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It remains an open question as to whether experiments involving collision-induced dissociation (CID) can provide a viable approach for monitoring spatially resolved deuteration levels in electrosprayed polypeptide ions. A number of laboratories reported the successful application of CID following solution-phase H/D exchange (HDX), whereas others found that H/D scrambling precluded site-specific measurements. The aim of the current work is to help clarify the general feasibility of HDX-CID methods, using a 22-residue zinc-bound protein domain (Zn-ZBD) as model system. Metal binding in Zn-ZBD should confer structural rigidity, and the presence of several basic residues should sequester mobile charge carders in the gas phase. Both of these factors were expected to suppress the extent of scrambling. HDX was carded out by employing rapid on-line mixing, thereby mimicking conditions typically encountered in kinetic pulse-labeling studies. Quadrupole time-of-flight MS/MS of pulse-labeled Zn-ZBD provides high sequence coverage. However, the measured fragment deuteration levels do not correlate with the known H-bonding pattern of Zn-ZBD, suggesting the occurrence of extensive scrambling. Instead of showing a uniform distribution, the fragment ions reveal a distinct nonrandom pattern of deuteration levels. In the absence of prior information, these data could erroneously be ascribed to the presence of protected sites. However, the observed patterns clearly originate from other factors; possibly they are caused by modulations of the amide CID efficiency by kinetic isotope effects. It is concluded that scrambling does not represent the only conceptual problem in HDX-CID studies and that control experiments on uniformly labeled samples are essential for ruling out interpretation artifacts.

The adverse effects of nonvolatile salts on the electrospray (ESI) mass spectra of proteins and other biological analytes are a major obstacle for a wide range of applications. Numerous sample cleanup approaches have been devised to facilitate ESI-MS analyses. Recently developed microdialysis techniques can shorten desalting times down to several minutes, the bottleneck being diffusion of the contaminant through a semipermeable membrane. This work introduces an approach that allows the on-line desalting of macromolecule solutions within tens of milliseconds. The device does not employ a membrane; instead, it uses a two-layered laminar flow geometry that exploits the differential diffusion of macromolecular analytes and low molecular weight contaminants. To maximize desalting efficiency, diffusive exchange between the flow layers is permitted only for such a time as to allow full exchange of salt, while incurring minimal macromolecule exchange. Computer simulations and optical studies show that the device can reduce the salt concentration by roughly 1 order of magnitude, while retaining ~70% of the original protein concentration. Application of this approach to the on-line purification of salt-contaminated protein solutions in ESI-MS results in dramatic improvements of both the signal-to-noise ratio and the absolute signal intensity. However, efficient desalting requires the diffusion coefficients of salt and analyte to differ by roughly 1 order of magnitude or more. This technique has potential to facilitate high-throughput analyses of biological macromolecules directly from complex matrixes. In addition, it may become a valuable tool for process monitoring and for on-line kinetic studies on biological systems.

Mass spectrometry (MS)-based techniques have enormous potential for kinetic studies on enzyme-catalyzed processes. In particular, the use of electrospray ionization (ESI) MS for steady-state measurements is well established. However, there are very few reports of MS-based studies in the pre-steady-state regime, because it is difficult to achieve the time resolution required for this type of experiment. We have recently developed a capillary mixer with adjustable reaction chamber volume for kinetic studies by ESI-MS with millisecond time resolution (Wilson, D. J.; Konermann, L. Anal. Chem. 2003, 75, 6408-6414). Data can be acquired in kinetic mode, where the concentrations of selected reactive species are monitored as a function of time, or in spectral mode, where entire mass spectra are obtained for selected reaction times. Here, we describe the application of this technique to study the kinetics of enzyme reactions. The hydrolysis of p-nitrophenyl acetate by chymotrypsin was chosen as a simple chromophoric model system. On-line addition of a 'makeup solvent' immediately prior to ionization allowed the pre-steady-state accumulation of acetylated chymotrypsin to be monitored. The rate constant for acetylation, as well as the dissociation constant of the enzyme-substrate complex obtained from these data, is in excellent agreement with results obtained by conventional stopped-flow methods. Bradykinin was chosen to illustrate the performance of the ESI-MS-based method with a nonchromophoric substrate. In this case, the unfavorable rate constant ratio for acylation and deacylation of the enzyme precluded measurements in the pre-steady-state regime. Steady-state experiments were carried out to determine the turnover number and the Michaelis constant for bradykinin. The methodologies used in this work open a wide range of possibilities for future ESI-MS-based kinetic assays in enzymology.

A novel continuous-flow apparatus for on-line kinetic studies of (bio)chemical solution-phase processes by electrospray ionization mass spectrometry (ESI-MS) is described. The device is based on two concentric capillaries. Fluid is released from the inner capillary into the intercapillary space, where it mixes with solution flowing through the outer capillary, thus initiating the reaction of interest. Gas-phase analyte ions are formed near the tip of the outer capillary by pneumatically assisted ESI. This setup allows the mixer to be placed directly within the ion source, thus providing a minimal dead volume of ~8 nL. Time-resolved data can be recorded in both 'spectral' and 'kinetic' modes. In the former case, the position of the inner capillary is fixed at various points, such that entire mass spectra can be recorded for selected reaction times. For experiments in kinetic mode, the mass spectrometer monitors the signal intensity at selected m/z values, while the inner capillary is continuously pulled back, thus providing intensity-time profiles for specific reactive species. A theoretical framework is developed that allows the measured kinetics to be analyzed by taking into account the effects of laminar flow within the reaction capillary. Failure to take these effects into account results in erroneous rate constants. Studies on the demetalation kinetics of chlorophyll reveal that the apparatus can reliably measure rate constants up to at least 100 [s.sup.-1]. This represents a substantial improvement over previous ESI-MS-based kinetic methods. Spectral mode experiments on the refolding of ubiquitin show the changing proportions of denatured and tightly folded protein sub-populations in solution. When monitored in kinetic mode, the refolding process was found to proceed with a rate constant of 5.2 [s.sup.-1].