16,121 Views
•
11:14 min
•
April 14, 2015
DOI:
The overall goal of this procedure is to monitor the structure of a protein at high resolution using emit hydrogen deuterium exchange and side chain lytic labeling coupled with mass spectrometry. This is accomplished by first lyophilize, the protein in the presence of different excipients. In solid state hydrogen deuterium exchange, the lyophilized protein is exposed to deuterium oxide vapor in a sealed desiccate under controlled relative humidity and temperature.
For lytic labeling, the protein is lyophilized with excipients and a photoreactive agent. The vial is then irradiated with the UV light to covalently attach the agent to the protein. For each method, the samples are reconstituted and analyzed using a mass spectrometer at both intact protein and peptide level resolution.
These two methods provide complementary information. Formulations in which protein structure is highly retained, show decreased deuterium uptake and increased lytic labeling, whereas those with perturbed structure show increased deuterium uptake and decreased lytic labeling. Chemical labeling, such as hydrogen deuterium exchange and photo cross linking coupled with mass spectrometric analysis has recently been adapted to study protein structure and interactions in lively supports.
The main advantage of these techniques over existing methods like CD and FDIR spectroscopy is that protein structure and environment can be probed with high resolution in the solid state. To begin, place 200 milliliters of deuterium oxide in the lower compartment of a desiccate and add 400 grams of potassium carbonate To make a saturated solution, place the porcelain desiccate plate inside and seal the desiccate airtight. Allow it to equilibrate at five degrees Celsius to reach a stable relative humidity close to 43%Next place the uncapped vials containing lyophilized protein in the upper compartment of the desiccate seal the desiccate and incubate at five degrees Celsius.
To initiate the hydrogen deuterium exchange reaction to collect a sample, cap the vial immediately after removing it from the desiccate and then quench the reaction by flash freezing the vial and liquid nitrogen. Store the vial at negative 80 degrees Celsius until mass spectrometric analysis. Use high resolution mass spectrometry to analyze the samples to minimize back exchange.
How is the liquid chromatographic system inside a refrigerated box? To set up the instrument first, connect the sample loop and the protein trap to the valve that controls the desalting and the elution processes. Next, calibrate the system by injecting the low concentration tuning mix into the mass spectrometer and setting the mass to charge ratio range of 200 to 3, 200.
Then cool the refrigerated system to a stable operating temperature of about zero degrees Celsius. Prepare the quenching buffer containing 0.2%formic acid and 5%methanol in water and chill it on ice. After transferring the samples to liquid nitrogen, carefully remove a vial and reconstitute the sample by the addition of two milliliters of quenching buffer.
Next load an appropriate HPLC and mass spectrometry method. To analyze the sample here, desalt 20 picomoles of myoglobin in the protein trap for 1.7 minutes with 5%acetyl nitrile and 0.1%formic acid. Then elute the protein over a 3.3 minute gradient, increasing to 80%aceto nitrile, and 0.1%formic acid.
Inject the sample and collect the mass spectra over a 200 to 3, 200 mass to charge ratio range. To determine the mass of the intact protein as a reference, use the same method with an undue rated protein sample. Obtain the protein mass by deconvoluting the raw spectra using data analysis software For myoglobin sample analysis, set the mass range from 15 to 18, kilodaltons the mass resolution to one Dalton and the peak kite to 90%For peptide analysis, prepare the samples using the same protocol as for intact proteins.
When samples are ready to be analyzed, connect an immobilized pepin column and an analytical column to the valve and replace the protein trap With a peptide trap. Calibrate the system for peptides has performed for intact proteins, but by setting the mass to charge ratio range of 100 to 1700, then cool the refrigerated system to a stable operating temperature of about zero degrees. Once the system is calibrated, program the appropriate HPLC and mass spectrometry method.
Here digest 20 picomoles of myoglobin on the Pepin column with 0.1%formic acid in water program the method to trap and desalt the peptides in the peptide trap for 1.7 minutes with 10%acetyl nitrile and 0.1%formic acid, and to elute the peptides in four minutes with a gradient increasing to 60%acetyl nitrile and 0.1%formic acid. Next, inject the sample and collect the mass spectra over a mass to charge ratio range of 100 to 1700. Analyze an undated peptide sample with tandem mass spectrometry to identify the peptic fragments.
