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Methods for Generating Protein Molecular Ions in ToF-SIMS. Sally L. McArthur, Marie C. Vendettuoli, Buddy D. Ratner, and David G. Castner. Langmuir 20...
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Static Secondary Ion Mass Spectrometry of Adsorbed Proteins David S. Mantust and Buddy D. Ratner' Department of Chemical Engineering, BF-10,University of Washington, Seattle, Washington 98195

Brad A. Carlson and John F. Moulder Perkin-Elmer Physical Electronics Division, 6509 Flying Cloud Drive, Eden Prairie, Minnesota 55344

Static secondary ion mass spectrometry (SIMS) was used to analyze proteins adsorbed to biomaterial surfaces. A spectral interpretation protocol was established by examining homopolymers of 16 amino acids. This protocol allows for the assignment of peaks unique to the various amino acids. Static SIMS was used to analyze plasma proteinsadsorbedto titanium. The various factors that contributed to the relative intensities observedin the spectra wereexplored. The potential application of the technique for studying proteinfouled materials was investigated by analyzing a fouled sensor membrane. INTRODUCTION The interaction of proteins with artificial surfaces is critical to material performance in a wide variety of biomedical and industrial situations.' Adsorbed proteins can communicate biological response, change surface properties, and initiate material fouling and degradation. There is an extensivebody of knowledge on the behavior of proteins on surfaces from solution experiments.2 These experiments have revealed the sensitivity of the adsorption process to changes in surface chemistry and have established a number of interestingresults regarding the behavior of proteins once adsorbed. For example, it has been observed that the strength with which proteins are attached to a surface increases with time.3 This observation is most likely a result of changes in the conformation of the protein at the surface. While circular dichroism and infrared measurements have been used to study adsorbed proteins, there have been few studies employing modern methods of surface analysis.pe X-ray photoelectron spectroscopy (XPS) has been used to study adsorbed protein^.^-^ Information on the extent and nature of the protein coverage could be extracted from the data. However, XPS is limited in ita ability to provide detailed molecular information. This is particularly true when atoms are in a wide variety of

* To whom correspondence should be addressed.

Current address: Procter & Gamble Pharmaceuticals, P.O. Box 191, Wood Corners Laboratories, Norwich, NY 13815. (1)Ratner, B. D.; Castner, D. G.; Horbett, T. A.; Lenk, T. J.; Lewis, K. B.; Rapoza, R. J. J. Vac. Sci. Technol. A 1990,8,2306-2317. (2)Proteins at Interfaces; Brash, J. L., Horbett, T. A., Eds.; ACS Symposium Series 343;American Chemical Society: Washington, DC, 1987. (3) Bohnert, J. L.; Horbett, T. A. J. Colloid Interface Sci. 1986,111 (2). -,, 269-372. - - - - . -. (4)Lenk,T. J.;Ratner,B. D.; Gendreau,R. M.;Chittur, K. K. J.Biomed. Mater. Res. 1989,23,549-569. (5)McMillin, C. R.; Walton, A. G. J. Colloid Interface Sci. 1974,48 (2),345-349. (6)Morrisey, B. W.; Stromberg, R. R. J. Colloid Interface. Sci. 1974, 6 (l), 152-164; (7) Ratner, B. D.; Horbett, T. A.; Shuttleworth, D.; Thomas, H. R. J. Colloid Interface Sci., 1981,83 (l),630-642. (8)Paynter, R. W.; Ratner, B. D.; Horbett, T. A. Thomas, H. R. J. Colloid Interface Sci., 1984,101 (l),233-245. (9)Sundgren, J.-E.; Bodo, P.; Ivarsson, B.; Lundstrom, I. J. Colloid Interface Sci. 1986,113,530-543. +

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0003-2700/93/0365-143i$04.00/0

chemical states, as exist in a protein. Static secondary ion mass spectrometry(SIMS)is both more surfacesensitive ( -20 A) and more chemically selective than XPS. Static SIMS has been widely applied for the analysis of polymer surfaces. This includes the use of the technique for the determination of the surface chemistry of polymers of biomedical importance.'0-13 However, there has been little effort to use this powerful technique for the study of the interaction of polymerswith biological moleculesand systems. It should be noted that there are two basic areas of application of the static SIMS technique. As a mass spectrometric method, static SIMS can be used to desorb and ionize biomoleculesfrom specially prepared surfaces. Benninghoven et al.lP16 pioneered this use of static SIMS, and the method has seen widespread use for the analysis of thermally labile molecules. The value of the method for practical mass spectrometric analysis is exemplified in the work of such researchers as Standing and co-~orkers.'~The surface sensitivity of the static SIMS technique is not fully exploited in these purely mass spectrometric applications. As a surface analysis method, static SIMS is unrivaled in ita molecular selectivity, because of ita basis in mass spectrometry. Static SIMS has been used to analyze a wide variety of "real" surfaces,18ranging from semiconductor materials to complex copolymers. The spectra produced reflect the surface chemistry of the material but, in general, do not contain large (mlz = >500) molecular ions or fragments. However, useful information is readily extracted from the fragment ions in this lower mass range. The ability of static SIMS to produce a surface-sensitivemass spectrum gives it great potential as a probe of proteins on surfaces. In order to use static SIMS as a surface analytical tool to study systems consisting of large biomolecules,such as proteins, on complex biomaterial substrates, we must develop a protocol for the interpretation of the resulting spectra. Homopolymers of amino acids represent the simplest chemical analogs for proteins and can be employed as model systems for the development of a spectral interpretation protocol. (10)Hearn, M. J.; Ratner, B. D.; Briggs, D. Macromolecules 1988,21, 2950-2959. (11)Wilding, I. R.; Melia, C. D.; Short, R. D.; Davies, M. C.; Brown, A. Appl. Polym. Sci. 1990,39,1827-1835. (12)Grainger, D. W.; Okano, T.; Kim, S.W.; Castner, D. G.; Rntner, B. D.; Briggs, D. J. Biomed. Mater. Res. 1990,24,547-571. (13)Gardella, J. A., Jr.; Pireaux, J. J. Anal. Chem. 1990,62,64514661A. (14)Benninghoven, A.; Jaspers, D.; Sichtermann, W. Appl. Phys. 1976, 11, 35-39. (15)Benninghoven, A.; Sichtermann, W. Org. Mass Spectrom. 