Photodissociation and Infrared Spectroscopy of Uranium-Nitrogen Cation Complexes.

Laser vaporization of uranium in a pulsed supersonic expansion of nitrogen is used to produce complexes of the form U+(N2)n (n = 1-8). These ions are mass selected in a reflectron time-of-flight spectrometer and studied with visible and UV laser fixed-frequency photodissociation and with tunable infrared laser photodissociation spectroscopy. The dissociation patterns and spectroscopy of U+(N2)n indicate that N2 ligands are intact molecules and that there is no insertion chemistry resulting in UN+ or NUN+. Fixed frequency photodissociation at 532 and 355 nm indicate that the U+-N2 bond dissociation energy varies little with changing coordination. The photon energy and the number of ligands eliminated allow an estimate of the average U+-N2 dissociation energy of 12 kcal/mol. Infrared bands are observed for these complexes near the N-N stretch vibration via elimination of N2 molecules. These resonances are observed to be shifted about 130 cm-1 to the red from the free-N2 frequency for complexes with n = 3-8. Density functional theory indicates that U+ is most stable in the sextet state in these complexes and that N2 molecules bind in end-on configurations. The fully coordinated complex is predicted to be U+(N2)8, which has a cubic structure. The vibrational frequencies predicted by theory are consistently lower than those in the experiment, independent of the isomeric structure or spin state of the complexes. Despite its failure to reproduce the infrared spectra, theory provides an average U+-N2 dissociation energy of 11.8 ± 0.5 kcal/mol, in good agreement with the value from the experiments.

Vibrational spectroscopy of anionic nitrate complexes of UO22+ and Eu3+ isolated in the gas phase.

Wavelength-selective infrared multiple photon photo-dissociation (IRMPD) was used to generate spectra of anionic nitrate complexes of UO(2)(2+) and Eu(3+) in the mid-infrared region. Similar spectral patterns were observed for both species, including splitting of the antisymmetric O-N-O stretch into high and low frequency components with the magnitude of the splitting consistent with attachment of nitrate to a strong Lewis acid center. The frequencies measured for [UO(2)(NO(3))(3)](-) were within a few cm(-1) of those measured in the condensed phase, the best agreement yet achieved for a comparison of IRMPD with condensed phase absorption spectra. In addition, experimentally-determined values were in good general agreement with those predicted by DFT calculations, especially for the antisymmetric UO(2) stretch. The spectrum from the [UO(2)(NO(3))(3)](-) was compared with that of [Eu(NO(3))(4)](-), which showed that nitrate was bound more strongly to the Eu(3+) metal center, consistent with its higher charge. The spectrum of a unique uranyl-oxo species having an elemental composition [UO(9)N(2)](-) was also acquired, that contained nitrate absorptions suggestive of a [UO(2)(NO(3))(2)(O)](-) structure; the spectrum lacked bands indicative of nitrite and superoxide that would be indicative of an alternative [UO(2)(NO(3))(NO(2))(O(2))](-) structure.

Mid-infrared vibrational spectra of discrete acetone-ligated cerium hydroxide cations.

