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Langmuir 2007, 23, 50-56
Comparison of Native Extracellular Matrix with Adsorbed Protein Films Using Secondary Ion Mass Spectrometry† Heather E. Canavan,*,‡,| Daniel J. Graham,‡,| Xuanhong Cheng,§,| Buddy D. Ratner,‡,§,|,# and David G. Castner‡,§,|,# National ESCA and Surface Analysis Center for Biomedical Problems, UniVersity of Washington Engineered Biomaterials, and Departments of Bioengineering and Chemical Engineering, Box 355061 UniVersity of Washington, Seattle, Washington 98195 ReceiVed August 7, 2006. In Final Form: NoVember 7, 2006 In the past decade, the temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) has come to be recognized as a convenient method for the nondestructive harvest of confluent cell layers. Recently, we have utilized this nondestructive cell harvest method as a means to ascertain the nature of the extracellular matrix (ECM) secreted from cells. In this work, we compare the ECM obtained after cell liftoff to individual ECM proteins adsorbed directly onto RF-plasma-deposited pNIPAM (ppNIPAM). Using X-ray photoelectron spectroscopy, we find that the composition of ppNIPAM post-cell liftoff surfaces is consistent with those of the ppNIPAM post-protein adsorption surface, both of which differ from control surfaces. Using principal component analysis of positive-ion time-of-flight secondary ion mass spectrometry (ToF-SIMS) data, we show that the major ECM proteins examined can effectively be identified from their amino acid compositions. By comparing the positive-ion ToF-SIMS data from each of the ppNIPAM post-protein adsorption surfaces to that of ppNIPAM post-cell liftoff, we find that ppNIPAM post-cell liftoff surfaces are distinctly separate from fibronectin (FN). This result is consistent with our previous observation using immunoassay that FN is clearly associated with the cell sheet after low-temperature liftoff from ppNIPAM.
Introduction In the past decade, the temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) has come to be recognized as a convenient method for the nondestructive harvest of confluent cell layers.1-4 Instead of requiring the use of harsh cell removal methods such as enzymatic digestion or physical scraping, it is possible to rapidly recover intact cell sheets from pNIPAM culture surfaces using only a modest temperature drop as the means of detachment.5 In addition, recent work by Okano and co-workers demonstrated that cell sheets detached from pNIPAM surfaces appear to retain their function upon transfer to another growth surface.2 This has allowed for the fabrication of functional, three-dimensional tissue, and has led to a new technology named “cell sheet engineering”.3,6-8 The ability of cell layers harvested from pNIPAM surfaces to re-adhere and grow was credited to the concurrent detachment † Part of the Stimuli-Responsive Materials: Polymers, Colloids, and Multicomponent Systems special issue. * Corresponding author. Current address: Department of Chemical and Nuclear Engineering, MSC 01 1120, University of New Mexico, Albuquerque, New Mexico 87131-0001. E-mail:
[email protected]. Tel: 505-277-8026. Fax: 505-277-0594. ‡ National ESCA and Surface Analysis Center for Biomedical Problems. § University of Washington Engineered Biomaterials. | Department of Bioengineering, University of Washington. # Department of Chemical Engineering, University of Washington.
(1) Kikuchi, A.; Okuhara, M.; Karikusa, F.; Sakurai, Y.; Okano, T. J. Biomater. Sci., Polym. Ed. 1998, 9, 1331-1348. (2) Nandkumar, M. A.; Yamato, M.; Kushida, A.; Konno, C.; Hirose, M.; Kikuchi, A.; Okano, T. Biomaterials 2002, 23, 1121-1130. (3) Shiroyanagi, Y.; Yamato, M.; Yamazaki, Y.; Toma, H.; Okano, T. Tissue Eng. 2003, 9, 1005-1012. (4) von Recum, H. A.; Kim, S. W.; Kikuchi, A.; Okuhara, M.; Sakurai, Y.; Okano, T. J. Biomed. Mater. Res. 1998, 40, 631-639. (5) Canavan, H. E.; Cheng, X.; Graham, D. J.; Ratner, B. D.; Castner, D. G. J. Biomed. Mater. Res. 2005, 75A, 1-13. (6) Shimizu, T.; Yamato, M.; Kikuchi, A.; Okano, T. Biomaterials 2003, 24, 2309-2316. (7) Harimoto, M.; Yamato, M.; Kikuchi, A.; Okano, T. Macromol. Symp. 2003, 195, 231-235. (8) Yamato, M. Eur. Cells Mater. 2001, 6, 26-27.
