprotected samples. This indicated that when the trehalose coating was applied (either prior to drying for protected samples or after drying for unprotected samples) affected the amount of trehalose deposited onto the protein films. These results indicated the 0.5 wt % trehalose solution was too concentrated for static ToFSIMS analysis, since the trehalose coating became too thick and prevented detection of the protein peaks. This was consistent with trehalose experiments on gold discussed earlier. Previous studies have shown that ToF-SIMS can classify proteins by their type,30,32 while this work demonstrated that ToF-SIMS could also classify proteins by differences in their conformation. Thus, it is important to determine which produces larger varia-

tions in the ToF-SIMS spectra, different protein types of different protein conformations. Figure 7 shows a comparative study of adsorbed bovine serum albumin (BSA) films vs a series of adsorbed antiferritin samples, which varied in protein conformation. In Figure 7, PC1 readily separates the BSA samples from antiferritin samples, while the various antiferritin samples are not differentiated from each other. This indicated the type of protein was more important than protein conformation for classifying adsorbed protein samples with PCA. However, if only one type of protein was examined, PCA can differentiate samples by protein conformation. Furthermore, the results in Figure 7 included antiferritin samples that were coated with different concentrations of trehalose. This confirmed that the trehalose coating method from solutions containing e0.1 wt % trehalose did not interfere with the ToF-SIMS analysis of adsorbed protein films.

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Figure 8. SPR response upon introduction of a 50 µg/mL IgM solution to a film of anti-IgM adsorbed onto a gold surface. The three curves represent adsorbed anti-IgM films that were in aqueous, dried (trehalose sugar protected), and dried (unprotected) conditions, respectively.

Figure 6. (a) PC1 scores plot from PCA of the positive ion spectra from 0.5 wt % trehalose-protected and unprotected antiferritin adsorbed from 50 µg/mL solutions. (b) PC1 loadings plot for the scores plot shown in Figure 6a. Hydrophobic amino acid fragments are labeled with an [o]. The peak at m/z ) 70 labeled with an asterisk (/) contains contributions from several different amino acids.

Figure 7. PC1 scores plot from PCA of the positive ion spectra of adsorbed bovine serum albumin (BSA) and adsorbed antiferritin (a-f) samples. Both trehalose-protected (p) and unprotected (u) protein films are included.

3.3. Anti-IgM. To determine whether the results described above were unique to just antiferritin, SPR and static ToF-SIMS analysis of another antibody, anti-IgM, were also done. Figure 8 shows a comparison of the SPR wavelength shift upon injection of 100 µg/mL solution of IgM to aqueous, protected and unprotected films of adsorbed anti-IgM. These results clearly show that biological functionality was lost on drying and that trehalose protection did allow the protein to retain a significant portion of its functionality. Figure 9a shows the scores plot for unprotected vs 0.1% trehalose-protected anti-IgM adsorbed onto a gold surface. This plot shows PC1 clearly separates the two sample types. The corresponding loadings plot in Figure 9b show

Figure 9. (a) PC1 scores plot from PCA of the positive ion spectra from 0.1 wt % trehalose-protected and unprotected antiIgM adsorbed from 50 µg/mL solutions. (b) PC1 loadings plot for the scores plot shown in Figure 9a. Hydrophilic amino acid fragments are labeled with an [i], hydrophobic amino acid fragments are labeled with an [o], and polar amino acid fragments are labeled with a [p]. The peak at m/z ) 70 labeled with an asterisk (/) contains contributions from several different amino acids.

the protected samples had relatively high positive loadings for hydrophilic amino acids peaks (Arg at m/z ) 73 and His at m/z ) 81, 82, 110), while the unprotected samples had high negative loadings for hydrophobic amino acids (Val at m/z ) 72 and Leu/Ile at m/z ) 86). The presence of a highly negative loaded peak from Lys at m/z ) 84 was unexpected since Lys has a hydrophilic side chain. Further experiments were done to investigate the reason for this negatively loaded Lys peak. The ToF-SIMS spectra of anti-IgM were compared with spectra of antiferritin. Parts a-c of Figure 10 show the PCA comparisons between the

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drying), respectively. Upon examination, it was clear that Lys loads predominantly in the anti-IgM spectra, indicating that relative to antiferritin there is an abundance of Lys in anti-IgM. It was also apparent that the Lys loading is highest in the two unprotected samples. This suggests that Lys may be present in hydrophobic regions of antiIgM, which would explain why the Lys peak loaded with the hydrophobic amino acids in Figure 9b. Further support for this explanation requires knowledge of the amino acid sequence of anti-IgM, which is currently not available. 4. Conclusions SPR and static ToF-SIMS were used to determine how drying affected the conformation and functionality of adsorbed protein films. The important results were as follows: 1. Drying causes a significant loss of protein functionality. A decrease in the antigen-binding affinity of dried antibody films was detected by SPR. 2. Trehalose reduced the loss of protein functionality in dried adsorbed protein films. SPR showed that the trehalose-protected antibodies retained a significant portion of their antigen-binding affinity. 3. Using the combination of PCA and static ToF-SIMS analysis, structural differences were observed between trehalose-protected dried protein films and unprotected dried protein films. Trehalose-protected proteins retain a greater proportion of their original conformation. 4. Trehalose protection can be used for static ToF-SIMS analysis of adsorbed protein films to obtain structural information that is more relevant to the structure of the proteins in aqueous conditions. 5. Static ToF-SIMS has the capability to distinguish conformational differences in adsorbed protein films. Many properties of the proteins (e.g., amino acid sequence) characterized in this study are currently unavailable. Thus, it is desirable to extend this study to proteins with well-defined and characterized properties, so the relationship between the static ToF-SIMS results and protein structure can be more completely investigated. In future studies, it would also be useful to evaluate proteins that are specifically bound to a surface in well-defined orientations, rather than randomly adsorbed. Both the SPR and ToF-SIMS responses should be significantly higher if proteins are attached in a uniform orientation.

Figure 10. PC1 loadings plots from PCA of the positive ion spectra of adsorbed antiferritin vs adsorbed anti-IgM samples containing no trehalose (a), 0.1% trehalose (coated prior to drying) (b), and 0.1% trehalose (coated after drying) (c).

proteins of samples containing no trehalose, 0.1% trehalose (coated after drying), and 0.1% trehalose (coated before

Acknowledgment. This research was supported by NIH Grant RR-01296 to the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO) from the National Center for Research Resources and NSF Grant EEC-9529161 to the University of Washington Engineered Biomaterials center (UWEB). Jennifer Shumaker-Parry and Dr. Charles T. Campbell are thanked for providing access to the SPR system used in this study. Dr. Stephen Golledge is thanked for his technical expertise with the static ToF-SIMS experiments. Matthew S. Wagner is thanked for his assistance with the principal component analysis. LA020022U