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

Summary

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.

Abstract

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.

Introduction

Over the past several decades, significant advancements have been made in the development of analytical techniques designed to measure protein structure and dynamics^1,2,3,4. 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 popularity^6,7.

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 µM 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 solution^9,10. 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 α-helices and β-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) approach^15,16. This detailed protocol will apply TRESI-HDX to study tau, an IDP, in both it's native form as well as it's pathogenic hyperphosphorylated state. While native tau is one of the most well studied IDPs, little is known about its amyloidogenic counterpart¹³.

Protocol

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 importance to use High Performance Liquid Chromatography (HPLC) grade reagents whenever possible, with all being of ACS grade or higher to decrease interfering contaminants during analysis.

1. Preparation of the Microfluidic Device

1. Construction of the PMMA Microfluidic Platform
   1. Obtain a standard PMMA block (8.9 x 3.8 x 0.6 cm) and laser-ablate an input channel for introducing the reagents, a proteolysis chamber, and an output channel using a laser engraver^17,18.
      NOTE: Etch the proteolysis chamber in an elongated oval shape (30 x 5 x 0.05 mm). The input and output channel must extend to the end of the PMMA block and be no larger than 75 µm in both width and depth in order to accommodate a 30 ga capillary.
   2. Cut a 30 ga stainless steel metal capillary into two pieces of approximately 10 cm each using a rotary tool with a 1/64" thick cut-off disc. Use sandpaper to smooth the ends of the capillaries (this can be facilitated by viewing under a light microscope).
   3. Melt the capillaries into the etched poly(methyl methacrylate) block using a soldering iron. The input channel will be connected to automated syringe pumps, and the output channel will be used for coupling into the MS.
2. Construction of the Continuous-Flow Time-Resolved Kinetic Mixer
   1. Obtain a fused silica glass capillary (ID: 75 µm, OD: 150 µm) of approximately 40 cm, and insert it into a 28 ga stainless steel metal capillary of approximately 15 cm. Depending on the reaction time required, use a longer outer metal capillary or one with a larger ID.
      NOTE: Determining the 'true' inner diameter of the metal capillary is critical for obtaining accurate reaction times. This can be done by attaching the metal capillary to an HPLC, flowing solvent though the capillary, and recording the back pressure. A backpressure to inner diameter calculation can be made, and the true inner diameter of the metal capillary determined. This can be calculated using the Molecular Weight Calculator software (for Windows Version 6.49).
   2. Produce a 2 mm notch using low power laser engraver settings on one end of the inner glass capillary and seal this end of the inner glass capillary (Figure 2). Alternatively, make the notch using a ceramic glass capillary cutter tool or a rotary grinder with a fine cutting blade.
   3. Line-up the inner glass capillary with the end of the metal capillary (this can be facilitated by viewing under a light microscope).
   4. Attach this kinetic mixer to one end of the mixing tee (Figure 2).
   5. Attach a fused silica glass capillary (ID: 75 µm, OD: 150 µm) of approximately 40 cm to the opposite end of the kinetic mixer on the mixing tee. This is used to deliver acid (5% acetic acid, pH 2.4) to quench the reaction.
      NOTE: Reactants are supplied to the device with gas-tight syringes through polytetrafluoroethylene (PTFE) tubing using automated infusion pumps.
3. Pepsin Activation
   1. Weigh out 20 mg of pepsin from porcine gastric mucosa and suspend it in 1 mL of coupling buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 6.0).
   2. Weigh out 50 mg of N-hydroxysuccinimide (NHS)-activated agarose beads, add to the re-suspended protease and rotate gently overnight at 4 °C.
   3. Spin down at 1,000 x g for 2 min at room temperature to collect the resin.
   4. Aspirate the unbound protease.
   5. Incubate the agarose in 1 mL of blocking buffer (1 M Tris-HCl, pH 6.0) and rotate gently at room temperature for 1 h.
   6. Spin down at 1,000 x g for 2 min at room temperature to collect the resin.
   7. Aspirate the blocking buffer.
   8. Incubate the pepsin-agarose with 1 mL of 5% acetic acid, pH 2.4 for 5 min.
   9. Spin down at 1,000 x g for 2 min to collect the resin.
   10. Aspirate the supernatant.
   11. Repeat steps 1.3.8 - 1.3.10 for a total of three washes.
   12. Store the beads in 1 mL acetic acid at 4 °C for long-term use.
4. Device Assembly
   1. Fill the proteolysis chamber with the slurry of activated pepsin-agarose beads in 5% acetic acid using a sterile spatula.
   2. Place the PMMA microfluidic platform in between two blank PMMA blocks as a cover to seal the device, lined with silicone rubber to create a liquid tight seal.
   3. Use metallic clamping plates to pressure-seal the device.
      NOTE: The clamp was custom made in order to fit the microfluidic platform and is composed of two plates measuring 10.1 cm x 7.4 cm x 1.2 cm.
   4. Flow 5% acetic acid, pH 2.4 at a rate of 10 µL/min. Flow 50 mM ammonium acetate buffer (pH 6.9) though the protein line at a rate of 1 µL/min.
      NOTE: It is extremely important that the acetic acid is continuously flowing through the device throughout the entirety of the experiment. Ensure that there are no leakages and that fluid is exiting only from the output channel, which will serve as the ESI source.
   5. Couple the device to the front end of a modified quadrupole time-of-flight (Q-TOF) mass spectrometer using an adjustable insulated stage to achieve optimal electrospray conditions.
      NOTE: A bypass switch is introduced in order to simulate the presence of a commercial ESI source.¹⁷

2. Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange

1. Acquisition of Pepsin and Protein Only Spectra
   NOTE: ESI-MS acquisition is carried out in positive ion mode with a voltage of +4,500 to +5,000, 60-V declustering potential, and 250-V focusing potential. Spectra are acquired over a range of 350-1,500 m/z with a scanning rate of 1 s^-1.
   1. Acquire a pepsin only spectrum. Any peaks appearing in this spectrum are subtracted once the protein is added.
   2. Introduce 50-100 µM tau/phospho-tau protein (purify and prepare as previously described^13,19) at a rate of 1 µL/min where the 50 mM ammonium acetate buffer was previously flowing.
   3. Acquire a protein only spectrum.
2. Acquisition of Time Points
   1. While 100 µM tau/phospho-tau protein is flowing at 1 µL/min, introduce D₂O at a rate of 3 µL/min via a tee connector and allow to react in the kinetic mixer. Allow for the system to equilibrate for at least 10 min before acquisition of the spectrum.
      NOTE: After the exchange, the labelling reaction is quenched by the flow of acetic acid pH 2.4 at 10 µL/min and digestion of the labelled protein occurs in the proteolytic chamber.
   2. In order to increase the labelling time, manually pull back the position of the inner glass capillary to achieve mixing times of 42 ms to 8 s. Allow for the system to equilibrate for at least 10 min in between each pull-back.

3. Data and Statistical Analysis

1. Identifying Peptides and Calculating the Percentage of Deuterium Exchange
   1. Perform MS spectra analyses using mMass software, version 5.5.0²⁰.
   2. Identify peptides using the ExPASy FindPept proteomic server and confirm by collision-induced dissociation (CID) when required²¹.
      NOTE: Here, deuterium uptake incorporation was measured using an in-house developed FORTRAN software for isotopic distribution analysis^22,23.
   3. Calculate the theoretical intrinsic rates based on the primary sequence using the SPHERE web tool^22,24.
   4. Fit the data using single exponential non-linear regression and normalize using a graphing and statistical software (e.g., SigmaPlot).
      NOTE: The ratio of k_int / k_obs yields the Protection Factor (PF), a semi-quantitative measure of the degree that a particular region is structured within the conformational ensemble.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Poly(methyl methacrylate) or PMMA	Professional Plastics	SACR.250CCP	8.9 cm x 3.8 cm x 0.6 cm
Fused Silica Glass Capillary	Polymicro Technologies	106815-0018	ID: 75 µm, OD: 150 µm
Metal Capillaries	McMaster-Carr	28 ga – 89875K97 30 ga  - 89875K99
Fluorinated Ethylene Propylene (FEP) Tubing	IDEX	1477 1548	ID: 0.007”, OD: 1/16” ID: 0.020”, OD: 1/16”
Standard Polymer Tubing Cutter	IDEX	A-327	for 1/16” and 1/8” OD tubing
Micro Static Mixing Tee	IDEX	M-540	for 1/16” OD tubing
or
Stainless Steel Tee, 0.25 mm Bore	Valco Instruments Co., Inc. (VICI)	ZT1C	for 1/16” OD tubing
PEEK Tee for 1/16” OD Tubing	IDEX	P-727
10-32 Female to Female Luer	IDEX	P-659
10-32 PEEK Double-Winged Nut	IDEX	F-300
Ferrule for 1/16” OD Tubing	IDEX	F-142
100 Series Rotary Tool	Dremel	F013010001
Cut-Off Discs	Jobmate		1/64” thickness
Stereomaster Digital Zoom Microscope	Fisher Scientific	12-563-411
Soldering Iron	Mastercraft	58-6301-2
VersaLaser	Universal Laser
Syringes	Hamilton	81220	500 µL capacity
Syringe Pumps	Harvard Apparatus	70-4501
Name	Company	Catalog Number	Comments
Reagents
NHS-Activated Agarose	Fisher Scientific	26196
Pepsin from Porcine Gastric Mucosa	Sigma-Aldrich	P6887-250MG
Deuterium Oxide	Sigma-Aldrich	151882-10X0.6ML
Acetic Acid	Sigma-Aldrich	695092-100ML
HPLC Grade Water	Fisher Scientific	W5-4
Ammonium Acetate	Sigma-Aldrich	A7330-500G
Sodium Phosphate	Fisher Scientific	S369-500
Sodium Chloride	Fisher Scientific	S671-3
Name	Company	Catalog Number	Comments
Software/Online Tools
CorelDraw X3	Corel
Molecular Weight Calculator		Version 6.49	Open Source MS Tool
mMass		Version 5.5.0	Open Source MS Tool
ExPASy FindPept	Swiss Institute of Bioinformatics
SigmaPlot	Systat Software	Version 11.0
PyMOL	Schrödinger	Version 1.5.0.4
Name	Company	Catalog Number	Comments
Instruments
QStar Elite Hybrid Q-TOF Mass Spectrometer	AB SCIEX