of extracellular matrix (ECM) proteins such as fibronectin (FN),9,10 which have been demonstrated to promote cell adhesion, migration, and differentiation via their interactions with cellsurface receptors and other ECM molecules.11-14 Since then, we have shown that, although low-temperature liftoff of a monolayer of bovine aortic endothelial cells (BAECs) from plasmapolymerized NIPAM surfaces (ppNIPAM) is accompanied by the majority of the components of the ECM, some protein is left at the surface after liftoff.15 Furthermore, we demonstrated that using low-temperature liftoff to remove cell layers is less damaging to the underlying ECM than other techniques.5 This provides unprecedented access to the ECM underlying the cell sheet, perhaps allowing the identities of the individual ECM proteins deposited on a substrate to be identified. To date, it has been difficult or impossible to detect irreversibly adsorbed proteins at surfaces (such as ECM on a biomaterial) by traditional methods such as matrix-assisted laser desorption ionization (MALDI).16 However, we previously demonstrated that using unique amino acid fragmentation patterns in time-of-flight secondary ion mass spectrometry (ToF-SIMS) positive-ion spectra, it is possible to identify proteins adsorbed from single protein solutions.17-21 (9) Kushida, A.; Yamato, M.; Konno, C.; Kikuchi, A.; Sakurai, Y.; Okano, T. J. Biomed. Mater. Res. 1999, 45, 355-362. (10) Yamato, M.; Konno, C.; Kushida, A.; Hirose, M.; Utsumi, M.; Kikuchi, A.; Okano, T. Biomaterials 2000, 21, 981-986. (11) Akiyama, S. K.; Yamada, K. M. In Extracellular Matrix: A Practical Approach, 1st ed.; Haralson, M. A., Hassel, J. R., Eds.; Oxford University Press: New York, 1995; pp 175-185. (12) Hynes, R. In Guidebook to the Extracellular Matrix, Anchor, and Adhesion Proteins, 2nd ed.; Kreis, T., Vale, R., Eds.; Sambrook & Tooze at Oxford University Press: Oxford, UK, 1999; pp 422-425. (13) Hynes, R. O. Cell 1992, 69, 11-25. (14) Olsen, B. R.; Ninomiya, Y. In Guidebook to the Extracellular Matrix, Anchor, and Adhesion Proteins, 2nd ed.; Kreis, T., Vale, R., Eds.; Sambrook & Tooze at Oxford University Press: Oxford, UK, 1999; pp 395-398. (15) Canavan, H. E.; Cheng, X.; Ratner, B. D.; Castner, D. G. Langmuir 2005, 21, 1949-1955. (16) Griesser, H. J.; Kingshott, P.; McArthur, S. L.; McLean, K. M.; Kinsel, G. R.; Timmons, R. B. Biomaterials 2004, 25, 4861-4875.