1977, 12,595-597. (16)Benninghoven, A.; Sichtermann, W. K. Anal. Chem. 1978,50, 1180-1184. (17)For example: Tang, X.; Ens, W.; Standing, K. G.; Westmore, J. B. Anal. Chem. 1988,60,1791-1799.Main, D.E.; Ens, W.; Beavis, R.; Schueler, B.; Standing, K. G.; Westmore, J. B.; Wong, C. M. Biomed. Enuiron. Mass Spectrom. 1987,14(2),91-96.Beavis, R. C.; Bolbach, G.; Ens, W.; Main, D. E.; Schueler, B.; Standing, K. G. J. Chromatogr. 1986, 359,489-497. Ens, W.; Standing, K. G.; Westmore, J. B.; Ogilvie, K. K.; Nemer, M. J. Anal. Chem. 1982,54,960-966. (18)Benninghoven, A. J. Vac. Sci. Technol. A 1986,3(3),451-460. 0 1993 American Chemlcal Society

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

Poly(amino acids) are a class of polymers with a rich scientific history and a still developing technological importance. Homopolymers and copolymers of the naturally occurring and derivatized amino acids have been used in a wide variety of applications. They served as models for proteins in the earliest studies of solid-state protein structure1g and continue to serve as protein analogs in such fields as the study of prebiotic evolutionz0 and protein biosynthesis.z1 Recent technological applications of poly(amino acids) have included use as biosensors,22 membranes and supports for enantioselective separations,23>24drug delivery systems,25*26 catalysts for organic synthesi~,~7 magnetic resonance imaging enhancement agents,28 artificial skin,Z9 and sutures.30 Poly(amino acids) have also been used as analytical models for the study of proteins by infrared spectro~copy~~ and XPS.32 Proteins are composed of 20 amino acids. The molar 5% C, 0, and N in most proteins is remarkably constant.33 Proteins, except for a few specific structural proteins, have similar amino acid contents. In a study comparing the frequency of occurrence of each amino acid residue in the primary structure of 207 unrelated proteins, the number of times an amino acid is observed in a randomly chosen portion of the molecule is related onlyto its frequency; that is, proteins do not exhibit characteristic sequences a t the local leve1.34 However, a concern for specific higher order distributions (apolar, polar, charged, etc.) is expressed, because such distributions lead to specific chain folding and protein c~nformation.~~ It is manifestations of this higher order distribution, related to protein orientation and conformation at surfaces, that is of interest in the biological reaction of proteins at interfaces. This study provides some of the groundwork needed to explore amino acid distributions within the outermost 20 A of a protein layer. In this work, homopolymers of 16amino acids are examined with static SIMS. The results are used to establish a spectral interpretation protocol for protein surfaces. This protocol is used to describe the static SIMS results from a number of systems consisting of adsorbed proteins on artificial surfaces. These examples are chosen for their relevance to potential applications of the technique. Because of the molecular specificity of the technique, the spectrum produced can include substrate ions (if the film is thin enough or discontinuous), ions indicative of protein, and fragment ions from other molecules that either coadsorb or are part of a protein

aggregate. Static SIMS could also be useful for the examination of peptide oligomers artificially attached to surfaces. Short sequences of amino acids have been shown to elicit strong biological responses to materials a t surface concentrations well below the detection limits of XPS.35 Static SIMS should be able to detect ions indicative of these peptides at very low concentrations and in the presence of complex polymer substrates. The analysis of fouled materials should also be possible with static SIMS. The molecular specificity of the method should allow us to probe the nature of contamination on materials and devices in contact with various biological systems, such as sensors in processing tanks, etc. The relative intensity of the ions in static SIMS spectra of adsorbed proteins may be due to a number of factors. The intensity of ions indicative of individual amino acids will depend on the relative stability of the ions and the number of residues present in the protein. The intensities of ions may also be related to the mobility of amino acid units within the peptide, and this mobility may in turn be associated with how tightly the protein is bound to the surface and how spread or globular the protein is at the surface. Mobility effects in static SIMS have been suggested in a recent study.36 The possibility that the way in which a protein is adsorbed to a surface can contribute to the relative intensity of the ions in the spectrum is both intriguing, and useful. Since the sampling depth of static SIMS is smaller than the physical dimension of many proteins, under the proper conditions only the outermost amino acid residues of an adsorbed protein may be sampled. The spectra would be sensitive to the conformation and positioning of the protein (and its domains of hydrophilic and hydrophobic amino acids) on the surface. Since static SIMS analyses are performed in an ultrahigh vacuum environment, air-drying of protein fiims will probably alter their conformation. Using static SIMS to explore fine details of adsorbed protein conformationnecessitates sample preparation procedures aimed a t preserving the adsorbed protein in a state resembling that at the solution-solid i n t e r f a ~ e . ~In~this - ~ ~paper, protein films were air-dried so that only broad conclusions about conformation are possible. Still, suggestions were obtained from this work that conformation might be probed. Many of the issues, in addition to conformation, that might contribute to the intensity of static SIMS spectra of adsorbed proteins are explored in this work.

