A Capillary Mixer with Adjustable Reaction Chamber Volume for Millisecond Time-Resolved Studies by Electrospray Mass Spectrometry

Abstract

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-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 subpopulations in solution. When monitored in kinetic mode, the refolding process was found to proceed with a rate constant of 5.2 s-1.

A versatile microfluidic chip for millisecond time-scale kinetic studies by electrospray mass spectrometry

“…Data analysis might also be convoluted by Taylor dispersion-based distortion of the laminar flow velocity profile. However, on the scale of the reaction channel (155 m ϫ 155 m), significant distortion of the laminar flow profile occurs about seconds [3], much longer than the timescale of our kinetic experiments (final timepoint ϭ 0.65 s). The effects of Taylor dispersion can therefore also be neglected.…”

“…The data were fit to expression 3 using a FORTRAN program developed previously [3] adjusted for square channel laminar flow as described in theory section. Fits are shown as solid lines in Figure 5.…”

“…The complementary advantages of microfluidic liquid handling and ESI-MS based analysis make these devices a natural answer to the challenges of proteomic studies. One 'underrepresented' application is rapid microfluidic mixing for time-resolved studies by ESI-MS. Time-resolved ESI-MS (TR ESI-MS) is a powerful approach for the characterization of shortlived, 'transient' intermediates in biochemical reactions [3][4][5][6][7][8][9][10], which are critical to the biological activity of proteins, from enzymatic reactions [7,11] to protein folding [12,13].…”

“…This motivated the development of 'Stopped Flow ESI-MS' by Kolakowski and Konermann in 2000 [9,10]. In 2003, Wilson and Konermann introduced a continuous flow capillary mixer for TR ESI-MS that facilitated full time-course acquisitions on time-scales comparable to optical stopped flow experiments [3]. This approach has enabled numerous investigations [3-5, 14 -17], but the concentric capillary design is limiting in terms of reproducibility of fabrication and is not well suited to the inclusion of directly coupled sample preparation or separation steps.…”

“…The device incorporates an internal capillary mixer that is similar to the one in reference [3], but formed by laser ablation with greatly improved reproducibility. Device performance is characterized by monitoring acid induced cytochrome c unfolding kinetics.…”