10.1021/la062330o CCC: $37.00 © 2007 American Chemical Society Published on Web 12/27/2006
Comparison of NatiVe ECM with Protein Films
In this work, we compare the ECM obtained post-cell liftoff from ppNIPAM to that of proteins adsorbed directly onto ppNIPAM from single protein solutions. Using X-ray photoelectron spectroscopy (XPS), we compare the composition of ppNIPAM post-cell liftoff surfaces with those of ppNIPAM postprotein adsorption. Next, we compare the positive-ion ToF-SIMS data from each of the ppNIPAM post-protein adsorption surfaces to that of ppNIPAM post-cell liftoff. We find that, although the elemental compositions of the ECM proteins we studied are identical, they can effectively be identified from their amino acid compositions using principal component analysis (PCA). The resulting data from this comparison is then used to build a model ECM into which ppNIPAM post-cell liftoff surfaces are projected. We find that ppNIPAM post-cell liftoff surfaces share significant similarities with bovine serum albumin (BSA) and laminin (LN), and are distinctly separate from FN. This result is consistent with our previous observation using immunoassay that FN is clearly associated with the cell sheet after lowtemperature liftoff from ppNIPAM.9,15 Materials and Methods Materials. Collagen type I (Coll I) from rat tail was a gift from Dr. Shaoyi Jiang (University of Washington, Seattle, Washington). BSA from bovine serum, LN from human placenta, and FN from bovine plasma were obtained from Sigma (St. Louis, MO), and were used without further purification. The cell culture media were purchased from Gibco Invitrogen Corporation (Carlsbad, CA) and filtered though 0.2 µm filters before use. BAECs were a generous gift from Dr. Cecilia Giachelli (University of Washington, Seattle, Washington). TCPS 48-well plates were from Falcon (BD Biosciences, Franklin Lakes, NJ). N-isopropyl acrylamide (NIPAM, 97% +) monomer was purchased from Aldrich (Sigma-Aldrich, St. Louis, MO) and was used as received. Methods. To minimize sample variance, both cell culture and protein adsorption experiments were carried out in 48-well plates. Furthermore, samples for both analytical techniques (XPS and ToFSIMS) were obtained from the same plate. ppNIPAM Deposition. pNIPAM has been immobilized as a cell culture surface using many techniques.22-26 In this work, we use the vapor-phase deposition of ppNIPAM on tissue culture polystyrene (TCPS) 48-well plates following a procedure previously described in a paper by Pan, et al.22 We have found that this technique affords a one-step, solvent-free, vapor-phase method for the deposition of a sterile film compatible with cell culture.27 Cell Culture and Detachment. The cell culture and detachment were performed using previously described protocols.5,15 Briefly, BAECs were cultured in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 4.5 g/L of glucose, 10% fetal bovine serum, 0.1 mM of MEM non-essential amino acids, 1 mM of MEM sodium pyruvate, 100 U/mL of penicillin, and 100 µg/mL of (17) Wagner, M. S.; Horbett, T. A.; Castner, D. G. Langmuir 2003, 19, 17081715. (18) Wagner, M. S.; Castner, D. G. Langmuir 2001, 17, 4649-4660. (19) Wagner, M. S.; Horbett, T. A.; Castner, D. G. Biomaterials 2003, 24, 1897-1908. (20) Wagner, M. S.; Tyler, B. J.; Castner, D. G. Anal. Chem. 2002, 18241835. (21) Tidwell, C. D.; Castner, D. G.; Golledge, S. L.; Ratner, B. D.; Meyer, K.; Hagenhoff, B.; Benninghoven, A. Surf. Interface Anal. 2001, 31, 724-733. (22) Pan, Y. V.; Wesley, R. A.; Luginbuhl, R.; Denton, D. D.; Ratner, B. D. Biomacromolecules 2001, 2, 32-36. (23) Ista, L. K.; Mendez, S.; Perez-Luna, V. H.; Lopez, G. P. Langmuir 2001, 17, 2552-2555. (24) Zhang, G. Z. Macromolecules 2004, 37, 6553-6557. (25) Bohanon, T.; Elender, G.; Knoll, W.; Koberle, P.; Lee, J. S.; Offenhausser, A.; Ringsdorf, H.; Sackmann, E.; Simon, J.; Tovar, G.; Winnik, F. M. J. Biomater. Sci., Polym. Ed. 1996, 8, 19-39. (26) Zhuang, Y. F.; Yang, H.; Wang, G. W.; Zhu, Z. Q.; Song, W. Q.; Zha, H. D. J. Appl. Polym. Sci. 2003, 88, 724-729. (27) Cheng, X. H.; Canavan, H. E.; Stein, M. J.; Hull, J. R.; Kweskin, S. J.; Wagner, M. S.; Somorjai, G. A.; Castner, D. G.; Ratner, B. D. Langmuir 2005, 21, 7833-7841.
