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.
