Measuring Dynamics in Weakly Structured Regions of Proteins Using Microfluidics-Enabled Subsecond H/D Exchange Mass Spectrometry

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• 作者：Tamanna Rob ; Peter Liuni ; Preet Kamal Gill ; Shaolong Zhu ; Naresh Balachandran ; Paul J. Berti ; Derek J. Wilson
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：April 17, 2012
• 年：2012
• 卷：84
• 期：8
• 页码：3771-3779
• 全文大小：435K
• 年卷期：v.84,no.8(April 17, 2012)
• ISSN：1520-6882

文摘

This work introduces an integrated microfluidic device for measuring rapid H/D exchange (HDX) in proteins. By monitoring backbone amide HDX on the millisecond to low second time scale, we are able to characterize conformational dynamics in weakly structured regions, such as loops and molten globule-like domains that are inaccessible in conventional HDX experiments. The device accommodates the entire MS-based HDX workflow on a single chip with residence times sufficiently small (ca. 8 s) that back-exchange is negligible (鈮?%), even without cooling. Components include an adjustable position capillary mixer providing a variable-time labeling pulse, a static mixer for HDX quenching, a proteolytic microreactor for rapid protein digestion, and on-chip electrospray ionization (ESI). In the present work, we characterize device performance using three model systems, each illustrating a different application of 鈥榯ime-resolved鈥?HDX. Ubiquitin is used to illustrate a crude, high throughput structural analysis based on a single subsecond HDX time-point. In experiments using cytochrome c, we distinguish dynamic behavior in loops, establishing a link between flexibility and interactions with the heme prosthetic group. Finally, we localize an unusually high 鈥榖urst-phase鈥?of HDX in the large tetrameric enzyme DAHP synthase to a 鈥榤olten globule-like鈥?region surrounding the active site.