Langmuir, Vol. 23, No. 1, 2007 51 streptomycin. BAEC cells used in the experiments were between passage 7 and 15. BAECs were plated onto ppNIPAM-treated 48well plates at the cell density of 2.5 × 104 cells/well. They were then incubated at 37 °C in a humidified atmosphere with 5% CO2, and cultured until confluence. To perform cell liftoff, the culture media was removed and replaced with serum-free DMEM, and the cells were incubated at room temperature for 2 h. Protein Adsorption. Single protein solutions containing each of the ECM proteins were made at 2× concentration [where Coll, FN, and LN 1× ) 0.1 mg/mL; BSA 1× ) 1.0 mg/mL]. Next, 250 µL of phosphate-buffered saline (PBS) was pipetted into each well of a 48-well dish. The 48-well plate was incubated for 1 h at 37 °C to allow for equilibration of the polymer, as the adsorption of protein on ppNIPAM-treated surfaces differs greatly between room temperature and 37 °C.28 The 48-well plate was then removed from incubation, and 250 µL of the 2× protein solution was pipetted into each well, making the final concentration in each well 1×. The 48-well plate was then returned to the incubator for 1 h at 37 °C to allow for protein adsorption. Following adsorption, the 48-well plate was removed from incubation, and the protein solutions were pipetted off from each well via dilution-displacement with fresh PBS. Sample Preparation. To obtain the samples used for analysis in the XPS and ToF-SIMS ultrahigh-vacuum chambers, each well in the 48-well plate was washed with ultrapure water (18 MΩ resistivity) three times, and soaked with ultrapure water for at least 24 h to reduce the remaining free ions from the buffer. Next, the well bottoms were removed from the 48-well plates using a punch and hammer. The surfaces were then packed in inert containers backfilled with N2, sealed, and stored until analysis. XPS Analysis. XPS data were acquired on a Surface Science S-Probe instrument. This system is equipped with a monochromatized aluminum KR X-ray source, an electron flood gun for charge neutralization, and a hemispherical electron energy analyzer. All survey and detail scans for compositional analyses were acquired at a pass energy of 150 eV, and all high-resolution scans were acquired at a pass energy of 50 eV. Compositional analyses (0-1100 eV) and high-resolution scans of the C 1s region were carried out on all samples. Binding energies for high-resolution spectra were referenced to the C 1s (C-C/C-H) peak at 285.0 eV to account for binding energy shifts inherent to insulator samples. At least four replicates of each sample were analyzed, with three survey spectra and one high-resolution carbon spectrum acquired on each replicate. Data treatment was performed on Service Physics ESCAVB data reduction software. Core-level spectra were peak-fit using the minimum number of peaks possible to obtain random residuals. A 100% Gaussian line shape was used to fit the peaks, and a Shirley function was used to model the background. ToF-SIMS 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, chevron type multichannel plates, and a time-to-digital converter. Positive secondary ions mass spectra were acquired over a mass range of m/z ) 0-450. Negative-ion ToF-SIMS spectra were not considered in this study due to their lower information content and lack of unique peaks for different amino acids.18 The area of analysis for each spectrum was 100 µm × 100 µm, and 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 in the positive spectra was typically between 4000 and 6000. The ion beam was moved to a different spot on the sample for each spectrum. Positive spectra were calibrated using the CH3+, C2H3+, C3H5+, and C7H7+ peaks before further analysis. At least four replicates were prepared for each sample type, with five spectra acquired on each replicate. PCA. Although a detailed description of PCA is not warranted here, the interested reader is referred to more complete discussions of PCA by Jackson or Wold.29,30 Previous work by our group (28) Cheng, X.; Canavan, H. E.; Castner, D. G.; Ratner, B. D. Biointerphases 2006, 1, 61-72. (29) Jackson, J. E. J. Qual. Technol. 1980, 12, 201-213.