Cerium(iii) hydroxy reactive sites are responsible for several important heterogeneous catalysis processes, and understanding the reaction chemistry of substrate molecules like CO, H(2)O, and CH(3)OH as they occur in heterogeneous media is a challenging task. We report here the first infrared spectra of model gas-phase cerium complexes and use the results as a benchmark to assist evaluation of the accuracy of ab initio calculations. Complexes containing [CeOH](2+) ligated by three- and four-acetone molecules were generated by electrospray ionization and characterized using wavelength-selective infrared multiple photon dissociation (IRMPD). The C[double bond, length as m-dash]O stretching frequency for the [CeOH(acetone)(4)](2+) species appeared at 1650 cm(-1) and was red-shifted by 90 cm(-1) compared to unligated acetone. The magnitude of this shift for the carbonyl frequency was even greater for the [CeOH(acetone)(3)](2+) complex: the IRMPD peak consisted of two dissociation channels, an initial elimination of acetone at 1635 cm(-1), and elimination of acetone concurrent with a charge separation producing [CeO(acetone)](+) at 1599 cm(-1), with the overall frequency centered at 1616 cm(-1). The increasing red shift observed as the number of acetone ligands decreases from four to three is consistent with transfer of more electron density per ligand in the less coordinated complexes. The lower frequency measured for the elimination/charge separation process is likely due to a combination of: (a) anharmonicity resulting from population of higher vibrational states, and (b) absorption by the initially formed photofragment [CeOH(acetone)(2)](2+). The C-C stretching frequency in the complexes is also influenced by coordination to the metal: it is blue-shifted compared to bare acetone, indicating a slight strengthening of the C-C bond in the complex, with the intensity of the absorption decreasing with decreasing ligation. Density functional theory (DFT) calculations using three different functionals (VWN, B3LYP, and PBE0) were used to predict the infrared spectra of the complexes. Calculated frequencies for the carbonyl stretch are within 40 cm(-1) of the IRMPD of the three-acetone complex measured using the single acetone loss, and within 60 cm(-1) of the measurement for the four-acetone complexes. The B3LYP functionals provided the best agreement with the measured spectra, with the VWN modestly lower and PBE0 modestly higher. The C-C stretching frequencies calculated using B3LYP are higher in energy than the measured values by approximately 30 cm(-1), and reproduce the observed trend which shows that the C-C stretching frequency decreases with increasing ligation. Agreement between C-C frequency and calculation was not as good using the VWN functional, but still within 70 cm(-1). The results provide an evaluation of changes in the acceptor properties of the metal center as ligands are added, and of the utility of DFT for modeling f-block coordination complexes.

Influence of "alternative" C-terminal amino acids on the formation of [b3 + 17 + Cat]+ products from metal cationized synthetic tetrapeptides.

The aim of this study was to investigate the dissociation patterns, and in particular the relative abundance of [b(3) + 17 + Cat](+), for peptides with C-termini designed to allow transfer of the -OH required to generate the product ion, but not necessarily as the most favored pathway. Working with the hypothesis that formation of a five-membered ring intermediate, including intramolecular nucleophilic attack by a carbonyl oxygen atom, is an important mechanistic step, several model peptides with general sequence AcFGGX were synthesized, metal cationized by electrospray ionization and subjected to collision-induced dissociation (CID). The amino acid at position X was one that either required a larger ring intermediate (beta-alanine, gamma-aminobutyric acid and epsilon-amino-n-caproic acid to generate six-, seven- or nine- membered rings, respectively) to transfer -OH, lacked a structural element required for nucleophilic attack (aminoethanol) or prohibited cyclization because of the inclusion of a rigid ring (p- and m-aminobenzoic acid). For Ag(+), Li(+) and Na(+) cationized peptides, our results show that amino acids requiring the adoption of larger ring intermediates suppressed the formation of [b(3) + 17 + Cat](+), while amino acids that prohibit cyclization eliminated the reaction pathway completely. Formation of [b(3) - 1 + Cat](+) from the alkali metal cationized versions was not a favorable process upon suppression or elimination of the [b(3) + 17 + Cat](+) pathway: the loss of H(2)O to form [M - H(2)O + Cat](+) was instead the dominant dissociation reaction observed. Multiple-stage dissociation experiments suggest that [M - H(2)O + Cat](+) is not [b(4) - 1 + Cat](+) arising from the loss of H(2)O from the C-terminus, but may instead be a species that forms via a mechanism involving the elimination of an oxygen atom from an amide group.

Gas-phase investigation of Pd(II)-alanine complexes with small native and derivatized peptides containing histidine.

We report here the generation of gas-phase complexes containing Pd(II), a ligand (deprotonated alanine, A-), and/or N-terminus derivatized peptides containing histidine as one of the amino acids. The species were produced by electrospray ionization, and their gas-phase reactions were investigated using ion-trap tandem mass spectrometry. Pd(II) forms a stable diaqua complex in the gas phase of the formula, [Pd(A-) (H(2)O)(2)]+, (where A- = deprotonated alanine) along with ternary complexes containing A- and peptide. The collision-induced dissociation (CID) patterns of the binary and ternary complexes were investigated, and the dissociation patterns for the ternary complexes suggest that: (a) the imidazole ring of the histidine side group may be the intrinsic binding site of the metal ion, and (b) the peptides fragment primarily by cleavage of the amide bond to the C-terminal side of the histidine residues. These observations are in accord with previous solution-state studies in which Pd(II) was shown to cause hydrolysis of an amide bond of a peptide at the same position.

