4444
Langmuir 2002, 18, 4444-4448
Quantification of Protein-Polymer Interactions by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Ji Zhang and Gary R. Kinsel* Department of Chemistry and Biochemistry, University of Texas at Arlington, Box 19065, Arlington, Texas 76019-0065 Received September 26, 2001. In Final Form: February 11, 2002 The use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to quantitate surfaceprotein binding interactions is explored using the peptide-porcine insulin and a variety of commercially available polymers. We show that a standard-additions plot of porcine-insulin surface concentration versus porcine-insulin MALDI ion signal is linear over a range of surface concentrations from 100 ng/cm2 to 800 ng/cm2. Further, similar to previous studies, we demonstrate that the positive x-intercepts of these standardadditions plots correlate with the polymer-porcine insulin retention affinities as established using 125Ilabeled-porcine insulin. Additional novel studies demonstrate that the polymer-porcine insulin retention affinities determined from the standard-additions plots are independent of both the MALDI matrix and the desorption/ionization wavelength. Finally, it is shown that the polymer-porcine-insulin adsorption affinity can be established by measuring the porcine-insulin MALDI ion signal resulting from adsorbed peptide and by using the standard-additions plot for quantitation. Cumulatively, these studies suggest MALDI mass spectrometry can be effectively employed to quantitate polymer-protein binding interactions.
Introduction Protein adsorption on solid surfaces has been investigated for several decades, but the mechanism of this process remains poorly understood.1 The aim of research in this field is to develop a quantitative mechanistic model of protein adsorption behavior. Research in this field is critically dependent upon the development of experimental techniques for the accurate quantification of surfacebound proteins. Currently, experimental techniques that have been developed to allow the quantification of surface-bound proteins include the use of radiolabeled proteins,2-4 total internal reflectance fluorescence (TIRF) spectroscopy,5-7 electron spectroscopy for chemical analysis (ESCA),7 surface-plasmon resonance (SPR) spectroscopy,8,9 ellipsometry,10-12 chromatography,13,14secondary * To whom correspondence should be addressed. Department of Chemistry and Biochemistry, The University of Texas at Arlington, Box 19065, Arlington, TX 76019-0065. Phone: (817) 272-3541. FAX: (817) 272-3808. E-mail:
[email protected]. (1) Interfacial Phenomena and Bioproducts; Brash, J. L., Wojciechowski, P. W., Eds.; Marcel Dekker: New York, 1996. (2) Butler, J. E.; Lu, E. P.; Navarro, P.; Christiansen, B. J. Mol. Recogn. 1997, 10, 36-51. (3) Johansson, J.; Yasuda. H. K.; Bajpai, R. K. Appl. Biochem. Biotech. 1998, 70-72, 747-763. (4) Schmitt, A.; Varoqui, R.; Uniyal, S.; Brash, J. L.; Pusiner, C. J. Colloid Interf. Sci. 1983, 92(1), 23-34. (5) Robeson, J. L.; Tilton, R. D. Langmuir 1996, 12, 6104-6113. (6) Roth, C. M.; Lenhoff, A. M. Langmuir 1995, 11, 3500-3509. (7) Go¨lander, C.-G.; Hlady, V.; Caldwell, K.; Andrade, J. D. Colloids Surf. 1990, 50, 113-130. (8) Knoll, W. Annu. Rev. Phys. Chem. 1998, 49, 569-638. (9) Rao, J.; Yan, L.; Xu, B.; Whitesides, G. M. J. Am. Chem. Soc. 1999, 121, 2629-2630. (10) Freij-Larsson, C.; Nylander, T.; Jannasch, P.; Wesslen, B. Biomaterials 1996, 17, 2199-2207. (11) Krisdhasima, V.; Vinaraphong, P.; McGuire, J. J. Colloid Interf. Sci. 1993, 161, 325-334. (12) Elwing, H.; Nilsson, B.; Svensson, K.; Askendahl, A.; Nilsson, U. R.; Lundstron, I. J. Colloid Interf. Sci. 1988, 125(1), 139-145. (13) Basiuk, V. A.; Gromovoy, T. Y.; Khil’Chevskaya, E. G. Origins Life Evol. B. 1995, 25, 375-393. (14) Boppana, V. K.; Miller-Stein, C. Anal. Chim. Acta 1997, 352, 61-69.