52 Langmuir, Vol. 23, No. 1, 2007
CanaVan et al.
Figure 1. Confluent cell layers cultured on ppNIPAM have been observed to spontaneously detach as an intact sheet when the temperature is dropped below the LCST of the polymer. identified unique amino acid fragmentation patterns in the ToFSIMS positive-ion spectra capable of identifying proteins present at a surface.18,20 Those fragments were used to construct a limited peak set to compare the positive ToF-SIMS spectra from each sample type (ppNIPAM-coated TCPS, ppNIPAM-coated TCPS post-cell liftoff, and ppNIPAM-coated TCPS post-protein adsorption). ToFSIMS spectra containing a sodium ion peak of >1% of the total intensity of the selected protein peaks were discarded due to the matrix effects of the sodium ion on the SIMS fragmentation process.18 Prior to multivariate analysis, all spectra were mean-centered. Selected peaks were then normalized to the intensity of the sum of selected peaks to account for fluctuations in secondary ion yield between different spectra. Next, PCA was used to capture the linear combination of peaks that described the majority of variation in the dataset (the principal components, or PCs). From this input, an output of both a “scores” and a “loadings” plot were obtained. In order to determine which of the adsorbed proteins were most similar to the ppNIPAM surface after cell liftoff, a PCA model was constructed using the data from the pure proteins adsorbed onto ppNIPAM. Two PCs were retained in this model, which captured 74% of the variance in the data. The data from the ppNIPAM substrate after cell liftoff was then projected into this model to determine score values for the new data. All the data were then compared on one scores plot. PCA was performed using the PLS Toolbox v. 2.0 (Eigenvector Research, Manson, WA) for MATLAB (the MathWorks, Inc., Natick, MA).
Results and Discussion Surfaces treated with pNIPAM have been observed to undergo a transition at the lower critical solution temperature (LCST) ∼31 °C. Above the LCST (i.e., at the cell culture temperature of 37 °C), the surfaces are more hydrophobic (as observed by water contact angle), and many cell types will adhere and proliferate. Below the LCST (i.e., at room temperature), the surface rapidly hydrates, and cell monolayers will spontaneously detach as a sheet (see Figure 1).1-4,31,32 Previously, surface characterization of ppNIPAM-coated TCPS showed that, after cell liftoff, although the majority of the ECM stayed with the cell sheet, some protein still remains at the surface.15 We further showed that the proteinaceous species remaining on the dish are capable of promoting new cell growth, indicating that the surface resembles viable ECM.5 This provides unprecedented access to the ECM underlying the cell sheet for surface analytical methods, such as XPS and ToF-SIMS. In this work, we seek to compare ppNIPAM surfaces after exposure to single protein solutions containing three biologically relevant ECM proteins: LN, FN, and Coll.11-14,33 Each of these proteins have demonstrated diverse biological functions and can impact cell adhesion, morphology, migration, and differentiation via their interactions with cell-surface receptors and other ECM (30) Wold, S.; Esbensen, K.; Geladi, P. Chemom. Intell. Lab. Syst. 1987, 2, 37-52. (31) Doorty, K. B.; Golubeva, T. A.; Gorelov, A. V.; Rochev, Y. A.; Allen, L. T.; Dawson, K. A.; Gallagher, W. M.; Keenan, A. K. CardioVasc. Pathol. 2003, 12, 105-110. (32) Taillefer, J.; Brasseur, N.; van Lier, J. E.; Lenaerts, V.; Le, Garrec, D.; Leroux, J. C. J. Pharm. Pharmacol. 2001, 53, 155-166. (33) Hudson, B. G.; Gunwar, S.; Chung, A. E.; Burgeson, R. E. In Extracellular Matrix: A Practical Approach, 1st ed.; Haralson, M. A., Hassel, J. R., Eds.; Oxford University Press: New York, 1995; p 404.
