An Improved Rapid Mixing Device for Time‐Resolved Electrospray Mass Spectrometry Measurements

Zinck, Nicholas; Stark, Ann-Kathrin; Wilson, Derek J.; Sharon, Michal

doi:10.1002/open.201402002

Cited by 16 publications

(11 citation statements)

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“…Information about reaction kinetics, including protein folding − and unfolding, is often obtained by rapidly mixing two or more solutions. Conventional mixing devices include chaotic, turbulent, and laminar , flow mixers.…”

Section: Introductionmentioning

confidence: 99%

“…Mass spectrometry (MS) is an excellent detector for measuring reaction products resulting from rapid mixing experiments owing to its high sensitivity, high chemical specificity, and rapid speed of analysis . Several different in-line mixers have been coupled with MS including stopped, continuous, , and laminar , flow devices. The shortest reaction time achieved using a conventional mixer coupled with MS is 200 μs with a laminar flow mixer and a 10 μL/s flow rate …”

Section: Introductionmentioning

confidence: 99%

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Ultrafast (1 μs) Mixing and Fast Protein Folding in Nanodrops Monitored by Mass Spectrometry

2016

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The use of theta-glass emitters and mass spectrometry to monitor reactions that occur as fast as one μs is demonstrated. Acidified aqueous solutions containing unfolded proteins are mixed with aqueous ammonium acetate solutions to increase the solution pH and induce protein folding during nanoelectrospray ionization. Protein charge-state distributions show the extent to which folding occurs, and reaction times are obtained from known protein folding time constants. Shorter reaction times are obtained by decreasing the solution flow rate, and reaction times between 1.0 and 22 μs are obtained using flow rates between 48 and 2880 pL/s, respectively. Remarkably similar reaction times are obtained for three different proteins (Trp-cage, myoglobin, and cytochrome c) with folding time constants that differ by more than an order of magnitude (4.1, 7, and 57 μs, respectively), indicating that the reaction times obtained using rapid mixing from theta-glass emitters are independent of protein identity. A folding time constant of 2.2 μs is obtained for the formation of a β-hairpin structure of renin substrate tetradecapeptide, which is the fastest folding event measured using a rapid mixing technique. The 1.0 μs reaction time obtained here is about an order of magnitude lower than the shortest reaction time probed using a conventional mixer (8 μs). Moreover, this fast reaction time is obtained with a 48 pL/s flow rate, which is 2000-times less than the flow rate required to obtained the 8 μs reaction time using a conventional mixer. These results indicate that rapid mixing with theta-glass emitters can be used to access significantly faster reaction times while consuming substantially less sample than in conventional mixing apparatus.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Ultrafast (1 μs) Mixing and Fast Protein Folding in Nanodrops Monitored by Mass Spectrometry

2016

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“…not the folded core which has HDX half lives > hours) by measuring the structural dynamics in solution at physiologically relevant pH (7.0). Previously it was determined that the subunit interface of the dimer has two main contact regions on opposite sides of the enzyme (1): one between the cap region (residues [35][36][37][38][39][40][41][42][43][44][45][46] and the α1-α2 loop, β7 and the α2' helix (47-78) of the opposite subunit. The other contact is antiparallel association of the two tower helixes, α7 and the immediately adjacent structural elements.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, there has been growing interest in measuring HDX at the sub-second time-scale to address our inability to measure the structural dynamics and stability of classes of molecule that are currently intractable (including peptide hormones, neurotransmitters and intrinsically disordered proteins/regions) (22,23). A small number of academic laboratories have reported experimental systems and approaches to obtain millisecond HDX data (24,25): these include microfluidic chips (26)(27)(28)(29)(30)(31), a quench flow apparatus (16,32) employed for analysis of disordered proteins, a completely online quench flow setup (11,33) and a capillary mixer for characterizing biological reactions (34,35).…”