ion mass spectrometry(SIMS)15 and various colorimetric methods.16-20 While each of these methods offer various advantages and disadvantages, there remains a need for further development of simple, efficient, and broadly applicable methods for the quantification of surfacebound proteins. In recent years, matrix-assisted laser desorption/ ionization mass spectrometry (MALDI MS) has become a mainstream tool in the analysis of biomolecules.21 This method offers extremely high mass resolution and very low detection limits for a wide variety of biomolecules. While this technique has been traditionally used to characterize the molecular weights of peptides, proteins, etc., a number of features of MALDI MS are attractive for the study of surface-protein binding interactions.22 MALDI MS can be extremely sensitive, with the detection of subfemtomolar quantities23 and submonolayer surface coverages24 of protein having been reported. Mass spectrometric detection of adsorbed proteins also offers the opportunity for the study of competitive protein binding and/or the detection and identification of unexpected surface-bound proteins from complex biological fluids.25 Finally, within the constraints of available instrumenta(15) Lhoest, J.-B.; Wagner, M. S.; Tidwell, C. D.; Castner, D. G. J. Biomed. Mater. Res. 2001, 57, 432-440. (16) Hashim, M. A.; Chu, K. H.; Tsan, P. S. Chem. Eng. Comm. 1997, 161, 45-63. (17) Fukuzaki, S.; Urano, H.; Nagata, K. J. Ferment. Bioeng. 1995, 80(1) 6-11. (18) Beeskow, T.; Kroner, K. H.; Anspach, F. B. J. Colloid Interf. Sci. 1997, 196, 278-291. (19) Rabinow, B. E.; Ding, Y. S.; Qin, C.; McHalsky, M. L.; Schneider, J. H.; Ashline, K. A.; Shelbourn, T. L.; Albrecht, R. M. J. Biomat. Sci.Poly. E. 1994, 6(1), 91-109. (20) Wahlgren, M.; Arnebrant, T. Trends Biotechnol. 1991, 9, 201208. (21) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A. (22) Griesser, H. J.; Kingshott, P.; McArthur, S. L.; McLean, K. M.; Kinsel, G. R.; Timmons, R. B. Biomaterials, in press. (23) Zhang, H.; Per, E.; Caprioli, R. M. J. Mass Spectrom. 1995, 30, 1768-1771. (24) Walker, A. K.; Land, C. M.; Kinsel, G. R.; Nelson, K. D. J. Am. Soc. Mass Spectrom. 2000, 11, 62-68.