McLafferty-type rearrangement in the collision-induced dissociation of Li+, Na+ and Ag+ cationized esters of N-acetylated peptides.

In this study we investigated the multi-stage collision-induced dissociation (CID) of N-terminally acetylated di-, tri- and tetrapeptides in the form of C-terminal ethyl, n-propyl, isopropyl, n-butyl and tert-butyl esters and cationized by the attachment of Li(+), Na(+) and Ag(+). While methyl ester versions of the metal cationized peptides primarily eliminate H(2)O following collisional activation and dissociation, the ethyl, propyl and butyl ester versions of the peptides exhibit a dissociation pathway consistent with gamma-hydrogen transfer to the C-terminal carbonyl group, with associated elimination of an alkene, in a McLafferty-type rearrangement. The rearrangement leaves a metal cationized, free-acid form of the peptide, as confirmed by comparing the multi-stage CID of rearrangement products generated from peptide esters with the CID of corresponding metal cationized free-acid peptides. The transfer of a gamma-hydrogen in the rearrangement reaction was confirmed by investigating the CID of ethyl esters for which the terminal methyl group was labeled with deuterium. We found that the rearrangement product was significantly more abundant, relative to other product ions, when derived from isopropyl and tert-butyl esters than from ethyl, n-propyl or n-butyl ester analogues.

Formation of [b(n) + 17 + Ag]+ product ions from Ag+ cationized native and acetylated peptides.

We compared the tandem mass spectra of a range of native and acetylated Ag(+) cationized peptides to determine the influence of the derivatization step on the abundance of the [b(n) + 17 + Ag](+) product ions. Using tripeptides, the smallest for which the mechanisms to generate [b(2) - 1 + Ag](+) and [b(2) + 17 + Ag](+) products are both operative, we found that in most cases acetylation causes an increase in the abundance of the C-terminal rearrangement ion, [b(2) + 17 + Ag](+), relative to the rival N-terminal rearrangement ion, [b(2) - 1 + Ag](+). The presence of a free amino group to bind to the metal ion significantly influences the relative abundances of the product ions. We propose a mechanism for the formation of the [b(n) + 17 + Ag](+) that is based on the formation of a five-membered oxazolidin-5-one and tetrahedral carbon intermediate that may collapse to a peptide upon release of CO and an imine, aided by the fact that the ring formed during C-terminal rearrangement is both a hemiacylal and hemiaminal. We also identified an influence of amino acid sequence on the relative abundances of the [b(n) + 17 + Ag](+) and [b(n) - 1 + Ag](+) product ions, whereby bulky substituents located on the alpha-carbon of the amino acid to the C-terminal side of the cleavage site apparently promote the formation of the [b(n) + 17 + Ag](+) product over [b(n) - 1 + Ag](+) when the amino acid to the N-terminal side of the cleavage site is glycine. The latter ion is the favored product, however, when the bulky group is positioned on the alpha-carbon of the amino acid to the N-terminal side of the cleavage site.

Influence of a ring substituent on the tendency to form H(2)O adducts to Ag(+) complexes with phenylalanine analogues in an ion trap mass spectrometer.

In a previous report we showed that certain binary Ag(+)-amino acid complexes formed adduct ions by the attachment of a single water and methanol molecule when stored in an ion trap mass spectrometer: complexes with aliphatic amino acids and with 4-fluorophenylalanine formed the adduct ions whereas complexes with phenylalanine and tryptophan did not. In this study we compared the tendency of the Ag(+) complexes derived from phenylalanine, 4-fluorophenylalanine, 4-hydroxyphenylalanine (tyrosine), 4-bromophenylalanine, 4-nitrophenylalanine and aminocyclohexanepropionic acid to form water adducts when stored, without further activation, in the ion trap for times ranging from 1 to 500 ms. Because the donation of pi electron density to the Ag(+) ion is a likely determining factor in complex reactivity, our aim in the present study was to determine qualitatively the influence of para-position substituents on the aromatic ring on the formation of the water adducts. Our results show that the reactivity of the complexes is influenced significantly by the presence of the various substituents. Decreases in [M + Ag](+) ion abundance, and increases in adduct ion abundance, both measured as a function of storage time, follow the trend -NO(2) > -Br > -F > -OH > -H. The complex of Ag(+) with 4-nitrophenylalanine was nearly as reactive towards water as the Ag(+) complex with aminocyclohexanepropionic acid, the last being an amino acid devoid of pi character in the ring system. Collision induced dissociation of the [M + Ag](+) species derived from the amino acids produces, among other products, Ag(+) complexes with a para-substituted phenylacetaldehyde: complexes that also form adduct species when stored in the ion trap. The trends in adduct ion formation exhibited by the aldehyde-Ag(+) complex ions were similar to those observed for the precursor complexes of Ag(+) and the amino acids, confirming the influence of the ring substituent.