(19)SyntheticPolypeptides;Bamford,C. H.,Elliot,A., Hanby, W. F., Eds., Academic Press: New York, 1956. (20) Liebl, V.; Bejsovcova, L. Origins Life Euol. Biosphere 1990,20, 269-277. (21)Hanabusa, K.; Tsutsumi, H.; Kurose, A.; Shirai, H.; Hayakawa, T.; Hojo, N. J.Polym. Sci., Sect. A: Polym. Chem. 1989,27,1665-1673. (22) Abeysekara, A. M.; Grimshaw, J. J.Chem. Soc., Chem. Commun. 1987,13,100C-1002. (23)Torii, M.; Sanui, K.; Ogata, N. Macromolecules 1990,23,27482752. (24)Kiniwa, H.; Doi, Y.; Nishikaji, T. Makromol. Chem. 1987,188, 1841-1850. (25)Kopecek, J. Biomaterials 1984,5 , 19-25. (26)Zunino, F.;Pratesi, G.; Micheloni, A. J. Controlled Release 1989, IO, 65-73. (27) Itsuno, S.; Sakakura, M.; Ito, K. J. Org. Chem. 1990,55, 60476049. (28) Spaltro, S. M.; Foster, N. J. Appl. Polym. Sci. 1990,41,12351249. (29)Dickinson, H. R.; Hiltner, A.; Gibbons, D. F.; Anderson, J. J. Biomed. Mater. Res. 1981,15, 577-589. (30)Miyama, T.; Mori, S.; Takeda, Y. U.S.Pat. 3,371,069, February 27,1968. (31)Carmona, P.; Molina, M.; Martinez, P.; Ben Altabef, A. A p p l . Spectrosc. 1991,45, 977-982. (32)Bomben, K. D.;Dev, S. B. Anal. Chem. 1988,60, 1393-1397. (33)Paynter, R. W.; Ratner, B. D. In Surface andInterfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum: New York, 1985; Vol. 2,pp 189-216. (34)Klapper, M. H. Biochem. Biophys.Res. Commun. 1977,78,10181024.

EXPERIMENTAL SECTION Homopolymers. Homopolymers of the 16 amino acids listed in Table I were obtained from Sigma Chemical Co. (St. Louis, MO). All of the polymers had average molecular masses in the range 5-20 000 amu. Table I includes the R group indicative of each amino acid and the commonly used three-letter and oneletter abbreviations. A majority of the polymers were dissolved in trifluoroacetic acid (TFA),although ethanol and acetonitrile were used to dissolve poly(L-tyrosine)and poly(L-tryptophan), respectively. Poly(L-serine),the hydrobromide salts of poly(Llysine) and poly@-ornithine),the hydrochloride salt of poly(Larginine), and sodium salts of poly@-glutamicacid) and poly(Laspartic acid) were dissolved in water. Solution concentrations were typically 1% (w/w). Films of polymers dissolved in nonaqueous solvents were made by centrifugal casting onto glass ~

~~~~~~~~

(35)For example: Massia, S. P.; Hubbell, J. A. J . Cell. Biol. 1991,114 (5),1089-1100. Massia, S. P.; Hubbell, J. A. Ann. N.Y. Acad. Sci. 1990, 589,261-270. Massia, S. P.; Hubbell, J. A. Anal. Biochem. 1990,187, 292-301. (36)Chilkoti, A.; Lopez, G. P.; Ratner, B. D.; Hearn, M. J.; Briggs, D. Macromolecules, in press. (37)Ratner, B. D.;Weathersby, P. K.; Hoffman, A. S.; Kelly, M. A.; Scharpen, L. H. J. Appl. Polym. Sci. 1978,22,643-664. (38)Paynter, R. W.; Ratner, B. D.; Horbett, T. A.; Thomas, H. R. J. Colloid Interface Sci. 1984,101, 233-245. (39)Lewis, K. B.;Ratner, B. D. J. Colloid Interface Sci., submitted for publication.

ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

Table 1. Summary of the Poly(amino acids) Used in This Study. amino acid abbrev R m/z of R glycine 1 Gly, G 15 alanine Ala, A Val, v valine 43 57 leucine Leu, L Ser, S serine 31 methionine Met, M 75 glutamic acid Glu, E 73 aspartic acid 59 ASP, D lysine 72 LYS,K ornithine Om, 0 58 arginine 100 Arg, R Pro, P proline 42

phenylalanine

Phe, F

91

tyrosine

Tyr, Y

107

tryptophan

Trp, W

130

histidine

His, H

81 H

The polymers have the general structure (-NH-CHR-CO-). Corresponding R groups, abbreviations,and the mlz of the R group are listed for each amino acid. (I

disks. A 20-pL volume was deposited on a 9- or 12-mmglass disk and spun at 4000 rpm for 20 s using a Metron Systems, Inc. (Allamuchy,NJ) LS-8OOO laboratory spinner. Films of the other polymers were made by vacuum drying of small volumes (20-40 pL) of solution on glass disks. All of these films were visible to the naked eye and had an estimated thickness of many microns. Bulk Film of Bovine Serum Albumin. Bovine serum albumin (Fraction V, "fatty acid free", ICN, Costa Mesa, CA) was dissolved at a concentration of 2 % (w/v)in deionized water, and a 20-40-pL droplet was spread on a 9-mm glass disk. The sample was allowed to dry in vacuum before SIMS analysis. This film was visible to the naked eye, and again a film thickness of many microns was estimated. Bovine Fibrinogen and Bovine Serum Albumin Adsorbed to Ti. Bovine fibrinogen and bovine serum albumin (ICN, Costa Mesa, CA) were adsorbed to 98.7% pure Ti foil, 0.025 mm thick (Alfa, Ward Hill, MA), using the following procedure. Onecentimeter-square pieces of Ti were ultrasonically cleaned in acetone, followed by methanol and 2-propanol. The Ti samples were then placed in 2-mLvials. Phosphate-buffered saline (PBS) (1 mL) was added to the vials to completely cover the Ti, and the samples were allowed to equilibrate at room temperature for 1h. A 1mg/mL solution of protein was prepared in PBS. Protein solution (1 mL) was added to the vials and mixed once by repipetting. Adsorption was allowed to proceed for 2 h. Rinsing proceeded via two steps. Each vial was rinsed with 40 mL of clean PBS, allowing the vial to overflow during rinsing. This process of displacement and dilution ensures that the sample is never exposed to the layer of denatured protein at the air-water interface. Subsequently, each vial was rinsed with 20 mL of deionized water, using the same dilution-displacement method. The water remaining in each vial was then removed using a clean Pasteur pipet connected to a vacuum aspirator. The samples were dried in vacuum overnight before SIMS analysis. Adsorbed Bovine Insulin and Chain Fragments. Bovine insulin (Sigma) was dissolved in a dilute NaOH solution. The ammonium salts of the oxidized chains that comprise insulin, chain A and chain B (Sigma), were each dissolved in deionized water. The substrate used for adsorption was a piece of polished Si that had been ultrasonically cleaned in acetone, followed by methanol and 2-propanol. The Si was covered with a sufficient

