Microsecond electrophoresis
- Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology and the Center for Nano- and Molecular Science and Engineering, University of Texas, Austin, TX 78712
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Edited by Royce W. Murray, University of North Carolina, Chapel Hill, NC, and approved January 22, 2003 (received for review November 26, 2002)
Abstract
Although analysis strategies exist for probing a diverse array of molecular properties, most of these approaches are not amenable to the study of reaction intermediates and other transient species. Separations in particular can provide detailed information on attributes not readily measured by spectroscopy but typically are performed over time scales much longer than the life span of highly unstable compounds. Here we report the development of an electrophoretic strategy that dramatically extends the practical speed limit for fractionations and demonstrate its utility in examining transient hydroxyindole photoproducts. Fluorescent reaction intermediates are optically generated in femtoliter volumes within a flowing reagent stream and are differentially transported at velocities as large as 1.3 m⋅s−1, thereby minimizing band variance and allowing multicomponent reaction mixtures to be resolved over separation paths as short as 9 μm. Analyte migration times and band variances do not deviate significantly from basic theory for separations performed with fields that exceed 0.1 MV⋅cm−1, indicating that effects from Joule heating are minor. We demonstrate the feasibility of achieving baseline resolution of a binary mixture in <10 μs, nearly 100-fold faster than previously possible. Application of this approach to the study of a range of short-lived molecules should be feasible.
Footnotes
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↵ * To whom correspondence should be addressed. E-mail: jshear{at}mail.utexas.edu.
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↵ ‖ A variety of recent technologies now make it possible to initiate protein folding on microsecond or faster time scales, including high-speed mixing (18), photolysis of intramolecular tethers (19, 20), and photoexcited electron transfer (21).
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This paper was submitted directly (Track II) to the PNAS office.
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See commentary on page 3545.
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↵ † Generation of visible-emitting hydroxyindole photoproducts depends on absorption of three to four near-IR photons, leading (in nonsaturating conditions) to an intensity dependence of I 3 to I 4 (9–11) Because fluorescence excitation of products occurs via two-photon absorption, this process ideally scales as I 2 and can be accomplished by using significantly lower laser powers than are required for efficient photoreaction.
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↵ ‡ High-intensity optical gating of 5HTrp (a neutral zwitterion at pH 7.1) creates a photobleached “hole” that migrates at the velocity of the unreacted indole and results in a negative-going peak in the baseline level of UV fluorescence. On time scales ranging from ≈40 to 200 μs, 5HTrp comigrates with its fluorescent photoproduct, indicating that the 5HTrp photoproduct is neutral (and the 5HT photoproduct is positively charged). Shorter times were not examined here, because the poor signal-to-noise ratio for hole measurements required the use of long gate durations.
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↵ § This value represents an average of the gate and probe foci dimensions, which are likely to be somewhat different as a result of the different dependencies on laser intensity of the photoreaction and detection events and the possibility that these processes are saturated to different degrees.
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↵ ¶ 5HTrp mobility was determined in a conventional CE analysis by using 5 mM phosphate separation buffer (containing 500 μM 5HT and 500 μM 5HTrp) and used to estimate the field in this fractionation based on an assumption that analyte mobility is constant.
- Abbreviations:
- CE,
- capillary electrophoresis;
- 5HT,
- serotonin;
- 5HTrp,
- 5-hydroxytryptophan
- Copyright © 2003, The National Academy of Sciences