10.1021/la015594d CCC: $22.00 © 2002 American Chemical Society Published on Web 04/25/2002
Protein-Polymer Interactions by MALDI MS
tion, MALDI MS offers one of the simplest and most efficient methods for the analysis of surface-bound proteins. In principle, only the addition of a drop of MALDI matrix solution to the protein-coated surface is required for sample preparation prior to mass spectrometric analysis. In previous studies, we have shown that a MALDI MS method of standard additions can be used to quantitate the amount of peptide strongly bound to (retained by) a variety of polymer surfaces.24 The approach is based on the observed inverse relationship between the magnitude of a peptide MALDI ion signal and the surface-peptide binding affinity, as determined using 125I-radiolabeled peptides.26 In this report, we extend the MALDI MS standard additions method to determine the surfaceprotein retention affinities for porcine insulin applied to a variety of commercial polymers. In addition, we report new results examining the influence of changes in both the MALDI matrix and desorption/ionization wavelength on the determination of the surface-protein retention affinity. Finally, we report a new application of the MALDI MS standard additions plots as simple calibration plots to quantitate surface-protein adsorption affinity. Experimental Section Chemicals and Materials. The materials poly(etheretherketone) (PEEK, 0.125 mm thickness), lowdensity poly(ethylene) (LDPE, 0.25-mm thickness), poly(etherimide) (PEI, 0.25-mm thickness), poly(ethersulfone) (PES, 0.125-mm thickness), and poly(tetrafluoroethylene) (PTFE, 0.10-mm thickness) were obtained from Goodfellow Corp. (Berwyn, PA). Phosphate buffered saline (PBS), sodium dodecyl sulfate (SDS), and trifluoroacetic acid (TFA) were purchased from Sigma (St. Louis, MO). The PBS solution (pH ) 7.4) was prepared as directed by the manufacturer and the 0.3% SDS solution and 10% (v/v) TFA solution were prepared by dilution of the pure substances with distilled water. Porcine insulin, R-cyano4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), and 2,5-dihydroxybenzoic acid (gentisic acid) were purchased from Sigma and used without further purification. Trans-4-hydroxy-3-methoxycinnamic acid (ferulic acid) was obtained from Lancaster (Windham, NH). The 125I-porcine insulin was provided by the University of Texas Southwestern Medical Center (Dallas, TX). 125 I-Radiolabeled Peptide Binding. Quantification of peptide binding to the polymeric materials was investigated by exposure of the polymer samples to 125I-porcine insulin using a flow-through cell. After cleaning by sonication for 4 h in methanol, a total of 15 polymer samples (1.5 × 1.5 cm) were placed in the flow-through cell and initially equilibrated for 3 min in PBS. The PBS solution was then flushed from the cell by the introduction of 125I-porcine insulin (0.010 mg/mL in PBS, 1.0 µCi/mL) and the polymer samples were allowed to incubate for 20 min. Subsequently, the peptide solution was flushed from the cell by the introduction of 50 mL of PBS and the polymer samples were removed from the flow-through cell and place in individual test tubes. The polymer surface-125I-porcine-insulin adsorption affinity was determined by measuring the radioactivity of the samples using a Wallac (Gaithersburg, Md) model 1282 well-type γ counter. Quantification of the adsorbed (25) Kingshott, P.; St John, H. A. W.; Chatelier, R. C.; Griesser, H. J. J. Biomed. Mater. Res. 2000, 49, 36-42. (26) Walker, A. K.; Wu, Y.; Timmons, R. B.; Kinsel, G. R.; Nelson, K. D. Anal. Chem. 1999, 71, 268-272.
Langmuir, Vol. 18, No. 11, 2002 4445
Figure 1. 355 nm MALDI standard additions plot for porcine insulin applied to LDPE using ACHCA as the MALDI matrix.
peptide was established by comparison of the measured radioactivity with 200 µL of 125I-porcine-insulin solution having a known concentration. Typically, five samples of each commercial polymer were examined to establish the average quantity of adsorbed 125I-porcine insulin and the standard deviation in the data set. The polymer surface125 I-porcine-insulin retention affinity was assessed by vigorously washing the polymer samples for 3 min using 0.3% SDS. The washed polymer samples were placed in fresh test tubes and the radioactivity was recounted. The average and standard deviation of the retained 125I-porcine insulin was again established by comparison of the measured radioactivity for five samples of a given commercial polymer with that of a standard solution. MALDI Analysis. Porcine-insulin solutions of varying concentration were prepared by dissolving 1 mg of porcine insulin in 1 mL of PBS and diluting with additional PBS as necessary. A 2-µL aliquot of a porcine-insulin solution was applied to a 4.8 mm diameter disk of the previously cleaned (4 h sonication in methanol) polymer under investigation and allowed to dry under ambient conditions (drying time approximately 45 min). Subsequently, 2 µL of 10% TFA and 2 µL of a matrix solution (15-mg/mL in methanol) were deposited on the porcine-insulin coated