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oxide to provide a hydrophilic surface, as determined by its wettability. The procedure for adsorption was as describedabove for the plasma proteins on Ti. The insulin was adsorbed at 1.0 mg/mL, and chain A and chain B were adsorbed at 0.25 mg/mL due the small quantity of these peptides available. Adsorption was allowed to occur for 2 h at room temperature. The samples were each rinsed with 20 mL of deionizedwater using the dilution displacement method described above. Adsorbed Multichain Amino Acid Copolymer. A multichain copolymer of alanine and lysine was obtained from Sigma. This polymer consists of two large (MW >10 OOO) homopolymer chains linked together. The ratio of the alanine chain length to the lysine chain length was 14to 1. The copolymer was dissolved in deionized water. A number of different substrates were used for the adsorption experiments. Clean Si and Ti, prepared as described above, were used as hydrophilic surfaces. A clean, smooth polyethylene (PE) film was prepared by hydraulic pressing. The sheet was pressed at -100 "C, for 3 min, under 3 metric tons of force. The final film thickness was -0.5 mm. Poly(tetrafluoroethy1ene) (PTFE) (Berghof/America,Concord, CA) substrates were ultrasonically cleaned in acetone, followed by methanol and 2-propanol. The copolymer was adsorbed at a concentration of 0.5 mg/mL for 2 h at room temperature. The samples were each rinsed with 20 mL of deionized water using the dilution-displacement method described above. A bulk film of the copolymer was produced by spreading a 20-pL drop of solution on a 12-mm glass disk and drying in vacuum. Fouled Screen Membrane. Polycarbonate screen material (Poretics Corp., Livermore, CA) was used as received. The membrane was 37 mm in diameter and 6 pm thick, and was tracketched with 300-A pores to a density of 6 X 108pores/cm2. Poly(N-vinylpyrrolidone)was added to the membrane material as a wetting agent. The membrane was immersed in whole milk for 2 h, followed by rinsing in deionized water. The membrane was dried under vacuum prior to SIMS analysis. A bulk film of whole milk was produced by spreading a drop of milk on a 12-mm glass disk and drying under vacuum. Static SIMS Analyses. Static SIMS analyses of the 16 homopolymersand the bulk film of bovine serum albumin were performed on a Perkin-Elmer Model 3700 system with a 1-nA, 4-keV Xe+ ion beam rastered over a 4 X 4 mm2area, at an angle of 50" to the sample plane. In this instrument, the sample is held normal to the entrance lens to the mass analyzer. The mass analyzer consists of a 90" electrostatic analyzer for energy filtration and a quadrupole spectrometer with a mass range of 0-1024 amu. A total acquisition time of -4 min or less was required for each sample to ensure static conditions for all spectra.40 Charge neutralization was achieved with a low-energy (0-30-eV) electron gun that was impinged at an angle of -5" from the sample plane. Static SIMS analyses of the adsorbed proteins and peptides, and the fouled membrane were performed using a 3.5-keV, 0.5nA Xe+primary beam, impinged at a 45" angle from the sample plane from a differentially pumped Leybold-Heraeus ion gun. The beam size and raster were adjusted to cover a 5 X 5 mm2 area. Secondary ions were detected with a modified QMG 511 Balzers quadrupole massspectrometer with an adjustable energy filter. The entrance lens was normal to the sample plane. Again, the total acquisition time was less than or equal to 4 min. When charge neutralization was necessary, an electron gun (0-200 eV) impinged at an angle to 45O from the sample plane was used.

RESULTS AND DISCUSSION Homopolymers. Overview. Insight intothe interpretation of the static SIMS spectra of the poly(amino acids) can be obtained by studying characteristic ions and mechanistic pathways in the general mass spectrometry literature. For positive ion spectra, particularly applicable work includes that of Arpino and McLafferty41 on oligopeptide mass (40) Heyn, M. J.; Briggs, D. Surf. Interface Anal. 1986,9, 411. (41) Arpino, P.. J.; McLafferty, F. In Determination of Organic

Structure byPhysicalMethods;Nachod,F.C., Zuckerman, J. J.,Randall, E. W., Eds.; Academic Press: New York, 1976; Vol. 6, Chapter 1.

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

Table 11. Summary of Major Positive Ions in the SIMS Spectra of Homopolymers of 16 Amino Acid@

AA G~Y Ala Val Leu Pro Phe TYr TrP His Ser Met LYS Arg Orn ASP Glu

other

I

I-Hz

30 (100) 44 (100) 72 (100) 86 (100) 70 (100) 120 (43) 136 (40)

28 (8) 42 (12) 70 (6) 84 (10) 68 (24) 118 (6) 134 (8)

110 (91) 60 (78) 104 (18)

108 (9) 58 (24) 102 (11)

129 (3)

127 (4)

88 (80) 102 (15)

86 (12)

44 (32)

30 (33)

91 (100) 107 (100) 130 (56) 82 (62) 44 (100) 91 (23) 84 (18) 128 (4) 70 (100) 72 (100) 84 (64)

77 (36) 91 (83) 77 (100) 81 (100) 30 (46) 61 (100) 68 (3) 97 (25) 44 (17) 62 (69) 56 (100)

58 (47) 77 (72) 44 (62) 44 (8)

56 (26) 44 (36) 30 (46) 30 (15)

44 (35) 30 (30)

42 (50)

30 (28)

56 (53) 56 (24) 73 (32) 30 (95) 44 (19) 44 (86)

44 (16) 44 (9) 70 (100)

42 (17) 30 (100) 60 (25)

30 (15) 44 (48)

30 (53)

30 (18) 30 (69)

Relative intensities are given in parentheses. Peaks indicative of hydrocarbons, residual solvent, and sodium have been excluded from this summary. I = immonium ion.

