Time-resolved ESI

Monitoring protein folding dynamics by time-resolved ESI-MS... 

Monitoring real-time phosphorylation kinetics with time-resolved ESI-TOF-MS 682... 

Figure 2 Time-resolved ESI-TOF-MS. (a) Diagram with the experimental design, (b) Chymotrypsin reaction with p-nitrophenyl acetate under single-turnover conditions monitored by time-resolved ESI-TOF-MS. The decay of chymotrypsin and formation of the acetyl-chymotrypsin intermediate is observed over a time course of 45 s. (c) Kinetic analysis of chymotrypsin decay and acetyl-chymotrypsin formation. For both traces the rate constant was 0.1 s . ...

Time-Resolved ESI-TOF-MS to Detect an Intermediate in the KD08P Synthase Reaction... 

This work demonstrates the feasibility of the time-resolved ESI-TOF—MS for the detection of low abundance, short-lived, and chemically unstable enzyme intermediates and opens up the potential of utilizing MS for performing real-time mechanistic enzymology. 

Figure 8 Analysis of phosphorylation kinetics by time-resolved ESI-TOF-MS. (a) FGFR1 autophosphorylation. A selection of MS spectra collected by time-resolved ESI-TOI MS showing the formation of mono and diphosphorylated FGFR1 species at increasing reaction times. OP, 1P, and 2P represent the FGFR1 in unphosphorylated, mono, and diphosphoryl states.

Figure 12.1 (a) Experimental apparatus for the time-resolved ESI-MS experiments. Arrows... 

In an interesting work, Liuni et al. [72] investigated pre-steady state conformational dynamics in an active enzyme using combined time-resolved ESI-MS and sub-second HDX. Here, biocatalytic processes were monitored as time-dependent intensity changes of reaction intermediates. Conformational dynamics was analyzed by the rate and magnitude of deuterium uptake [72],... 

Enzyme reaction intermediates can be characterized, in sub-second timescale, using the so-called pulsed flow method [35]. It employs a direct on-line interface between a rapid-mixing device and a ESI-MS system. It circumvents chemical quenching. By way of this strategy, it was possible to detect the intermediate of a reaction catalyzed by 5-enolpyruvoyl-shikimate-3-phosphate synthase [35]. The time-resolved ESI-MS method was also implemented in measurements of pre-steady-state kinetics of an enzymatic reaction involving Bacillus circulans xylanase [36]. The pre-steady-state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase were determined. The MS results were in agreement with those obtained by stopped-flow ultraviolet-visible spectroscopy. In a later work, hydrolysis of p-nitrophenyl acetate by chymotrypsin was used as a model system [27]. The chymotrypsin-catalyzed hydrolysis follows the mechanism [27] ... 

Figure S.2 Schematic representation of a continuous-flow mixing setup for kinetic measurements by time-resolved ESI-MS. Syringes 1 and 2 deliver a continuous flow of reactants mixing of the two solutions initiates...

Figure 9.16. Denaturation of holomyoglobin (hMb) in water/ methanol (75 25 v/v) at pH 11.2 monitored by time-resolved ESI MS in the negative-ion mode. These spectra were recorded 0.08 s (A), 0.43 s (B), 1.2 s (C), and 5 min (D) after initiation of denaturation. Notation h is tiMb a is apomyoglobin (aMb), H is heme. Also indicated are the charge stales of some protein ions. (Reprinted with permission from Sogbein, O. Simmons, D. A. Konermann, L. J. Am. Soc. Mass Spectrom. 2000, //, 312-319.)...

P 47] The capillary-in-capillary mixer was especially developed for millisecond time-resolved studies by ESI-MS. Since this ranges involves an application and characterization of the mixer itself was not performed, for further information on the... 

M 52] [P 47] The capillary-in-capillary mixer proved functionality for millisecond time-resolved studies by ESI-MS [133]. The experiments were performed in two modes of operation in a spectral mode with recording of entire mass spectra and in a kinetic mode where the intensity of selected ion signals can be monitored as a function of the average reaction time. This enabled new means of resolving kinetic data, i.e. to measure reliably first-order rate constants up to at least 100 s 1. This performance is four times better than for reported ESI-MS experiments. 

Van Zyl et al. reported on the diffusion of ipratropium through porcine bronchial epithelium tissue [74], In principle, ipratropium is administered via the respiratory tract by inhalation to treat pulmonary diseases associated with bronchoconstriction. Therefore, pulmonary absorption by bronchial tissue determines its local efficacy and was thus investigated in a diffusion cell in vitro. Bronchial epithelium was equilibrated in PBS and discs of 4 mm2 were mounted on that diffusion cell separating the donor and receiver compartment. The donor compartment contained the drug dissolved in PBS (1 mg/ml) and the receiving chamber was permanently flushed with a low flow (1.5 ml/h) of PBS thus allowing time-resolved fractionation for subsequent direct analysis by LC-ESI MS/MS in MRM mode. Transition to the product ion at m z 124 was monitored for quantification (Table 9). The transfer of ipratropium was characterized by the flux (about 220 ng/cm2/min) and the permeability coefficient calculated to be 1.6 x 10-8 cm/s. 

ESI and MALDI-MS are routinely used for both accurate mass information on intact proteins and their proteolytic digests. As a result these methods have already helped detect viral mutants, identify capsid proteins, and post-transla-tional modifications. Recent work has also included the detection of the first intact viral particles as well as a viral protein capsid. Other mass-based approaches like time-resolved proteolysis is giving new insight into the dynamics of viral capsid proteins in solution. This information, when combined with complementary information from X-ray crystallography studies is leading to a better understanding of viral structure and function. 

ESI Time-resolved phosphoproteomic study Verano-Braga et a/. 

Figure 4.3 Schematic cross-sectional diagram of the experimental apparatus used for time-re-solved ESI-MS experiments. Syringes I and 2 deliver a continuous flow of reactants mixing of the two solutions initiates the reaction of interest. The inner capillary can be automatically pulled back together with syringe I (as indicated by the dashed arrow), thus providing a means to control the average reaction time. Solid arrows indicate the directions of liquid flow. Small arrows in the ESI source region represent the directions of air flow [95]. Reprinted with permission from Wilson, D.J., Konermann, L. (2003) A Capillary Mixer with Adjustable Reaction Chamber Volume for Millisecond Time-Resolved Studies by Electrospray Mass Spectrometry. Anal. Chem. 75 6408-6414. Copyright (2003) American Chemical Society...

Experimental design of time-resolved mass spectrometry (TRMS) systems can greatly affect temporal resolution in the analysis of dynamic samples (see also Section 4.2). The two popular MS approaches - laser desorption/ionization (LDI)-MS and electrospray ionization (ESI)-MS - are suitable for studies of enzymatic reactions [8]. 

Current proteomics studies rely almost exclusively on 2D gel electrophoresis to resolve proteins before MALDI-TOF or ESI-MS/MS approaches. A drawback of the 2D gel approach is that it is relatively slow and work intensive. In addition, the in-gel proteolytic digestion of spots followed by mass spectrometry is a one-at-a-time method that is not well suited for high throughput studies. Therefore, considerable effort is being directed towards alternate methods for higher throughput protein characterization. 

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