polymer disk. The sample was then allowed to dry for at least 6 h. All MALDI mass spectra were acquired using a laboratory-built linear time-of-flight (TOF) mass spectrometer operated in continuous extraction (18 kV acceleration) mode. Laser desorption was performed using either an Oriel (Stratford, CT) nitrogen laser (337 nm) or a Continuum (Santa Clara, CA) Minilite Nd:YAG laser (355 nm). The laser irradiance was adjusted using a neutral density filter to achieve the largest protonated peptide ion signal while maintaining baseline separation between the protonated and sodiated porcine-insulin parent ion signals. In general, this laser intensity was slightly above threshold for ion formation (e.g. 10-20 mJ/cm2). After maximizing the protonated porcine-insulin ion signal a total of 20 twenty-shot mass spectra were collected from three samples of each analyte/surface combination. The singly protonated peptide-ion signals were then integrated and the average peak area and standard deviation determined. Results and Discussion Figure 1 shows the results of a MALDI MS standard addition experiment performed to evaluate the surfacepeptide retention affinity of LDPE for porcine insulin. The retention affinity of a polymer surface represents the quantity of porcine insulin that cannot be removed through
4446
Langmuir, Vol. 18, No. 11, 2002
washing of the surface with the SDS solution. Each of the points on the graph represent the average of the singly protonated porcine-insulin peak areas for a total of 20 MALDI mass spectra acquired on three separate samples. The error bars on each of the points represent (1 standard deviation of the data set (the relative standard deviation of a data set is typically 5-15%). To reduce the relative standard deviation of the data sets to the values shown, a number of constraints are placed on the acquisition of the data set. First, the area of the LDPE disk with which the 2 µL of porcine-insulin solution interacts must be determined via measurement of the droplet diameter on the polymer surface. This constraint is necessary to accurately assign the porcine-insulin surface concentration. Second, the porcine-insulin MALDI mass spectra are acquired by adjusting the desorption-laser intensity to maximize the protonated porcine-insulin signal while maintaining a user-specified ion signal resolution. It is our experience that this approach to data acquisition improves the reproducibility of the integrated peak area for a given surface concentration. For our laboratoryconstructed MALDI TOF MS, the user specified ion signal resolution is set to require baseline separation between the protonated and sodiated porcine-insulin parent ions. This latter requirement is satisfied by using desorptionlaser intensities slightly above threshold for ion formation and the intensity, once established, is generally applicable across the entire range of surface concentrations used in these experiments. The graph in Figure 1 clearly demonstrates that, if the above constraints are applied, the porcine-insulin MALDI ion signal versus porcine-insulin surface concentration is linear over a range of surface concentrations from approximately 100 ng/cm2 to 700 ng/cm2. These concentrations reflect a surface coverage ranging from approximately 0.5 monolayers to 3.5 monolayers assuming a smooth surface and close-packing of the porcine-insulin molecules.27 Furthermore, the graph clearly shows that the linear best-fit to the data set does not go through the origin of the plot but instead has a positive x-intercept i.e., the porcine-insulin ion signal goes to zero at a finite surface concentration. In our previous studies with angiotensin I, it was found that the x-intercept of this type of standard additions plot correlated with the surfacepeptide retention affinity (i.e. the quantity of tightly bound angiotensin that is not removed by an SDS wash) measured using radiolabeled 125I-angiotensin I.24 Table 1 lists the surface-peptide retention affinities (i.e. the quantity of tightly bound porcine insulin that is not removed by an SDS wash) of five different polymers, PEEK, LDPE, PEI, PES, and PTFE for 125I-radiolabeledporcine insulin. Also listed in Table 1 are the x-intercepts of MALDI standard addition plots derived from experiments in which porcine insulin was deposited on the five polymers and data acquired using the approach described. A comparison of the two sets of numbers clearly indicates the good agreement between the two approaches to measuring surface-peptide retention for this peptide. Beyond the surface on which the peptide is deposited, a variety of additional parameters may be varied in a given MALDI experiment including: the method of sample preparation, the matrix, the desorption/ionization wavelength, etc. In previous work, we have shown that reductions in peptide MALDI ion signals due to polymer surface-peptide binding interactions are remarkably insensitive to the method of sample preparation.28 Specifically, neither changes in the order of deposition of the (27) Gao, J.; Whitesides, G. M. Anal. Chem. 1997, 69, 575-580.