Multi-stage tandem mass spectrometry of metal cationized leucine enkephalin and leucine enkephalin amide.

We have examined the multi-stage collision induced dissociation (CID) of metal cationized leucine enkephalin, leucine enkephalin amide, and the N-acetylated versions of the peptides using ion trap mass spectrometry. In accord with earlier studies, the most prominent species observed during the multi-stage CID of alkali metal cationized leucine enkephalin are the [b(n) + 17 + Cat]+ ions. At higher CID stages (i.e. >MS(4)), however, dissociation of the [b2 + 17 + Cat]+ ion, a cationized dipeptide, results in the production of [a(n) -1 + Cat]+ species. The multi-stage CID of Ag+ cationized leucine enkephalin can be initiated with either the [b(n) -1 + Ag]+ or [b(n) + 17 + Ag]+ ions produced at the MS/MS stage. For the former, sequential CID stages cause, in general, the loss of CO, and then the loss of the imine of the C-terminal amino acid, to reveal the amino acid sequence. Similar to the alkali cationized species, CID of [b2 -1 + Ag]+ produces prominent [a(n) -1 + Ag]+ ions. The multi-stage CID of argentinated peptides is reminiscent of fragmentation observed for protonated peptides, in that a series of (b(n)) and (a(n)) type ions are generated in sequential CID stages. The Ag+ cation is similar to the alkali metals, however, in that the [b(n) + 17 + Ag]+ product is produced at the MS/MS and MS3 stages, and that sequential CID stages cause the elimination of amino acid residues primarily from the C-terminus. We found that N-acetylation of the peptide significantly influenced the fragmentation pathways observed, in particular by promoting the formation of more easily interpreted (in the context of unambiguous sequence determination) dissociation spectra from the [b2 + 17 + Li]+, [b2 + 17 + Na]+ and [b2 -1 + Ag]+ precursor ions. Our results suggest, therefore, that N-acetylation may improve the efficacy of multi-stage CID experiments for C-terminal peptide sequencing in the gas phase. For leucine enkephalin amide, only the multi-stage CID of the argentinated peptide allowed the complete amino acid sequence to be determined from the C-terminal side.

Determination of regioselectivity in ring opening of tert-butylaziridinones by a combination of (15)N labeling and electrospray ionization-ion trap mass spectrometry.

The ring opening of 1,3-di-tert-butylaziridinone by tert-butylamine and aniline was investigated by using electrospray ionization and collision-induced dissociation in an ion trap mass spectrometer in conjunction with (15)N labeling of the two amine nucleophiles. Using the MS(n) capabilities of the ion trap instrument, we were able to monitor the retention of the (15)N label through successive fragmentation steps. Both amines exhibited a remarkable degree of selectivity in that they both cleaved exclusively the 1,3-bond (the alkyl-nitrogen bond). This result is in contrast to that obtained previously with methylamine, which cleaved just the opposite bond, namely, the 1,2-bond (the acyl-nitrogen bond). These contrasting results could not have been predicted by previously published guidelines.

Antigen-induced translocation of PKC-theta to membrane rafts is required for T cell activation.

Protein kinase C-theta (PKC-theta) is essential for mature T cell activation; however, the mechanism by which it is recruited to the TCR signaling machinery is unknown. Here we show that T cell stimulation by antibodies or peptide-major histocompatibility complex (MHC) induces translocation of PKC-theta to membrane lipid rafts, which localize to the immunological synapse. Raft translocation was mediated by the PKC-theta regulatory domain and required Lck but not ZAP-70. In addition, PKC-theta was associated with Lck in the rafts. An isolated PKC-straight theta catalytic fragment did not partition into rafts or activate the transcription factor NF-kappa B, although addition of a Lck-derived raft-localization sequence restored these functions. Thus, physiological T cell activation translocates PKC-theta to rafts, which localize to the T cell synapse; this PKC-theta translocation is important for its function.

