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.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:244px;">Poly(methyl methacrylate) or PMMA</td>
			<td style="width:136px;">Professional Plastics</td>
			<td style="width:145px;">SACR.250CCP</td>
			<td style="width:159px;">8.9 cm x 3.8 cm x 0.6 cm</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Fused Silica Glass Capillary</td>
			<td style="width:136px;">Polymicro Technologies</td>
			<td style="width:145px;">106815-0018</td>
			<td style="width:159px;">ID: 75&#181;m, OD: 150&#181;m</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">Metal Capillaries</td>
			<td style="width:136px;">McMaster-Carr</td>
			<td style="width:145px;">28 ga &#8211; 89875K97 
			30 ga&#160; - 89875K99</td>
			<td style="width:159px;"></td>
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		<tr height="52">
			<td height="52" style="height:52px;width:244px;">Fluorinated Ethylene Propylene (FEP) Tubing</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">1477 
			1548</td>
			<td style="width:159px;">ID: 0.007&#8221;, OD: 1/16&#8221; 
			ID: 0.020&#8221;, OD: 1/16&#8221;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Standard Polymer Tubing Cutter</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">A-327</td>
			<td style="width:159px;">for 1/16&#8221; and 1/8&#8221; OD tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Micro Static Mixing Tee</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">M-540</td>
			<td style="width:159px;">for 1/16&#8221; OD tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">or</td>
			<td style="width:136px;"></td>
			<td style="width:145px;"></td>
			<td style="width:159px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Stainless Steel Tee, 0.25mm Bore</td>
			<td style="width:136px;">Valco Instruments Co., Inc. (VICI)</td>
			<td style="width:145px;">ZT1C</td>
			<td style="width:159px;">for 1/16&#8221; OD tubing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">PEEK Tee for 1/16&#8221; OD Tubing</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">P-727</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">10-32 Female to Female Luer</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">P-659</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">10-32 PEEK Double-Winged Nut</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">F-300</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ferrule for 1/16&#8221; OD Tubing</td>
			<td style="width:136px;">IDEX</td>
			<td style="width:145px;">F-142</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">100 Series Rotary Tool</td>
			<td style="width:136px;">Dremel</td>
			<td style="width:145px;">F013010001</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Cut-Off Discs</td>
			<td style="width:136px;">Jobmate</td>
			<td style="width:145px;"></td>
			<td style="width:159px;">1/64&#8221; thickness</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:244px;">Stereomaster Digital Zoom Microscope</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:145px;">12-563-411</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Soldering Iron</td>
			<td style="width:136px;">Mastercraft</td>
			<td style="width:145px;">58-6301-2</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">VersaLaser</td>
			<td style="width:136px;">Universal Laser</td>
			<td style="width:145px;"></td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Syringes</td>
			<td style="width:136px;">Hamilton</td>
			<td style="width:145px;">81220</td>
			<td style="width:159px;">500&#181;L capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe Pumps</td>
			<td style="width:136px;">Harvard Apparatus</td>
			<td style="width:145px;">70-4501</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:244px;">Name</td>
			<td style="width:136px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:159px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Reagents</td>
			<td style="width:136px;"></td>
			<td style="width:145px;"></td>
			<td style="width:159px;"></td>
		</tr>
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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. ...

