Name: time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry for studying protein structure and dynamics
Uploaded: 2017-04-17T12:30:00
Description: Watch this Scientific Journal Video about Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics at JoVE.com

We gratefully acknowledge Dr. Markus Zweckstetter for providing the pdb coordinate file for the 'native' tau ensemble predicted from his NMR work, with contributed analysis tools provided by Dr. Adnan Sljoka. Funding for this work was provided by the Natural Science and Engineering Research Council of Canada (NSERC) ENGAGE Grant program.

NOTE: Please consult all relevant material safety data sheets (MSDS) before use. Fumes produced by laser ablation of poly(methyl methacrylate) (PMMA) can be toxic. Be sure that the laser engraver is connected to a working ventilation system. Use all appropriate safety practices when building the microfluidic device including the use of engineering controls (fume hood, sharps container) and personal protective equipment (safety glasses, face mask, gloves, lab coat, full length pants, closed-toe shoes). It is of utmost imp...

<ol>
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While structural biology methods such as X-ray crystallography and NMR are advantageous because they provide extremely detailed structures of proteins, these pictures are often static. The characterization of transient species and weakly structured domains continues to be elusive when studied by these conventional methods. Therefore, in order to gain dynamic insights on these types of systems it is important to work at rapid time scales. We have successfully applied TRESI-HDX-MS to obtain detailed insights on the conform...

Over the past several decades, significant advancements have been made in the development of analytical techniques designed to measure protein structure and dynamics1,2,3,4. While X-ray crystallography remains the principle tool for determining protein structure, high concentrations of protein are needed and extensive optimization is required to produce diffraction quality crystals. Proteins that are difficult to crystallize, such as membrane-associated and intrinsically disordered proteins have classically been studied by hydrogen-deuterium exchange (HDX) NMR5. However, in recent decades, coupling of electrospray ionization mass spectrometry (ESI-MS) to HDX has rapidly gained popularity6,7.
Mass spectrometry offers a solution to many of the restrictions posed by X-ray crystallography and NMR. In particular, MS is highly sensitive (nM to &#181;M concentrations required), and there is virtually no limit on protein size. In addition, the high duty cycle of MS analysis allows for the possibility of studying proteins as they undergo enzymatic turnover, misfolding, complexation and other biologically-relevant processes. These processes often occur on the millisecond to second time scale and require rapid mixing of reagents prior to analysis.
The development of Time-Resolved ElectroSpray Ionization (TRESI) by Wilson and Konermann in 2003 allowed reactions to be monitored in pseudo-real time by ESI-MS. Their setup incorporated a capillary mixer with a continuously adjustable reaction chamber volume8. The device consists of two concentric capillaries, with the inner capillary sealed and a notch cut into its side to allow for mixing within the narrow inter-capillary space from the notch to the end of the inner capillary (typically 2 mm). When applied to HDX experiments, the inner capillary carries the protein of interest, the outer capillary carries the labeling D2O solution, which then undergoes mixing with the protein before entering the adjustable reaction chamber allowing for HDX labelling prior to direct transfer into the ESI source.
Briefly, HDX relies on backbone amide hydrogens undergoing exchange with deuterium atoms in solution9,10. The exchange is base-catalyzed at physiological pH, with acid-catalysis becoming prevalent at pH below approximately 2.6. The rate of exchange is based on four main factors: pH, temperature, solvent accessibility and intramolecular hydrogen bonding. As the former two factors are kept constant throughout the experiment, the rate of exchange, particularly at peptide backbone amide positions, is primarily dependent on protein structure11. Tightly folded regions with extensive, stable hydrogen bonding networks in &#945;-helices and &#946;-sheets will take up deuterium at substantially slower rates compared to loops and disordered regions (and sometimes not at all)12. This allows for global protein analysis, where perturbations in structure (e.g., upon aggregation or substrate binding) lead to differing deuterium uptake (Figure 1).
The kinetic capillary mixer can be incorporated into a microfluidic platform containing a proteolytic chamber for localization of the deuterium uptake. This proteolytic chamber is held at low pH in order to effectively quench the exchange reaction, and requires an immobilized acid protease in order to digest the protein into localized peptides (Figure 2). Monitoring backbone exchange at millisecond to second time scales is especially important for the characterization of conformational changes within difficult to characterize loop regions, molten globules, and intrinsically disordered proteins (IDPs)13,14. Alternatively, TRESI-HDX can also be used to characterize proteins that currently do not have a solved atomic structure through the methods of X-ray crystallography and NMR, using deuterium exchange coupled to the COREX algorithm (DX-COREX) approach15,16. This detailed protocol will apply TRESI-HDX to study tau, an IDP, in both it&#39;s native form as well as it&#39;s pathogenic hyperphosphorylated state. While native tau is one of the most well studied IDPs, little is known about its amyloidogenic counterpart13.

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time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry for studying protein structure and dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Digestion profiles of native and phospho-tau were similar, yielding a sequence coverage of 77.1 and 71.7% respectively. Deuterium uptake values of each peptide was determined by fitting the observed isotopic distributions with the theoretical distributions generated using an in-house developed FORTRAN software. The best fitting distributions are shown (Figure 3a) along with the associated deuterium uptake values. Uptake kinetic profiles are then generated, and were well d...

Watch this Scientific Journal Video about Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics at JoVE.com

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Research

JoVE Journal

Biochemistry

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