Gas phase attachment of water and methanol to Ag(I) complexes with alpha-amino acids in an ion trap mass spectrometer.

Electrospray ionization was used to generate gas phase complexes of Ag+ with selected alpha-amino acids. Following storage (isolation without collisional activation) in an ion trap mass spectrometer, the mass spectra produced from the complexes of Ag+ with alpha-amino acids such as alanine, valine and tert-leucine contained peaks consistent with the formation of water or methanol molecule adduct ions. The same adduct ions were not present, however, in the mass spectra generated from the Ag+ complexes with phenylalanine, tyrosine and tryptophan following isolation and storage under similar conditions. For those complexes that showed reactivity, the uptake of water and methanol increased with longer storage times in the ion trap. A preliminary molecular modeling study using phenylalanine demonstrated that the aromatic ring coordinates the Ag+ ion, and the interaction between the metal ion and pi-system, in part, is assumed to prohibit the binding of water or methanol during isolation in the gas phase. This conclusion is supported by a comparison of the adduct formation by the Ag+ complexes with phenylalanine, 4-fluorophenylalanine and alpha-aminocyclohexanepropionic acid. In addition, collision induced dissociation experiments involving the Ag+ complexes of phenylalanine, tyrosine and tryptophan suggest that limiting the coordination of the Ag ion by the complexing molecule (i.e. by loss of a coordinating functional group and/or change in structure due to dissociation) results in the binding of a water or methanol molecule during storage in the ion trap. Surprisingly, the bare Ag+ ion, when trapped and stored under identical experimental conditions, formed neither adduct species, suggesting that the attachment of water or methanol may be due to interactions with a molecular orbital within the Ag+/molecule complex.

Naïve CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation.

In defense of the host, the immune system must often raise an effective cytotoxic T lymphocyte (CTL) response from a small number of clonal precursors. The degree to which activation stimuli regulate the expansion and differentiation of naïve CTLs, however, remains unknown. Using an engineered antigen-presenting cell (APC) system that allows control over antigenic stimulation, we studied the signaling duration requirements for priming and clonal expansion of naïve CTLs. We found that naïve CTLs become committed after as little as 2 h of exposure to APCs and that their subsequent division and differentiation can occur without the need for further antigenic stimulation of the daughter cells, whether priming is in vitro or in vivo. These data show that after a brief interaction with stimulatory APCs, naïve CTLs initiate a program for their autonomous clonal expansion and development into functional effectors.

T- and B-cell nonresponsiveness to self-alphaB-crystallin in SJL mice prevents the induction of experimental allergic encephalomyelitis.

The myelin-associated stress protein alphaB-crystallin triggers strong proliferative responses and IFN-gamma production by human T cells and it is considered a candidate autoantigen in multiple sclerosis. In this study we examined the capacity of alphaB-crystallin or peptides derived thereof to induce experimental autoimmune encephalomyelitis (EAE) in SJL mice. Despite extensive efforts to induce EAE using active immunization with whole alphaB-crystallin, using adoptive transfer of lymphocytes or using peptide immunizations, no clinical or histological signs of EAE could be induced. SJL mice were unable to mount proliferative T-cell responses in vitro or delayed-type hypersensitivity responses in vivo to self-alphaB-crystallin. Also, immunization with self-alphaB-crystallin did not lead to any antibody response in SJL mice while bovine alphaB-crystallin triggered clear antibody responses within 1 week. Immunizations with alphaB-crystallin-derived peptides led to the activation of IL-4-producing Th2 cells and only a few IFN-gamma-producing Th1 cells. Peptide-specific T cells showed no cross-reactivity against whole alphaB-crystallin. The inability of SJL mice to mount proinflammatory T-cell responses against self-alphaB-crystallin readily explains the lack of EAE induction by immunization with whole protein or peptides derived from it. T- and B-cell nonresponsiveness is associated with constitutive expression of full-length alphaB-crystallin in both primary and secondary lymphoid organs in SJL mice, which is seen in other mammals as well, but not in humans.

Tolerance controls encephalitogenicity of alphaB-crystallin in the Lewis rat.