Then crosscheck the experimentally determined fragment with software generated predicted masses. Next, set the mass cutoff to 10 parts per million to eliminate masses with high error for peptides with matches. Note the peptide sequence charge state, and the retention time.
Use the compiled peptide data to calculate the average number of deuteros incorporated per peptic fragment Using the data analysis software First, lyophilize the protein with the excipient and lucine as indicated in the text protocol. After preparing the samples switch on a UV crosslinker containing 365 nanometer UV lamps. Allow the lamps to warm for five minutes.
Once the lamps have warmed, turn them off and place the uncapped vials containing the formulation inside of the crosslinker chamber. Irradiate the samples with UV light for 40 minutes and include samples without photo leucine as a control After irradiating cap and store the vials at negative 20 degrees Celsius until mass spectrometric analysis. Prepare the samples for analysis by adding mass spectrometry grade distilled water to yield a final protein concentration of two micromolar.
Next, analyze the samples using the same mass spectrometry method as for the intact deuterated proteins. But do not use the refrigerated lc system. Acquire mass spectrometry data for an unlabeled protein sample to determine the native mass of the protein.
Perform the data analysis as for the DERATED samples for peptide level analysis. First, perform solid state photo litic labeling with intact proteins and store them at negative 20 degrees Celsius. Next, reconstitute the sample with 100 millimolar ammonium bicarbonate buffer to yield a final protein concentration of 10 micromolar.
Mix the sample with trypsin in a 10 to one molar ratio of protein to trypsin and incubate this mixture at 60 degrees Celsius for 16 hours. In the meantime, connect the sample loop, the peptide trap and the analytical column to the HPLC system valve. After digesting the protein, quench the reaction by adding 0.1%formic acid in water to yield a final protein concentration of two micromolar.
Next program the system with the HPLC and the mass spectrometry methods. Here inject and desalt 20 picomoles of the digested myoglobin in the peptide trap for 1.5 minutes with 5%aceto nitrile and 0.1%Formic acid elute the peptides by increasing the gradient over 22 minutes to 55%acetyl nitrile and collect the mass spectra over a range from 100 to 1700 mass to charge ratio. Use online tool to calculate the theoretical mass of the peptide.
Photo leucine adduct with the numbers of photo leucine previously obtained from the intact protein analysis. Include at least four missed cleavages. Create a mass list in Excel for peptide photo leucine adduct.
Next, match the theoretical mass list with experimentally observed masses by setting a cutoff point to identify masses with low error. During hydrogen deuterium exchange, unfolding of the protein allows replacement of hydrogen with deuterium proteins that retain their structure are less prone to deuterium exchange. When solid state hydrogen deuterium exchange mass spectrometry was performed on talose containing and sorbitol containing myoglobin formulations, the sorbitol containing formulation showed 46%greater deuterium uptake, suggesting that the structure of myoglobin in sorbitol is more perturbed during the lytic process.
Photo leucine cross links to accessible amino acids side chains. Solid state photo lytic mass spectrometry analysis of the sorbitol and TLOs containing myoglobin formulations showed more photo eine uptake in the TLOs formulation than its counterpart. This suggests that the side chains remained accessible in the TLOs formulation.
Both hydrogen deuterium exchange and lytic labeling use commercially available regions and readily available MS instrumentation to characterize proteins in solid state for any given formulation. The total time record from sample preparation to complete data analysis is less than two weeks. After watching this video, you should have a good understanding of how to obtain high resolution information about protein structure and its importance in the design of stable lyophilized protein formulations.
Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.
07:49
Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa
Related Videos
80785 Views
09:18
Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics
Related Videos
9926 Views
08:23
2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes
Related Videos
11435 Views
09:30
Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy
Related Videos
9453 Views
07:19
Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering
Related Videos
12696 Views
07:33
Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry
Related Videos
14321 Views
11:32
A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes
Related Videos
8086 Views
10:36
Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry
Related Videos
5455 Views
15:35
Analyzing Large Protein Complexes by Structural Mass Spectrometry
Related Videos
24258 Views
08:48
High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water
Related Videos
1782 Views
Read Article
Cite this Article
Moorthy, B. S., Iyer, L. K., Topp, E. M. Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders. J. Vis. Exp. (98), e52503, doi:10.3791/52503 (2015).
Copy