Table 111. Summary of Major Negative Ion Peaks in the SIMS Spectra of Homopolymers of the Alkyl Amino Acid@

AA G~Y Ala Val Leu a

Ri-CRHC(NH)OR1= NHCOH R1= NH2 101 (4) 115 (6) 143 (4) 157 (16)

73 (17) 87 (22) 115 (22) 129 (31)

R1= H

OCN-

CN-

58 (8)

42 (100) 42 (100) 42 (100) 42 (100)

26 (56) 26 (64) 26 (77) 26 (65)

72 (5) 100 (6) 114 (6)

other 99 (26) 98 (13) 155 (18) 169 (10)

141 (17) 155 (30)

116 (39) 140 (8)

Relative intensities are given in parentheses. Peaks from residual solvent have been excluded from this summary.

spectrometry and that of Heerma and K ~ l i k 4on ~ the identificationof amino acids in spectra obtained from peptides in a liquid matrix using fast atom bombardment mass spectrometry (FABMS). There is less information available on the negative ion mass spectrometry of proteins; however, a number of recent studies using free amino acids have proved usefu1.43-45 It should be noted that the following discussions focus on the ultimate application of the interpretation protocol to the static SIMS of proteins with molecular masses of 100 OOO amu. The goal of such investigationswould not be desorption of intact molecules or sequence determination but rather detection and study of proteins on surfaces. These applications would depend on our ability to correlate mass spectral features with chemical features unique to proteins, such as amino acid content. In general, the static SIMS spectra of the homopolymers contain significant peaks extending near mlz = 300. As detailed below, many of the high-mass peaks arise from ions consisting of multiple repeat units from the polymers. Such peaks are of interest in terms of the spectrometry of the homopolymers but will not be analyticallyuseful for the study of proteins. We have focused our attention on identifying those peaks that arise from single amino acid residues. Tables 11-IV are summaries of the peak information for both the positive and negative ion spectra of the amino acid homopolymers. These summaries include only those ions that are believed to contain single amino acid residue information. In addition, the summaries attempt to identify those instances wherevarious amino acid peaks will interfere with each other at unit mass resolution. Peaks indicative of hydrocarbon

-

(42) Heerma, W.; Kulik, W. Biorned.Enuiron. Mass Spectrorn. 1988, 16, 155-159. (43) Voigt, D.; Schmidt, J. Biomed.Enuiron. Mass Spectrorn. 1978,5, 44-46. (44)Kulik, W.; Heerma, W. Biomed.Enuiron. Mass Spectrom. 1988, 15,414-427. (45) Eckersley, M.; Bowie, J. H.; Hayes, R. N. Int. J.Muss Spectrorn. Ion. Processes 1989, 93, 199-213.

contamination and inorganic ions (Na+,Cl-,etc.) have been excluded. The positive ion spectra of many of the homopolymers are dominated by the immonium ion (I). This ion has the generic structure H&=CH-R

The R group distinguishes the particular amino acid, and the immonium ion peak will be found at mlz = [R + 291. This is by no means the only important positive ion in the spectra of the homopolymers, and in fact the immonium ion is not observed in a number of instances. Table I1 contains a summary of the important positive ions, including the immonium ion, for all 16homopolymersstudied. The negative ion spectra of the homopolymers are not as interpretable and are far less intense than the correspondingpositive ion spectra. In all of the homopolymers, the CN- and OCN- ions are observed in the spectra with significant intensity. In the case of the alkyl amino acids, a distinct and interpretable pattern of peaks is observed, and these are summarized in Table 111. The spectra of the other amino acid homopolymers do not exhibit an easily interpretable pattern. Table IV contains those peaks unique to some of the non-alkyl amino acid homopolymer spectra. Unfortunately,little or no information could be derived from the negative ion spectra of the acidic and basic amino acids because these spectra were dominated by intense inorganic anions. A detailed description of the positive and negative ion spectra resulting from the various classes of amino acids follows. Gly,Ala, Leu, and Val. Homopolymers of these four alkyl amino acids exhibit static SIMS spectra with a rich variety of peaks. These peaks are also relatively straightforward to intepret. All of the spectra exhibit intense immonium ions. A number of higher mass peaks are observed that can be explained as belonging to two series arising from linear ions cleaved from the polymer chain and cyclic ions formed by

ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

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Table IV. Summary of Major Negative Ion Peaks in the SIMS Spectra of Homopolymers of Some Amino Acids. other AA OCNCNPro Phe Tyr

TrP His Ser Met

42 (100) 42 (61) 42 (97) 42 (95) 42 (30) 42 (100) 42 (100)

26 (98) 26 (90) 26 (83) 26 (78) 26 (100) 26 (60) 26 (46)

165 (10) 91 (10) 134 (26) 189 (19)

139 (11) 77 (100) 119 (37) 162 (20)

137 (7) 76 (58) 99 (18) 144 (43)

68 (6) 60 (91) 60 (16) 134 (27)

99 (32) 47 (87)

84 (16) 33 (22)

59 (48) 32 (27)

58 (10)

66 (18) 25 (24) 25 (92) 108 (100)

62 (13) 16 (100) 80 (11)

Relative intensities are given in parentheses. Peaks from residual solvent and inorganic anions such as C1- and HS04- have been excluded from this summary. rearrangement. The mechanisms leading to both of these series are not uncommon in peptide mass spectrometry.41 Ions belonging to the linear series have the general structure outlined below:

The immonium ion is the first member of this series at rnlz = [R + 293. Peaks at two mass unita below this series are due to the loss of neutral Hz. The loss of RH is also indicated. It is probable that the cyclic series includesthe six-membered diketopiperazine ion and ita smaller analogdl

83

111

124

2R

2R

3R

+

+

+

As in the positive ion spectra of the alkyl amino acids, the negative ion spectra exhibit two series of peaks, most likely arising from linear and cyclic ions. Neutral losses of H2 and RH are also indicated. The linear series most likely has the general structure

I k k

Pro. Poly(L-proline)is actually a poly(imino acid), but the static SIMS spectra of this polymer are easily interpreted in the same manner as the alkyl poly(amino acids). The positive ion spectrum of poly(L-proline) contains an intense peak at rnlz = 70, with a cyclic structure analogous to the immonium ion. This is indicative of proline in peptide mass spectrometry.40841Other ions at higher masses are analogous to the linear and cyclic series described for the alkyl poly(amino acids). However, the relative intensity of the peak at rnlz = 70 is far greater in poly(L-proline). The negative ion spectrum of poly@-proline) contains no particularly intense peaks. There is, however, a distinctive peak at rnlz = -66. A logical formula for this ion is C4H4N-, and a possible structure is

There are other structures that are also valid, and it is not entirely obvious whether the negative charge resides on a carbon or the nitrogen in this ion. Phe, Tyr, Trp, and His. The polymers of these amino acids are grouped together due to the aromatic nature of their R groups, as shown in Table I. Histidine is usually grouped with other amino acids with basic side chains,but ita inclusion here is due to a mass spectral similarity rather than a biochemical one. The positive ion static SIMS spectrum of most of the polymers of the aromatic amino acids contains an immonium ion and ions indicative of the R group. There is little evidence of peaks representative of ions with more than one R group such as is seen with the alkyl amino acids. The positive ion spectrum of poly(L-tryptophan) does not exhibit an immonium ion at rnlz = 159 as is seen in FAB MS data.40 A fragment ion indicative of the R group is observed at rnlz = 130, but no fragment at mlz = 117 is observed. Again, this is contrary to FAB MS resulta.40 The negative ion spectra of the aromatic poly(amin0acids) do not exhibit particularly intense peaks. Poly(pheny1alanine) does exhibit a peak at rnlz = -77, and the negative ion spectrum of poly(L-tyrosine) contains a peak at mlz = -119 with the probable structure below:

[lW+R]'

[113+2RI.