Zhang and Kinsel Table 1. Polymer-porcine Insulin Retention Affinitiesa as Determined by Radiolabeled 125I-porcine Insulin and by Using a MALDI Standard Additions Method 125I-porcine
insulin
355-nm MALDI
polymer
ng/cm2b
coveragec
ng/cm2d
slope × 105
R2
PEEK LDPE PEI PES PTFE
126 ( 6 39 ( 5 130 ( 20 91 ( 5 40 ( 10
58% 18% 60% 42% 18%
120 ( 20 33 ( 9 130 ( 30 90 ( 30 40 ( 10
15.5 7.23 19.4 8.65 7.28
0.961 0.997 0.983 0.984 0.998
a The quantity of tightly bound porcine insulin that is not removed by an SDS wash. b The values shown are the average retained 125Iporcine insulin for five samples and the errors in these values correspond to one standard deviation of the individual data sets. c Percent coverages are calculated using a cross-sectional area for porcine insulin27 of 4.4 × 10-14 cm2 and macroscopic dimensions of the polymer material. d The values shown represent the xintercepts of the 355 nm MALDI standard addition plots and the errors in these values as calculated according to ref 35.
Figure 2. 355 nm MALDI standard additions plots for porcine insulin applied to PEEK using ACHCA ((), DHB (9), SA (2) and FA (b) as MALDI matrixes.
matrix and peptide, nor changes in the matrix and peptide solvents were observed to eliminate the influence of surface-peptide binding on the peptide MALDI ion signals. The influence of changes in the MALDI matrix on the 355 nm standard additions plots for porcine insulin deposited on PEEK is illustrated in Figure 2. In these studies, four commonly used MALDI matrixes, R-cyano-4-hydroxycinnamic acid (ACHCA), ferulic acid (FA), sinapinic acid (SA), and gentisic acid (DHB) were employed for sample preparation while all other mass spectra acquisition parameters were held constant. From the plots shown it is clear that, while the slopes of the response curves may vary considerably, the x-intercepts remain essentially unchanged. Table 2 summarizes the results of a large number of experiments in which MALDI standard-addition experiments were performed using each of the four MALDI matrixes with porcine insulin deposited on two polymers, PEEK and LDPE, and using two desorption/ionization wavelengths, 337 and 355 nm. From these data it can be seen that there is generally good agreement between the x-intercepts of the individual standard-additions plots and the 125I-porcine-insulin retention affinities. The average x-intercept, over the four matrixes and two desorption/ ionization wavelengths, of the standard-additions plots for porcine insulin deposited on PEEK is 131 ng/cm2. This value may be compared with the 125I-porcine-insulin PEEK retention affinity of 126 ng/cm2 given in Table 1. The (28) Chen, C.; Walker, A. K.; Wu, Y.; Timmons, R. B.; Kinsel, G. R. J. Mass Spectrom. 1999, 34, 1205-1207.
Protein-Polymer Interactions by MALDI MS
Langmuir, Vol. 18, No. 11, 2002 4447
Table 2. Polymer-porcine Insulin Retention Affinities a as Determined by Using a MALDI Standard Additions Method, for Various MALDI Matrices and at Two Desorption/Ionization Wavelengths 337-nm MALDI slope × 105
125I-porcine
355-nm MALDI slope × 105
R2
0.998 120 ( 20 0.985 33 ( 9
15.5 7.23
0.982 0.998
2.43 1.82
0.998 152 ( 36 0.979 34 ( 23
4.34 1.72
0.948 0.987
Matrix: DHB PEEK 130 ( 20 LDPE 44 ( 11
2.23 1.66
0.947 110 ( 10 0.991 31 ( 18
6.48 2.10
0.995 0.993
Matrix: FA PEEK 140 ( 10 LDPE 46 ( 23
1.51 1.53
0.963 130 ( 20 0.991 48 ( 26
3.27 1.28
0.990 0.982
ng/cm2b Matrix: ACHCA PEEK 139 ( 7 LDPE 49 ( 21
1.18 1.54
Matrix: SA PEEK 129 ( 4 LDPE 49 ( 23
Table 3. Polymer-porcine Insulin Adsorption Affinitiesa as Determined by Using Radiolabeled 125I-porcine Insulin and by Using the MALDI Standard Additions Plots for Quantification of the Adsorbed Porcine Insulin.