molecules. In addition, we compare the surfaces to ppNIPAM surfaces exposed to a single protein solution containing BSA. XPS Results. XPS is a quantitative surface analytical tool, sensitive to the atomic composition of the outer 20-100 Å of a material. We first used XPS to characterize blank ppNIPAMtreated 48-well TCPS plates used as controls. As discussed in previous work,5,15 we found that the composition of ppNIPAMtreated 48-well TCPS plates differs to a small degree from that predicted from the stoichiometry of the NIPAM monomer and from coatings on silicon chips produced using our method. [ppNIPAM-treated 48-well TCPS plates: 78.8% C, 7.2% N, and 14.0% O; pNIPAM theoretical composition: 75.0% C, 12.5% N, and 12.5% O; ppNIPAM-treated Si chips: 76.2% C, 12.5% N, and 11.3% O.] We theorize that this difference is due to the geometry of the wells preventing the gas species in the plasma from coating the well bottom in an identical manner to that on flat substrates. Regardless, sufficient monomeric structure is retained in the plasma-polymerized film to demonstrate the physical, mechanical, and thermoresponsive properties of the polymer.27 After exposure to single protein solutions (post-protein adsorption), we find that, although the amino acid composition of the ECM proteins used varies widely, the average elemental compositions of FN and LN are extremely similar (71% C, 16% O, and 11% N). The elemental composition of collagen (76% C, 14.0% O, 10% N) is significantly different from that of FN or LN (see Table 2a). Most importantly, when we compare the composition of the ppNIPAM surfaces exposed to single protein solutions (postprotein adsorption) to those after cell culture (post-cell liftoff), we find that they are almost identical: ppNIPAM surfaces postcell liftoff are 70.2% C, 17.8% O, and 10.7% N, whereas ppNIPAM surfaces post-adsorption with LN is 71.2% C, 16.4% O, and 11.4% N (see Table 2b). Previously, we suggested that some ECM proteins remain after cell liftoff from ppNIPAM surfaces,5,15 and the XPS results are consistent with that idea. We next examine the chemical shifts evident in the highresolution C 1s spectra of FN, LN, and Coll I (see Figure 2a). Similar to the composition results, we find that the high-resolution C 1s spectra resulting from the XPS analysis of FN and LN are similar (59% C-H, 25% C-OH, and 16% N-CdO), while the composition of collagen (64% C-H, 23% C-OH, and 13% N-CdO) is slightly different (see Table 3a). If we next compare the high-resolution C 1s spectrum of a surface after exposure to a representative protein (FN, single protein solution) to those after cell culture (post-cell liftoff) and that of the ppNIPAM-coated TCPS control surface, we can see obvious differences in the control and post-cell liftoff: after exposure to single protein solution or cell culture (post-cell liftoff), the intensity of the C 1s alcohol/amine and amide peaks (shifts of approximately +1.5 and 3.5 eV, respectively) are more pronounced compared to that of the control (see Figure 2b). As with the composition results, we find that the composition of ppNIPAM surfaces post-cell liftoff (58% C-C/C-H, 26% C-N/ C-O, and 16% N-CdO) is similar to that of the post-protein adsorption surface (e.g., FN 59% C-C/C-H, 26% C-N/C-O, and 16% N-CdO). Both the BSA and ppNIPAM controls have
Comparison of NatiVe ECM with Protein Films
Langmuir, Vol. 23, No. 1, 2007 53
Table 1. Amino Acid Mass Fragment and Abundance in ECM Proteinsa
mass
species
amino acid
abbr.