Conformational flexibility plays a critical role in protein function and the overall goal of Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry is to probe rapid structural changes that drive function in ordered and disordered proteins. This method can help answer key questions in the structural biology field, such as the craterization of transient protein confirmations, that's not a minimal to classical high resolution techniques such as electrocystography and NMR. The main advantage of this technique is that reactions can be monitored on the millisecond time scale, which is important for characterizing loop ridges, Moltinglobyals, and intrinsically disordered proteins.The implications of this technique extend toward therapy of neurodegenerative disorders. As they often involve the misfolding, or aggregation of intrinsically disordered protein regions. Though this method can provide insight into weekly structured proteins, it can also be applied to other systems, such as enzymes undergoing catalytic turnover, or the characterization of protein, protein and protein, ligan, interactions.To begin this procedure, laser a plate through an input channel for introducing the reagents, a pro dialysis chamber and an output channel, on a standard PMM8 block, using a laser engraver. Following this, cut a 30 gauge stainless steel metal capillary into two 10 centimeter pieces using a rotary tool for the 164th inch thick cut off disk. Use sand paper to smooth the end of the capillaries, which can be facilitated by viewing under a light microscope.Now, melt that capillaries into the etched PMM8 block using a sauntering iron. To construct a continuous flow, time resolved kinetic mixer, insert a 40 centimeter fused cilica glass capillary into a 28 gauge, steel, metal capillary of approximately 15 centimeters. Next, produce a two millimeter notch, using low power laser engraver settings on one end of the inner glass capillary and seal this end of the inner glass capillary.This is a critical step that ensures orthogonal and efficient mixing of the protein, which exits through the notch which an on common deterior. The two millimeter inter capillary space between the notch and the end of the capillary is the mix and volume. Attach appropriate size fittings and tubing sleeves and incorporate a T connector, which will be used to deliver deterium.Next, line up the inner glass capillary with the end of the metal capillary which can be facilitated by viewing under a light microscope. Mark this as the zero millimeter position. Attach this kinetic mixer to one end of the mixing T.Then attach a 40 centimeter fused silica glass capillary to the opposite end of the kinetic mixer on the mixing T.For pepsin activation, suspend 20 milligrams of pepsin from Porsena gassic mucosa in one milliliter of coupling buffer.Add 50 milligrams of NHS activated agro speeds to the resuspended protiens and rotate gently over night, at four degrees Celsius. On the following day, spin down the mixture at one thousand times G for two minutes at room temperature to collect the resin. Then aspirate the unbound protiyes.Following this, incubate the agros in one milliliter of blocking buffer and rotate gently at roomtemperature for one hour. After collecting the resin and aspirating the blocking buffer, incubate the pepsin agguros with one milliliter of five percent acedic acid for five minutes. Spin down the mixture at 1, 000 times G, for two minutes at room temperature, to collect the resin, then aspirate the super natent.After repeating the previous steps, for a total of three washes, store the beads in one milliliter of acedic acid at four degrees Celsius for longterm use. To assemble the device, fill a protialysis chamber with a slurry of activated pepsin agguros beads in five percent acedic acid using a sterile spatula. Place the PMMA micro falitic platform in between two blank PMMA blocks as a cover to seal the device, lined with silicone rubber to create a liquid tight seal.Use metallic clamping plates to pressure seal the device. Then attach the mixing T, to the inlet of the device using appropriate fittings. Now flow five percent acedic acid thought the acid line at a rate of ten microliters a minute.Then flow 50 millimeter amonia acid buffer through the protein line at a rate of one microliter per minute. Couple the device to the front end of a modified, quadrapole time of flight, or Qtof mass spectrometer, using and adjustable insulated stage to achieve optimal electro spray conditons. After setting the ESI-MS conditions, acquire a pepsin only spectrum.Next, introduce 50, to 100 micromoler of tau, or phospho-tau protein at a rate of one microliter per minute, where the 50 millimeter amonium acitate buffer was previously flowing. After allowing the system to equilibrate for at least 10 minutes, before acquisition, acquire protein only spectrum. While the Tao, or phospho tao protein is flowing, introduce duteriumoxide at a rate of three microliters per minute, via the T connector, and allow to react in the kinetic mixer.In order to increase the labeling time, manually pull back the position of the inner glass capillary to achieve mixing times of 42 milliseconds to eight seconds, allowing the system to equilibrate for at least 10 seconds in between each pull back. This animation depicts the workflow for a typical experiment. Protein exits through the notch and mixes with the oncoming duterium.Backbone M8 hydrogens are exchanged during the allotted reaction time, which can be varied by pulling back the protein capillary. The reaction is then quenched and then digested on a micro fluidic platform containing an outlet ESI needle. The resulting spectrum contains digested protein.The percentage of deuterium uptake is calculated for individual peptides and then mapped onto the 3D protein structure. Digestion profiles of native and phospho-tau were similar, yielding a sequence coverage of 77.1 and 71.7 percent, respectively, the best fitting isotopic distributions and the associated deuterium uptake values for four simple peptides are shown here. Observed kinetic plot of percent to deuterium uptake, versus time, for each peptide are used to extract the observed experimental rate.The calculated random quale profiles are shown for comparison and are used to extract the theoretical intrinsic grade. As expected, no protection factors were observed in the range, normally associated with secondary structure elements, confirming that native tau exhibits weak internal hydrogen bonding. Significant protection is observed at the N and C termini, the central domain, and the aggregation proned regions.In hyper phospho related tau, a general increase was observed in deuterium uptake across the protein. There are significant increases at the N and C termini and in the hexapeptide two region. Degree of protection factors are colored as a rainbow scheme, as shown here.Once mastered, this technique can be done in a few hours, if performed properly. While attempting this procedure, it is important to use regions of HPC grade, or higher, to minimize contaminants as they can interfere with analysis. Following this procedure, other methods, such as covalent labeling, chemical cross linking, computational modeling and docustudies can be performed in order to match three dimensional protein structures, or confirm areas of protein protein and protein legin interaction.After its development, this technique paved the way for researchers in the field of structural biology, to explore intrinsically disordered proteins and domains, most common in nuerodegenerative disease. After watching this video, you should have a good understanding of how to use time resolved electro spray ionization, hydrogen deuterium exchange, mass spectometry, for the study of protein structure and dynamics.

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Biochemistry

9.7K visualizações.  York University. Conformational flexibility plays a critical role in protein function and the overall goal of Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry is to probe rapid structural changes that drive function in ordered and disordered proteins. This method can help answer key questions in the structural biology field, such as the craterization of transient protein confirmations, that's not a minimal to classical high resolution techniques such as electrocystography an...