Section: Introductionmentioning

confidence: 99%

Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution

Kish

Smith

Subramanian

et al. 2019

Preprint

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Glycogen phosphorylase (GlyP) was the first allosteric enzyme to be described. Yet, the precise dynamic changes in solution phase structure and stability that underpin functional regulation have remained elusive. We have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. This enabled measurements of the solution phase local structural dynamics involved in allosteric regulation of GlyP. The sensitivity of these measurements discerned that the 250's loop is natively disordered in the apo T-state, adopting a more ordered conformation in the active state. The quantitative change in stability of the 280s loop is identified, providing the first direct evidence of the entropic switch that sterically regulates substrate access to the active site. Here, we quantify GlyP structural dynamics in solution, describing correlated changes in structure in the activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of the enzyme. Significance StatementWe have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. Measurements of glycogen phosphorylase quantify the solution phase stability of local structure at near-amino acid structural resolution and with no appreciable lower limit of stability. This uncovered the highly-resolved local alterations in stability we provide direct evidence of the entropic mechanism by which access to the active site is gated by the 280s loop. Results Fully-automated and flexible pulse-labeling hydrogen/deuterium-exchange mass spectrometry with millisecond precisionOur goal was to quantify the structural switch in solution, along with other coincident dynamic changes in structure between activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of GlyP. To enable this, we developed a fully automated broadband bottom-up HDX-MS approach capable of accurate quantitative assessment of peptide stability. The newly developed ms2min system design ( Figure 1A) offers certain significant advances over the other designs previously employed. Notably, it allows fully-automated, software-selection of mixing times over six orders of magnitude (ms to hours) with 1 ms time resolution, flexible labeling temperature control (0 -25 C), quench temperature control (0 C), on-line connection for 'bottom-up' workflows, two-way communication for reciprocal control of labeling, washing, digestion, desalting and chromatography, automated digestion column wash injection, intercalated blank injections and sample list scheduling of multiple runs. Precision of D-labeling.To evaluate the measurement variability and labeling precision of the system we determined the repeatability of the quench-flow labeling method. On three separate days we measured the D uptake of 50, 70 and 150 replicates of Bradykinin and Leucine enkephalin at 100 ms mixing time. The short labeling period close to the limit of quantification, wa...

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“…Moreover, the modified device allows for easy mounting onto the mass spectrometer in place of the commercial ionization source. 46 Despite its limitations, the sensitivity and selectivity of MS make it a promising technique for monitoring millisecond time scale reactions. This review has focused on how TRESI-MS has been thoroughly applied to studying protein folding/unfolding conformations, pre-steady-state intermediates, and the dynamic analysis of weakly structured protein regions.…”

Section: Discussionmentioning

confidence: 99%

Time-resolved electrospray mass spectrometry — a brief history

2015

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This review describes the evolution of time-resolved electrospray ionization mass spectrometry (TRESI-MS), a technology that was developed in large part at Western University. TRESI-MS was initially designed to characterize rapid chemical and biochemical reactions occurring on the millisecond time scale without need for a chromophore. Early TRESI-MS setups usually consisted of continuous-flow rapid mixers with a fixed tee for analysis of a single time point, and later adjustable reaction chamber devices allowing for automatic tracking of the reaction over time. Advances in instrumentation design over the years have resulted in improved time resolution, with microfluidic device implementation allowing for coupling to hydrogen−deuterium exchange (HDX) experiments. Areas of application that will be discussed include the investigation of protein folding intermediates, identification of enzyme−substrate intermediates in the pre-steady state, and the use of time-resolved HDX to study the dynamics of weakly structured protein regions. While some limitations still persist with the method, the continued development of TRESI-MS and related approaches paves the way to a promising future and the study of unexplored application areas.Résumé : La présente étude décrit l'évolution de la spectrométrie de masse à ionisation par électronébulisation résolue en temps (TRESI-MS), une technologie qui fut élaborée en grande partie à la Western University. À l'origine, la TRESI-MS fut conçue pour caractériser les réactions chimiques et biochimiques rapides qui se produisaient à l'échelle de la milliseconde sans présence nécessaire d'un chromophore. Les premiers appareils de TRESI-MS consistaient généralement en des mélangeurs rapides à flux continu équipés d'un « tee » fixe permettant d'effectuer des analyses ponctuelles, puis, plus tard, en des dispositifs à chambres de réaction ajustables offrant la possibilité de réaliser le suivi automatique des réactions au cours du temps. Des progrès réalisés dans la conception de l'instrumentation au fil des ans ont permis d'améliorer la résolution temporelle, notamment la mise en place d'appareils de microfluidique qui permirent de réaliser des expériences de couplage avec l'échange hydrogène−deutérium (HDX). Nous présenterons, entre autres champs d'application, l'étude des intermédiaires du repliement protéique, l'identification des intermédiaires du complexe enzyme−substrat dans l'état de pré-équilibre et l'utilisation de HDX résolu en temps pour l'étude de la dynamique des régions protéiques faiblement structurées. Bien qu'elle montre encore quelques limites, l'amélioration continue de la TRESI-MS et des techniques auxquelles elle est associée ouvre la voie à un avenir prometteur et à l'étude de champs d'application inexplorés. [Traduit par la Rédaction] Mots-clés : spectrométrie de masse, spectrométrie de masse à ionisation par électronébulisation, résolution en temps, cinétique, repliement protéique, catalyse enzymatique, échange hydrogène−deutérium.

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An Improved Rapid Mixing Device for Time‐Resolved Electrospray Mass Spectrometry Measurements

Cited by 16 publications

References 57 publications

Ultrafast (1 μs) Mixing and Fast Protein Folding in Nanodrops Monitored by Mass Spectrometry

Ultrafast (1 μs) Mixing and Fast Protein Folding in Nanodrops Monitored by Mass Spectrometry

Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution

Time-resolved electrospray mass spectrometry — a brief history

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