The cyclic series most likely has a general structure similar to that for the positive cyclic ion series, but with the negative charge residing on the oxygen in the peptide link. It appears that the cyclic negative ions have a greater propensity to lose neutral H2and RH. It should be noted that there is no single intense ion that dominates the spectra, as the immonium ion does in the positive ion spectrum. However, there are negative ions that contain only a single amino acid fragment and are therefore potential analytical indicators.

This ion is also observed in the negative ion mass spectrometry of tyrosine from the free amino a~id.~33" Met and Ser. Methionine and serine are similar in that their side chains contain a heteroatom. The spectra of poly(L-serine) and poly@-methionine)resemble one another in that no cyclic or extended linear chain ions are observed. Both polymers exhibit a strong immonium ion. For poly(L-methionine),a peak at rnlz = 61arises from a fragment ion

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ANALYTICAL CHEMISTRY, VOL. 85, NO. 10, MAY 15, 1993

from the R group, with the likely structure [CH2=SCH31+. This ion is not observed in the FAB MS of methioninecontaining peptides.42 The peak at rnlz = 91 is most likely due to an aromatic contaminant. The negative ion spectra of poly@-serine) and poly@methionine) exhibit single, strong peaks indicative of the R group. A peak at rnlz = -59, from an ion with the likely formulaC3H302-,is observed in the spectrum of poly(L-serine). The negative ion spectrum of poly(L-methionine) contains a strong peak at rnlz = -47 from the CHB- ion. Clu and Asp. In the positive ion SIMS spectrum of poly(L-glutamic acid), the immonium ion is observable as a weak peak at rnlz = 102. The peak at mlz = 84 is due to the loss of neutral HzO from the immonium ion, giving an ion with the formula C4H6NO+ and a possible cyclic structure. Poly(L-asparticacid) exhibits only an immonium ion a t rnlz = 88. It may be that the shorter chain of the R group lessens the likelikhood of rearrangement, leading to loss of H2O. The negative ion spectra of poly@-asparticacid) and poly(L-glutamicacid) exhibit no strong and distinctive peaks that can be easily identified. The spectra tend to be dominated by inorganic anions. Lye, O m , and Arg. The homopolymers of the amino acids with basic side chains exhibit positive ion spectra without obvious immonium ions. Peaks from the immonium ions of lysine, ornithine, or arginine are either weak or not observed in the FAB MS of peptides containing these amino The only distinctive peak in the positive ion spectrum of poly@-lysine) is at rnlz = 84. This peak is observed in the mass spectrometry of lysine-containing peptide~,4l,~~ and the ion is believed to have the structure

E+

The only major peaks in the positive ion spectrum of poly(L-ornithine) are a t mlz = 70 and 30. This ion most likely has a five-membered ring structure and is analogous to the ion a t rnlz = 84 in the spectrum of poly(L4ysine). The positive ion spectrum of poly(L-arginine) contains peaks at rnlz = 112,97, and 70. The peak at mlz = 112 arises from the ion formed by loss of NH3 from the immonium This ion may have a cyclic structure analogous to the rnlz = 84 ion in the spectrum of poly(L-lysine). The peaks a t rnlz = 70 and 97 most likely represent ions from further fragmentation of the immonium ion. However, no ion is observed at rnlz = 97 for arginine in peptide FAB MS.42 Instead, a peak at rnlz = 87, with no obvious structural assignment, is observed. As stated earlier, no useful information could be gleaned from the negative ion mass spectra of poly(L-arginine),poly(L-ornithine), and poly(L-lysine). The polymers were examined in the form of HBr and HC1 salts, and the isotopic patterns of Br, and C1, dominate the spectra. Summary. The list of peaks in Tables 11-IV represent a spectral interpretation protocol for proteins based on the static SIMS investigation of the 16 poly(aminoacids). Many of the ions, and the immonium ions in particular, appear at masses that are not particularly common in the static SIMS spectra of polymer^.^^,^^ This fact is partially due to the presence of the nitrogen atom in many of these ions, which leads to peaks at even values of mlz. The summaries also highlight the limitations of quadrupole mass spectrometers with regard to (46) Briggs, D.; Brown, A.; Vickerman, J. C. Handbook of Static Secondarylon MassSpectrometry;John Wiley& Sons: Chichester, U.K., 1989. (47) Newman, J. G.; Carlson, B. A,; Michael, R. S.; Moulder, J. F.; Holt, T.A. Static SIMS Handbook of Polymer Analysis; Perkin-Elmer: Eden Prairie, MN, 1991.

50

70

go m,z

iio

130

150

Figure 1. Positive ion static SIMS spectrum of a bulk film of bovine serum albumin. Peaks indicative of soma of the amino acids are labeled with onaletter abbreviatlons (see Table I).

