Accurate determination of protein molecular mass to within 1 Da would be a boon to protein characterization. It would then become possible to (a) count the number of disulfide bridges (-S-S-is 2 Da lighter than 2 -SH); (b) identify deamidation (-NH 2 is 1 Da lighter than -OH); (c) identify such post-translational modifications as phosphorylation and glycosylation; (d) resolve and identify adducts; (e) identify variant amino acid sequences; etc. Determination of the molecular mass of a neutral protein to within 1 Da from measurement of the mass of its gas-phase ion might appear easy. After all, electrospray ionization can now routinely generate abundant multiply-charged gas-phase unhydrated quasimolecular ions, (M + nH) n+ , for most proteins, 1,2 and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry 3-7 can determine the ion mass to parts-per-million accuracy at typical electrosprayed protein multiply-charged ion mass-to-charge ratios, 500 e m/z e 2000. 8 However, the monoisotopic mass (see below) of a protein inferred from the mass(es) of its corresponding ions may still be wrong by up to several Dalton! There are three stages in the determination of the molecular weight of a neutral protein from its electrosprayed ion mass spectrum. 9 (Our electrospray FT-ICR mass spectra were obtained with a homebuilt instrument operating at 9.4 T, as described elsewhere. 10 ) First, electrospray ionization produces protein ions with various numbers of attached protons and thus several charge states. The first stage in protein mass analysis is therefore to separate the individual charge states (e.g., (M + zH) z+ , (M + (z+1)H) (z+1)+ , etc.). Second, since mass spectrometry reports mass-to-charge ratio, it is necessary to determine the ion charge in order to determine its mass. The massto-charge ratio spectrum of a protein of a given charge state exhibits numerous "isotopic" peaks (see below) spaced ∼1 Da apart (Figure 1). High-resolution FT-ICR mass spectrometry can resolve those peaks for proteins of molecular mass up to more than 100 000 Da, so that the charge state, z, may be determined simply as the reciprocal of the separation between two adjacent isotopic peaks differing in mass by ∼1 Da. 11 However, protein mass measurement accuracy is presently limited by the third stage of mass analysis: namely, knowledge of the isotopic composition (i.e., the constituent chemical formula(s) composing each mass spectral peak). For organic molecules of less than ∼1000 Da, determination of molecular weight from the singly-charged molecular (M + ) or quasimolecular (e.g., (M + H) + ) ion is relatively simple. Why then is it so much more difficult to determine the mass of a biological macromolecule? The problem is apparent from Figure 1 (top). The natural abundance of 13 C is 1.066-1.106% relative to 12 C as 100%. 8 However, for a molecule containing n carbons, the isotopic distribution is a binomial expansion (0.9889 + 0.0111) n , and it is ∼n% as likely that a given molecule will contain one 13 C as that all of the c...
This paper reports results of analysis of the OH stretch Raman spectra of aqueous solutions of electrolytes. Error analysis supports the view that the environment of the electrolyte in the liquid is that of a liquid crystalline hydrate. Fitting error analysis for a two-state model further suggests that the presence of the hydrated electrolyte perturbs the structure of the bulk water in the solution. This perturbation is consistent with the results of previous studies of the apparent density of water in aqueous solutions of electrolytes. The previous studies showed that electrolytes increase the hydrogen bond strength of the bulk water and thereby cause a shift in the water equilibrium, which results in a change of the apparent density of water in the solution.
Nuclear magnetic resonance chemical shifts are used to examine the perturbations in water structure that occur with concentration changes in aqueous KF, KCl, and LiOH solutions. Changes in the slope of ion chemical shifts as a function of solute concentration can be explained by changes in water structure. The equilibrium shift in water structure occurs as a result of changes in the hydrogen bond strength. The changes in hydrogen bond strength are a result of changes in electrolyte concentration and electron delocalization throughout the liquid. The location of the changes in slope with concentration is temperature dependent. A correlation of the changes in slope of chemical shifts to minima in specific heat capacity suggests the occurrence of a weak continuous transition in the solution structure at the critical concentration corresponding to the specific heat capacity minimum. By extrapolation the experiments reported here imply that there is a weak continuous transition associated with the heat capacity minimum for pure water. There must also be a structural relaxation time in the liquid associated with this transition. The results of these experiments provide confirmation for the model of aqueous solutions we recently proposed in which the solution is composed of regions of pure water and regions of liquid crystalline electrolyte hydrates. The subphase composed of structurally perturbed water is the part of the system that participates in the weak continuous phase transition that is evidenced by the NMR chemical shifts. In complete agreement with earlier Raman experiments it appears that the entire solution is a single electronic whole with exquisite electronic delocalization between the water and liquid crystalline subphases so that the ionic nuclei experience the electronic effects of the transition in the water subphase.
This paper presents the Raman depolarization ratio of degassed ultrapure water as a function of temperature, in the range 303.4-314.4 K (30.2-41.2 degrees C). The pressure of the sample was the vapor pressure of water at the measurement temperature. The data provide a direct indication of the existence of a phase transition in the liquid at 307.7 K, 5.8 kPa (34.6 degrees C, 0.057 atm). The minimum in the heat capacity, C(p)(), of water occurs at 34.5 degrees C, 1.0 atm (J. Res. Natl. Bur. Stand. 1939, 23, 197(1)). The minimum in C(p)() is shallow, and the transition is a weak-continuous phase transition. The pressure coefficient of the viscosity of water changes sign as pressure increases for temperatures below 35 degrees C (Nature 1965, 207, 620(2)). The viscosity minimum tracks the liquid phase transition in the P, T plane where it connects with the minimum in the freezing point of pure water in the same plane (Proc. Am. Acad. Arts Sci. 1911-12, 47, 441(3)). Previously we argued (J. Chem. Phys. 1998, 109, 7379(4)) that the minimum in the pressure coefficient of viscosity signaled the elimination of three-dimensional connectivity in liquid water. These observations coupled with recent measurements of the coordination shell of water near 300 K (Science 2004, 304, 995(5)) suggest that the structural component that changes during the phase transition is tetrahedrally coordinated water. At temperatures above the transition, there is no tetrahedrally coordinated water in the liquid and locally planar water structures dominate the liquid structure. Water is a structured liquid with distinct local structures that vary with temperature. Furthermore, liquid water has a liquid-liquid phase transition near the middle of the normal liquid range.
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