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A method is presented for characterizing primary cement interfaces of barnacles using in situ attenuated total reflection-Fourier transform infrared spectroscopy. Primary cement of the barnacle, Balanus amphitrite (Amphibalanus amphitrite), was characterized without any disruption to the original cement interface, after settling and growing barnacles directly on double sided polished germanium wafers. High-quality IR spectra were acquired of live barnacle cement interfaces, providing a spectroscopic fingerprint of cured primary cement in vivo with the barnacle adhered to the substratum. Additional spectra were also acquired of intact cement interfaces for which the upper portion of the barnacle had been removed leaving only the base plate and cement layer attached to the substratum. This allowed further characterization of primary cement interfaces that were dried or placed in D(2)O. The resulting spectra were consistent with the cement being proteinaceous, and allowed analysis of the protein secondary structure and water content in the cement layer. The estimated secondary structure composition was primarily beta-sheet, with additional alpha-helix, turn and unordered components. The cement of live barnacles, freshly removed from seawater, was estimated to have a water content of 20-50% by weight. These results provide new insights into the chemical properties of the undisturbed barnacle adhesive interface.

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Adhesion of micron-scale probes with model poly(dimethylsiloxane), PDMS, elastomers was studied with a depth-sensing nanoindenter under oscillatory loading conditions. For contacts between diamond indenters (radius R = 5 or 10 microm) and PDMS, force-displacement curves were highly reversible and consistent with Johnson-Kendall-Roberts (JKR) behavior. However, our experiments have revealed striking differences between the experimental measurements of tip-sample interaction stiffness and the theoretical JKR stiffness. The measured stiffness was always greater than zero, even in the reflex portion of the curve (between the maximum adhesive force and release), where the JKR stiffness is negative. This apparent paradox can be resolved by considering the effects of viscoelasticity of PDMS on an oscillating crack tip in a JKR contact. Under well described conditions determined by oscillation frequency, sample viscoelastic properties, and the Tabor parameter (with variables R, reduced elastic modulus, E*, and interfacial energy, deltagamma), an oscillating crack tip will neither advance nor recede. In that case, the contact size is fixed (like that of a flat punch) at any given point on the load-displacement cycle, and the experimentally measured stiffness is equal to the equivalent punch stiffness. For a fixed oscillation frequency, a transition between JKR and punch stiffness can be brought about by an increase in radius of the probe or a decrease in PDMS modulus. Additionally, varying the oscillation frequency for a fixed E*, R, and deltagamma also resulted in transition between JKR and punch stiffness in a predictable manner. Comparisons of experiments and theory for an oscillating viscoelastic JKR contact are presented. The storage modulus and surface energy from nanoscale JKR stiffness measurements were compared to calculated values and those measured with conventional nanoindentation and JKR force-displacement analyses.

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Intact avidin-biotin and avidin-biotin maleimide noncovalent complexes have been observed by electrospray ionization mass spectrometry (ESI-MS) by using an extended mass range quadrupole mass spectrometer. By utilizing mild ES1 interface conditions, the expected solution behavior of four biotin or biotin maleimide molecules noncovalently binding to each avidin tetramer can be preserved in the gas phase. The ESI-MS results show the appropriate mass additions of 973 ± 60 Da for biotin and 1802 ± 40 Da for biotin maleimide to the avidin tetramer species. These results support the hypothesis that substantial retention of higher order structure is possible in the gas phase by using gentle ESI conditions.

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In this article, we briefly highlight the use of capillary electrophoresis for sampling, manipulating, and separating extremely small sample sizes. The extraordinary sensitivity that can be obtained by combined capillary electrophoresis-mass spectrometry is then demonstrated using recent results. We briefly describe the ability to detect noncovalently associated complexes (e.g., double-stranded DNA) by electrospray ionization-mass spectrometry, and conclude with recent results that show the potential for using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry for characterization of biomolecules.

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Electrospray ionization collisionally activated dissociation (CAD) mass spectra of multiply charged human hemoglobin beta-chain variant proteins (146 amino acid residues, 15.9 kDa), generated in the atmospheric pressure/vacuum interface and in the collision quadrupole of a triple-quadrupole mass spectrometer, are shown and compared. Several series of structurally informative singly and multiply charged b- and y-mode product ions are observed, with cleavage of the Thr 50-Pro 51 CO-NH bond to produce the complementary y96 and b50 sequence ions as the most favored fragmentation pathway. The eight different beta-globin variants studied differ by a single amino acid substitution and can be differentiated from the observed m/z shifts of the assigned product ions. The overall fragmentation patterns for the variant polypeptides are very similar, with the exception of the Willamette form, in which Arg is substituted for Pro- 51, and multiply charged y96 product ions are not observed. Circular dichroism spectra of normal beta A and beta Willamette show very little difference under a variety of solvent conditions, indicating that fragmentation differences in their respective CAD mass spectra are substantially governed by primary rather than secondary structure.

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High mass-to-charge ratio ions (> 4000) from electrospray ionization (ESI) have been observed for several proteins, including bovine cytochrome c (M r 12,231) and porcine pepsin (M r 34,584), by using a quadrupole mass spectrometer with an m/z 45,000 range. The ESI mass spectrum for cytochrome c in an aqueous solution gives a charge state distribution that ranges from 12 + to 2 +, with a broad, low-intensity peak in the mass-to-charge ratio region corresponding to the [M + H](+) ion. the negative ion ESI mass spectrum for pepsin in 1% acetic acid solution shows a charge state distribution ranging from 7- to 2-. To observe the [M - H](-) ion, harsher desolvation and interface conditions were required. Also observed was the abundant aggregation of the protens with average charge states substantially lower than observed for their monomeric counterparts. The negative ion ESI mass spectrum for cytochrome c in 1-100 mM NH4OAc solutions showed greater relative abundances for the higher mass-to-charge ratio ions than in acuidic solutions, with an [M - H](-) ion relative abundance approximately 50% that of the most abundant charge state peak. The observation that protein aggregates are formed with charge states comparable to monomeric species (at fower mass-to-charge ratios) suggests that the high mass-to-charge ratio monomers may be formed by the dissociation of aggregate species. The observation of low charge state and aggregate molecular ions concurrently with highly charged species may serve to support a variation of the charged residue model, originally described by Dole and co-workers (Dole, M., et al. J. Chem. Phys. 1968, 49, 2240; Mack, L. L., et al. J. Chem. Phys. 1970, 52, 4977) which involves the Coulombically driven formation of either very highly solvated molecular ions or lower ananometer-diameter droplets.

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Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H](15+) ion being the most intense peak. With the addition of H2O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.