R2
ng/cm2a
a The quantity of tightly bound porcine insulin that is not removed by an SDS wash. b The values shown represent the x-intercepts of the MALDI standard additions plots and the errors in these values as calculated according to ref 35.
average x-intercept for porcine insulin deposited on LDPE is 42 ng/cm2; a value that may be compared with the 125Iporcine-insulin LDPE retention affinity of 40 ng/cm2 given in Table 1. These results clearly indicate that the MALDI standard-additions approach to determining surfacepeptide retention affinities is independent of the matrix and desorption/ionization wavelength for the systems under study. From these results it is apparent that the strongly surface-bound porcine-insulin molecules are poorly ionized in the MALDI process. This observation is not surprising considering recent studies which suggest that incorporation of the peptide analyte in the MALDI matrix crystals is a necessary requirement for efficient desorption/ ionization.29 A further point of interest is the variations in the slopes of the standard-additions plots given in Tables 1 and 2. While it must be emphasized that these slopes are influenced by many instrumental factors, including ion collection efficiency, detector response, operating vacuum, etc., there is a clear trend of steeper slopes (higher sensitivities) at the longer 355 nm wavelength than at the 337 nm wavelength. This result is somewhat surprising given that the molar absorptivities of each of the four matrixes is anywhere from a factor of two to a factor of five lower at 355 nm as compared with 337 nm.30 Thus, the sensitivity of the response does not appear to be directly correlated with the absorption efficiency of the matrix and may instead be specifically related to the photon energy. Recent studies of proton-transfer reactions in clusters performed in our laboratory have suggested that desorption/ionization wavelengths that introduce large excess energies above the ionization potential of the cluster tend to favor cluster dissociation without proton transfer, i.e., less efficient analyte ionization.31 This observation is consistent with the enhanced analyte ionization efficiency at the lower 355 nm photon energy. It is also apparent that the method used to acquire the porcine-insulin MALDI mass spectra represents a viable approach to quantification of modest concentrations of (29) Horneffer, V.; Dreisewerd, K.; Lu¨demann, H.-C.; Hillenkamp, F.; La¨ge, M.; Strupat, K. Int. J. Mass Spectrom. 1999, 185/186/187 859-870. (30) Sumner, L. W.; Gray, J. P.; Kirkpatrick, J. R.; Russell, D. H. Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, 1997, p. 854. (31) Land, C. M.; Kinsel, G. R. J. Am. Soc. Mass Spectrom. 2001, 12, 726-731.
insulin
355-nm MALDIc
polymer
ng/cm2b
ng/cm2
PEEK LDPE PEI PES PTFE
350 ( 50 380 ( 70 440 ( 60 320 ( 90 400 ( 80
270 ( 40 420 ( 90 330 ( 50 320 ( 70 280 ( 40
a The quantity of porcine insulin that is not removed by a PBS solution wash. b The values shown are the average adsorbed 125Iporcine insulin for 5 samples and the errors in these values correspond to one standard deviation of the individual data sets. c The errors in the adsorbed porcine insulin values are calculated according to ref 35.