resolution. The ultimate need for high-resolution data is evidenced by those peaks that could be due to more than one ion. A mass resolving power of 5000 MIAM would be more than adequate to resolve these peaks, and this resolution is easily achieved with many contemporary mass analyzers. It should be noted that the ions at rnlz = 70 from proline and ornithine are identical in structure and therefore not resolvable. The 16 homopolymers included in the study were the only commercially available polymers of the amino acids. While it might be possible to predict the spectral features arising from the other amino acids based on results from other forms of mass spectrometry, we will restrict our protocol to those amino acids actually analyzed. Bulk Film of Bovine Serum Albumin. Figure 1shows the positive ion SIMS spectrum of a thick film of bovine serum albumin, from rnlz = 50 to 150. Peaks have been identified using the protocol developed above and have been labeled with the one-letter abbreviation of the amino acid that most likely produced the peak. The one-letter abbreviations of the amino acids used are listed in Table I. Only those amino acids included in this study have been marked. Below mlz = 50 the great number of intense peaks precludes confident assignment of the glycine and alanine immonium ions, although there are peaks present at rnlz = 30 and 44. Methionine is not assigned to the peaks at rnlz = 61 and 104. The relative intensity of these weak peaks does not agree well with the intensities listed in Table 11. There is only a single tryptophan residue in the protein, and no peaks indicative of this amino acid are dete~table.~S Ornithine is not present in the protein and therefore is not included in the assignm e n t ~ .It~is~ readily apparent that many more peaks are present in the spectrum than can be easily accounted for by the amino acid fragments discussed here. Many of the odd mass peaks, which are usually associated with hydrocarbon ions, may be due to either contamination or long chain fatty acids that often aggregate with albumin. While the albumin used here is labeled fatty acid free, any residue of fatty acid would be highly surface active and concentrate at the proteinvacuum interface. Other peaks may originate from various biomolecules found in small quantities in the protein. The relative intensities of the peaks from the various amino acids do not directly correlate with the relative abundance of the amino acid residues in the protein.48 The possible origins of the static SIMS intensities for the amino acid peaks are discussed below. Unfortunately, the negative ion spectrum of bovine serum albumin is of low intensity. The average count rate is approximately 2 orders of magnitude lower than that for the (48) Paul, L.; Sharma,C. P. J. ColloidInterface Sci. 1981,84,546-549.

ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

1497

c

0

100

150

Flgwr 2. Poskive ion static SIMS spectrum of bovine serum albumin adsorbed to 1. Peaks lndlcethre of some of the amino acids are labeled with oneletter abbreviations (see Table I). The peak at mlz = 48 from 1 is also labeled.

- I

0

'0

m/z

100

150

Flgurr 3. Positive ion statlc SIMS spectrum of bovine flbrinogen adsorbed to TI. Peaks Indicative of some of the amino acids are labeled with oneletter abbreviations (see Table I).

positive ion spectrum (data not shown). Peaks indicative of alanine and leucine were discernible from the noise. Bovine Serum Albumin and Bovine Fibrinogen Adsorbed to Ti. These adsorbed plasma proteins were investigated to explore the usefulness of static SIMS to provide fundamental information from such systems. Figures 2 and 3 show the positive ion static SIMS spectra of adsorbed albumin and fibrinogen,respectively. Major peaks indicative of various amino acid residues in the proteins are marked accordingly. There are three main differences between the spectra, which may arise from physical and chemical differences in the two systems. A peak from the major isotope of titanium is observed at mlz = 48 for the adsorbed albumin, but not in the case of the adsorbed fibrinogen. This indicates that the overlayer of adsorbed albumin is either discontinuous or thinner than the sampling depth of static SIMS. The latter possibility is rather unlikely, and therefore a structural difference between the two adsorbed protein layers is indicated. The adsorbed fibrinogen is both dense enough and continuous enough over the -1-cm2 area sampled to completely shield the substrate. The relative intensity of the amino acid peaks versus the odd mass peaks in the lower portion of the spectrum is also different for the two adsorbed protein systems. The odd mass peaks (e.g., mlz = 27,29,31, 41, etc.) arise from hydrocarbon ions and may be indicative of lipid material. If this were the case, the fact that the hydrocarbon ions for the adsorbed albumin system are more intense may be due to coadsorbed lipids or lipids that aggregate with the protein. Lipid aggregation is relatively common with albumin. The increased hydrocarbon signal may also be due to contamination on the exposed Ti surfaces. The third difference between the adsorbed albumin and fibrinogenspectra is the relative intensitiesof the peaks arising

Flgwr 4. Positive ion static SIMS spectrum of bovlne insulin adsorbed to SI. Peaks indicative of some of the amlno acids are labeled with oneletter abbreviations (seeTable I).