The myelin-associated protein, alphaB-crystallin, is considered a candidate autoantigen in multiple sclerosis (MS). In the present study, we examined the potential of alphaB-crystallin to induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Attempts to induce EAE with either bovine, rat or murine alphaB-crystallin or alphaB-crystallin peptides consistently failed. Immunization with either autologous rat or murine alphaB-crystallin did not trigger any antigen-specific T cell response. Immunization with bovine alphaB-crystallin or a synthetic peptide representing the cryptic epitope 49-64 did trigger T cell responses but these failed to crossreact with autologous rat alphaB-crystallin. Examination of lymphoid tissues of the Lewis rat revealed constitutive expression of alphaB-crystallin in thymus, spleen, and peripheral lymphocytes. Our data show that in Lewis rats, constitutive lymphoid expression of alphaB-crystallin is associated with a state of nonresponsiveness to autologous alphaB-crystallin that effectively controls the development of EAE in response to this myelin antigen.

Mistaken self, a novel model that links microbial infections with myelin-directed autoimmunity in multiple sclerosis.

Several findings indicate that infectious events play a role in the pathogenesis of multiple sclerosis (MS). At the same time, T-cell autoimmunity to myelin antigens is widely believed to be crucial to the development of MS lesions. Several mechanisms have been put forward to explain the presumed link between microbial infections and myelin-directed autoimmunity. These include molecular mimicry, bystander activation including epitope spreading and superantigenic activation of T cells. Evidence that either one of these mechanisms actually occurs in MS patients, however, is still weak. Also, none of the above mechanisms explain why MS is unique to humans. We propose an alternative link between microbial infection and myelin autoimmunity, which we refer to as 'mistaken self'. In this mechanism, peripheral microbial infections of lymphoid cells prime the human T-cell repertoire not only to microbial antigens but also to the stress protein alpha B-crystallin that is expressed de novo in infected lymphoid cells. Subsequently, stress-induced accumulation of this self antigen in oligodendocytes/myelin can provoke pro-inflammatory responses as the recruited memory T-cell repertoire then mistakes the self protein for a microbial antigen. In this paper we review the currently available evidence that 'mistaken self' centering on alpha B-crystallin represents a powerful source of anti-myelin autoimmunity in a way that is unique to humans.

Encephalitogenic and immunogenic potential of the stress protein alphaB-crystallin in Biozzi ABH (H-2A(g7)) mice.

The stress protein alphaB-crystallin is an immunodominant antigen in multiple sclerosis (MS)-affected myelin for human T cells and is expressed at elevated levels in MS lesions. Using bovine alphaB-crystallin and synthetic peptides based on mouse alphaB-crystallin the ability of this stress protein to induce experimental allergic encephalomyelitis (EAE) was screened in Biozzi ABH (H-2A(g7)) mice. While whole alphaB-crystallin and the immunodominant T cell epitopes (49-64, 73-88, 153-168) failed to induce disease the subdominant or cryptic epitope (1-16) was weakly encephalitogenic. The lack of encephalitogenicity of whole protein and dominant epitopes may be due to the low constitutive expression of alphaB-crystallin in the CNS combined with a state of peripheral tolerance suggested by the constitutive expression of alphaB-crystallin in secondary lymphoid tissues in ABH mice. Further evidence for a role of alphaB-crystallin in the progression of chronic relapsing neurological disease is suggested by the development of T cell responses to alphaB-crystallin during MOG-induced relapsing EAE as myelin damage accumulates. Together our data indicate that normal tolerising mechanisms in ABH mice prevent the induction of EAE by alphaB-crystallin while the subdominant or cryptic epitope is able to circumvent these mechanisms and contribute to pathogenic myelin-directed autoimmunity following T cell activation.

Characterization of photooxidized self-assembled monolayers and bilayers by spontaneous desorption mass spectrometry.

We show that selected self-assembled monolayers (SAMs) and bilayers are readily characterized by the application of controlled photooxidation and spontaneous desorption mass spectrometry (SDMS) in the negative ion mode. Additionally, SDMS is used to characterize organic and inorganic anionic species adsorbed to the surface of a positively charged SAM surface, 2-aminoethanethiol (AET). Prominent peaks are observed that correspond both to the sulfonate form of each SAM and bilayer and to the anion form of each molecule adsorbed to AET. In addition, fragments of the oxidized thin films were also observed at m/z 80 (SO3-) and 97 (HSO4-). Other prominent fragment peaks more characteristic of the molecule are also seen in the mass spectra.