30.0343 43.0296 44.05 55.0184 57.0578 58.0293 59.0483 60.0449 61.011 68.05 69.034 70.02929 70.0657 71.0133 72.044 72.08132 73.06399 74.061 76.022 81.0453 82.0531 83.0469 84.0449 84.08132 85.0402 86.09697 87.0558 88.0399 95.0609 98.0242 100.0875 101.0953 104.0534 107.0497 110.0718 113.0351 115.0507 117.037 120.0813 121.0402 127.0984 130.0657 132.0575 136.076 159.0922 170.0606
CH4N CH3N2 C2H6N C3H3O C3H7N C2H4NO CH5N3 C2H6NO C2H5S C4H6N C4H5O C3H4NO C4H8N C3H3O2 C3H6NO C4H10N C2H7N3 C3H8NO C2H6NS C4H5N2 C4H6N2 C5H7O C4H6NO C5H10N C3H5N2O C5H12N C3H7N2O C3H6NO2 C5H7N2 C4H4NO2 C4H10N3 C4H11N3 C4H10NS C7H7O C5H8N3 C4H5N2O2 C4H7N2O2 C5H9OS C8H10N C6H5N2O C5H11N4 C9H8N C9H8O C8H10NO C10H11N2 C11H8NO
glycine arginine alanine/cysteine tyrosine lysine glycine arginine L-serine methionine proline threonine asparagine arginine/leucine/proline L-serine glycine valine arginine threonine cysteine histidine histidine valine glutamine lysine glycine leucine/isoleucine asparagine/glycine asparagine/aspartic acid histidine asparagine arginine arginine methionine tyrosine arginine/histidine glycine glycine methionine phenylalanine histidine arginine tryptophan phenylalanine tyrosine tryptophan tryptophan
Gly Arg Ala/Cys Tyr Lys Gly Arg Ser Met Pro Thr Asn Arg/Leu/Pro Ser Gly Val Arg Thr Cys His His Val Glu Lys Gly Leu/Ile Asn/Gly Asn/Asp His Asn Arg Arg Met Tyr Arg/His Gly Gly Met Phe His Arg Trp Phe Tyr Trp Trp
a
most prevalent in
Table 2. (a) Elemental Compositions Determined by XPS Survey Spectra of the ppNIPAM-treated TCPS Control and Post-ECM Protein Adsorption Surfaces.a,b (b) Elemental Compositions Determined by XPS Survey Spectra of Post-Protein Adsorption, Post-Cell Liftoff, and ppNIPAM Control Surfacesa,c
Coll LN
(a) average atomic %
FN BSA Coll LN FN Coll Coll FN LN FN Coll FN LN FN LN BSA BSA FN FN BSA Coll
BSA LN LN LN Coll FN Coll Coll Coll BSA BSA LN FN BSA FN FN FN
Adapted from Wagner et al.18,20
significantly more hydrocarbon species (ppNIPAM: 72% C-C/ C-H, 18% C-N/C-O, and 11% N-CdO; BSA: 68% C-C/ C-H, 20% C-N/C-O, and 12% N-CdO) (see Table 3b). ToF-SIMS Results. From the XPS results presented in the preceding section, we can conclude that the composition of the post-cell culture ppNIPAM surface is consistent with that of the post-protein adsorption surface. However, using XPS, we are not able to clearly identify which specific protein species are present at the surface. To do this, we use ToF-SIMS. Like XPS, ToF-SIMS is a surface-sensitive analytical tool. ToF-SIMS is considered to be a complementary technique to XPS, as the former yields detailed information regarding molecular species at interfaces, while the latter gives predominantly elemental information. Although the extensive fragmentation of the proteins by ToFSIMS makes it impossible to identify large chains of the protein amino acids directly by mass (such as in MALDI), previous work by our group defined unique amino acid fragmentation patterns in ToF-SIMS positive-ion spectra capable of identifying
surface
C 1s
O 1s
N 1s
ppNIPAM FN LN Coll I
78.8 71.9 71.2 76.0
14.0 16.0 16.4 14.0
7.2 11.6 11.4 10.0
(b) average atomic % surface
C 1s
O 1s
N 1s
ppNIPAM ppNIPAM post-cell BSA
78.8 70.2 75.9
14.0 17.8 14.3
7.2 10.7 9.5
a Relative atomic % of the major components are presented; trace amounts of Cl, Na, P, Ca, Zn, and K were present in ppNIPAM post-cell culture and post-protein adsorption (