from the amino acids. The source of these differences may be the distinct amino acid composition of the two proteins. However, the possibility that other factors influence the intensities of these peaks is explored in further detail in the following sections. Adsorbed Bovine Insulin and Chain Fragments. To study the relationship between the intensities of the peaks arising from the amino acids and the actual number of amino acids present in the protein, insulin and the two peptide chains that comprise insulin were examined. Insulin was chosen because it is a small protein with a well-known amino acid composition. Figure 4 shows a portion of the positive ion SIMS spectrum of insulin adsorbed to Si. The adsorbed layer was incomplete and a number of substrate peaks were observed. Regardless of the coverage, the relative intensities of the amino acid ions do not correspond to the known composition of the protein. In fact, the peaks indicative of tyrosine and arginine, which do exist in the protein, could arguably be dismissed as noise. The intensities of the spectra from the two constituent chains of insulin, chain A and chain B, also do not reflect their compositions (data not shown). The lack of quantitative agreement between the static SIMS intensities and the known chemical composition of the peptides may be due to a number of factors. The stability of ions is a major factor affecting peak intensities. Cyclic ions, ions with aromaticity, and ions with electron-withdrawing groups or heteroatoms, all exhibit relative intensities greater than what would be expected from simple stoichiometry. In addition, the fact that many of the amino acids exhibit multiple SIMS peaks that interfere with one another contributes to the lack of quantitation. The possibility that the geometry of the adsorption process can contribute to the relative intensity of peaks is discussed below. Adsorbed Multichain Amino Acid Copolymer. The adsorption of a multichain amino acid copolymer was studied to determine if static SIMS is sensitive to the way in which a peptide macromoleculeadsorbsto a surface. The copolymer consists of two long-chain homopolymers linked together. There are an average of 100 residues in an entire chain. The poly(alanine) portion of the chain is relatively hydrophobic, while the poly(1ysine)portion of the chain is hydrophilic. A spectrum of a bulk film of the copolymer exhibits strong peaks at mlz = 44 and 84, indicative of alanine and lysine, respectively (data not shown). The ratio of the peak intensities in the bulk film is 25 k 5. The ratio of the intensities of the mlz = 44-84 peaks for the copolymer adsorbed to various substrates is given in Table V. On hydrophobic surfaces, it would be expected that the poly(alanine) portion of the polymer would preferentially adhere to the surface, while the poly(1ysine) chain would partially shield the molecule. Conversely,on hydrophilic surfaces,the

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

Table V. Relative Intensities of the Positive Secondary Ion Peaks from Alanine ( m / z = 44) and Lysine ( m / z = 84) from a Multichain Copolymer of Alanine and Lysine, Adsorbed to a Variety of Substratesa substrate

type of surface

polyethylene poly(tetrafluoroethy1ene) titanium silicon (oxidized)

hydrophobic hydrophobic hydrophilic hydrophilic

m/z = 44/84 14*2 35f4 46 5 55 10

* *

a The relative ratios of the background peaks a t mlz = 44 and 84 from clean substrates ranged from 1.1 to 11. The ratio of mlz = 44/84for a bulk sample of the copolymer was 25 5. See text for details.

*

1.2

P, R

0

100

150

Flgure 5. Positive ion static SIMS spectrum of a polycarbonate membrane exposed to whole mllk. Peaks indicathre of some of the amino acMs are labeled with onaietter abbreviations (see Table I).

poly(1ysine)portion would adhere and the poly(alanine)chain would partially shield the molecule. The data in Table V support this hypothesis. On PE and PTFE, the relative signal from poly(alanine) is reduced, whereas it is enhanced on the Si and Ti surfaces. It should be noted that spectra of the clean substrates contain only small background peaks at these two values of mlz, with relative intensities ranging from 1.1 to 11. These results support the premise that the intensities of peaks in a static SIMS spectrum may be sensitive to the conformation of a macromolecule on a surface. Fouled Screen Membrane. Track-etched polycarbonate screen material is a common component of a number of commercial chemical sensors. Fouling of such materials is of great concern in a wide variety of applications. For example, the fouling of surfaces in the processing of milk can dramatically affect product quality.49 To demonstrate the potential utility of static SIMS for the study of biofouled materials, polycarbonatemembrane material exposed to whole milk was investigated. The static SIMS spectrum of the clean polycarbonate membrane exhibits peaks indicative of polycarbonate (mlz = 77, 91, 135) and the poly(N-vinylpyrrolidone) wetting agent (mlz = 41,69). Figure 5 showsthe positive ion spectrum of the polycarbonate membrane after exposure to whole milk. Major peaks indicative of amino acids are marked accordingly. Whole milk contains approximately 3.3% protein that is mostly casein.50 This is roughly the same concentration as lipids in milk. In the static SIMS (49)Lalande, M.;Rene, F. Fouling Sei. Tech. 1988, 557-573. (50)Newer Knowledge of Milk; National Dairy Council: Rosemont,

PA,1988.

spectra of bulk films of whole milk, peaks indicative of hydrocarbon ions are of greater intensity than those from the amino acids (data not shown). In the spectrum in Figure 5, the opposite is true. This would indicate that protein fouling is occurring, rather than fouling by other biomolecules.

CONCLUSIONS The goals of this work were to develop a strategy for the qualitative interpretation of static SIMS spectra of adsorbed proteins, to investigate the multiple factors responsible for the intensity of peaks in the spectra, and to demonstrate the potential utility of static SIMS for studying adsorbed proteins and polypeptides. The protocol for spectral interpretation described here, based upon experiments on homopolymers and known formation routes of ions in protein mass spectrometry, should be generally useful in static SIMS protein interpretation. In this paper, the protocol is successfully applied to static SIMS spectra of a variety of proteins and peptides. Some of the factors that affect the relative intensity of the peaks in the spectra of adsorbed proteins have been considered. They include the amino acid content of the protein, the relative stability of fragment ions, the extent and nature of the surface coverage, and possibly the conformation of the adsorbed protein. Conformationalsensitivity may be an especially useful tool, since it is difficult to get this information on adsorbed proteins by other means. To confidently explore protein conformational effects, further studies with model systems and systems of increasing complexity will be required. Low-temperature studies on protein films freeze-dried in situ may further enhance the interpretabilityof static SIMSstudies aimed at understanding adsorbed protein conformation.37-39 Static SIMS protocols for protein spectral interpretation may also be useful for studies on biofouling, immunoassays, and protein separation supports. While it is premature to predict the ability to identify specific proteins, the increased selectivity and sensitivity of static SIMS compared to current surface analysis spectroscopies (Le., FTIR, XPS)make this a method worthy of exploration, especially when coupled with contemporary pattern recognition algorithms. Enhanced instrumentation, including time-of-flight (TOF) SIMS, ion cyclotron resonance mass analyzers, desorption methods capable of producing larger fragment ions, postionization methods for increasing analytical sensitivity,and tandem mass spectrometry methods permitting more accurate identification of ion structures, should further advance the utility of static SIMS for the study of adsorbed proteins.

ACKNOWLEDGMENT This work was generously supported by Grant R01296 from the National Center for Research Resources. D.S.M. gratefully acknowledges the support of the Leopold Schepp Foundation. Graham Leggett is gratefully acknowledged for many fruitful discussions and his considerable insight into the interpretation of the static SIMS spectra. We also wish to thank Erika Johnston for providing the fouled polycarbonate membrane. RECEIVEDfor review December 21, 1992.

January

8, 1992.

Accepted