surface-deposited or surface-adsorbed peptides. Numerous experimental approaches to quantitative MALDI have been described in the literature, but this application of MALDI remains a challenge.32-34Our studies indicate that by applying the described constraints for MALDI mass spectra acquisition and by recognizing that, for any surface with a measurable surface-protein-retention affinity, the calibration plot will not go through the origin, a linear surface-concentration versus MALDI ion signal plot can be obtained. To evaluate the utility of the described methodology for quantification of low-levels of surface-deposited and/or surface-adsorbed protein, the standard-addition plots were used to determine the adsorption affinity of the polymer surfaces for porcine insulin. The adsorption affinity of a polymer surface represents the quantity of porcine insulin that cannot be removed through washing of the surface with PBS buffer solution and is expected to represent approximately a monolayer of protein. The polymer disks were placed in a flow-through cell and porcine insulin was introduced to the cell in a manner identical to that used in the 125I-porcine-insulin binding studies. Thus, we expect a similar quantity of porcine insulin to adsorb to the polymer surfaces as determined in the 125I-porcineinsulin binding studies (values given in Table 3). Following removal of the porcine-insulin coated polymer disks from the flow-through cell, the MALDI matrix was deposited on the disk and MALDI mass spectra were acquired and integrated using the identical approach to that used to generate the standard-addition plots. Table 3 lists the surface-peptide adsorption affinities (i.e. the quantity of porcine insulin that is not removed by a PBS solution wash) of the five different polymers for 125I-porcine insulin. Also shown in Table 3 are the surface concentrations of adsorbed porcine insulin based on the integrated porcine-insulin MALDI ion signals and the standard-addition plots used as quantification curves. A comparison of the two sets of values indicates there is generally good agreement between the two approaches, although several values only overlap within the extremes of one standard deviation. One factor that remains uncharacterized in these studies is the morphology of the polymer surfaces and the influence of the surface mor(32) Mirgorodskaya, O. A.; Kozmin, Y. P.; Titov, M. I.; Korner, R.; Sonksen, C. P.; Roepstorff, P. Rapid Commun. Mass Spectrom. 2000, 14, 1226-1232. (33) Gobom, J.; Kraeuter, K.-O.; Persson, R.; Steen, H.; Roepstorff, P.; Ekman, R. Anal. Chem. 2000, 72, 3320-3326. (34) Kan, M.-L.; Tholey, A.; Heinzle, E. Rapid Commun. Mass Spectrom. 2000, 14, 1972-1978. (35) Harris, D. C. Quanitative Chemical Analysis, 5th ed.; W. H. Freeman; New York, 1998; p 104.
4448
Langmuir, Vol. 18, No. 11, 2002
phology on the polymer surface-porcine-insulin adsorption affinity. A surface concentration of ca. 220 ng/cm2 would correspond to adsorption of a monolayer of porcine insulin on a perfectly smooth surface and this value can, therefore, be considered a lower limit for the adsorption affinity.27 All of the values shown in Table 3 clearly exceed this lower limit and it would be of interest to correlate the measured adsorption-affinity values with an assessment of the polymer surface roughness. Additional experiments are currently in progress to assess the MALDI method for quantification of modest concentrations of surface deposited/adsorbed peptides and to examine the influence of surface morphology on the values obtained. Conclusion These studies demonstrate that a MALDI method of standard additions can be used to establish surface-peptide retention affinities for porcine insulin deposited on PEEK, LDPE, PEI, PES, and PTFE. The implication of these results is that strongly surface-bound peptides are inefficiently ionized in a typical MALDI experiment, presumably due to poor incorporation of the peptide into the
Zhang and Kinsel
MALDI matrix crystals. This behavior is further shown to be independent of the MALDI matrix and wavelength used for desorption/ionization, although the slopes of the standard-additions plots do vary with these parameters. Surprisingly, the highest sensitivities are obtained at desorption/ionization wavelengths for which the matrix compounds have lower absorption coefficients. This result suggests that the photon energy is a more important parameter for efficient analyte protonation than is the ability of the bulk sample to absorb the laser radiation. Finally, it is demonstrated that the methodology developed to obtain the MALDI standard-addition plots can also be used to quantitate low concentrations of surface-deposited or surface-adsorbed peptides. Acknowledgment. This work was supported by the National Science Foundation (CHE-9876249). The authors wish to thank Dr. A. Constantinesceu for synthesis of the 125I-porcine insulin. LA015594D
