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Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins
氧敏感蛋白的氢氘交换质谱法   

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参见作者原研究论文

本实验方案简略版
Biochimica et Biophysica Acta
Oct 2017

Abstract

The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen sensitive chemistry. A problem when using HDX-MS to study anaerobic proteins is that there are many parts that require constant movement into and out of an anaerobic chamber. This can affect the seal, increasing the likelihood of oxygen exposure. Exposure to oxygen causes the cofactors bound to these proteins, a common example being FeS clusters, to no longer interact with the amino acid residues responsible for coordinating the FeS clusters, causing loss of the clusters and irreversible inactivation of the protein. To counteract this, a double vial system was developed that allows the preparation of solutions and reaction mixtures anaerobically, but also allows these solutions to be moved to an aerobic environment while shielding the solutions from oxygen. Additionally, movement isn’t limited like it is in an anaerobic chamber, ensuring more consistent data, and fewer errors during the course of the reaction.

Keywords: HDX-MS (HDX-MS), Mass spectrometry (质谱法), Anaerobic proteins (厌氧蛋白), H/D exchange (H/D交换), Protein dynamics (蛋白质动力学), Protein-protein interactions (蛋白-蛋白相互作用), Protein-ligand interactions (蛋白-配体相互作用)

Background

Many oxygen sensitive proteins are required for organisms to thrive in an anoxic environment. Some of these proteins provide an alternative supply of energy to anaerobic microbes through a process known as Flavin-based electron bifurcation (FBEB) (Lubner et al., 2017). FBEB generates reduced ferredoxin, which can be oxidized to produce energy. Proteins that are capable of reducing ferredoxin are of great interest and have been the focus of recent studies using HDX-MS (Demmer et al., 2016; Lubner et al., 2017; Berry et al., 2018). HDX-MS is a powerful technique for investigating protein stability, dynamics, and ligand binding providing information about the relationship between structure and function. HDX-MS uses the intrinsic property of amide hydrogens to exchange with hydrogens in solution to track changes in the structure and dynamics of a protein/protein complex. By preparing buffers with heavy water (D2O) instead of monoisotopic water (H2O), amide hydrogens on a protein will exchange with the deuterium in solution. The rate of exchange for a given amino acid is influenced by the stability of hydrogen bonds in the secondary structure, as well as the tertiary and quaternary interactions within a single protein or protein complex. Using mass spectrometry, deuterium incorporation is determined by measuring the shift in isotope distribution between deuterated and non-deuterated samples. HDX-MS has been applied to a large number of proteins and protein complexes across a wide range of conditions. To successfully study these proteins with HDX-MS, it was imperative to establish a means of performing this reaction on the benchtop to avoid heavy traffic into and out of an anaerobic chamber which is time consuming and burdensome. The problem was then how to allow manipulation of the sample while keeping the protein sample anaerobic for an extended period of time in an aerobic environment. To solve this problem, the reaction mixture and protein stock solutions were placed into a double vial system that allowed addition and removal of sample while maintaining strict anaerobic conditions. The logic behind the setup was to create an airlock. Vials are placed under positive pressure with nitrogen gas with a screw cap vial, inside a larger crimp vial that contains reductant. With this double barrier system, small volumes of air can be trapped in the outer vial and do not contact the sample.

Materials and Reagents

  1. VerexTM vial kit, 9 mm, screw top, polypropylene, 300 μl + PTFE/silicone cap, blue, 1,000/pk (Phenomenex, catalog number: AR0-9991-13 )
  2. Clear glass serum vial with 20 mm crimp top finish, 10 ml, 100/case (DWK Life Sciences, WHEATON, catalog number: 225278 )
  3. Curwood Parafilm MTM laboratory wrapping film (Bemis, catalog number: PM996 )
  4. Costar Microcentrifuge Tubes 0.65 ml, 500/bag (Corning, catalog number: 3208 )
  5. Onyx Monolithic C18 column, 100 x 2 mm (Phenomenex, catalog number: CH0-8467 )
    Alternative column (see Notes for a detailed explanation): Onyx Monolithic C18 column, 100 x 3 mm (Phenomenex, catalog number: CH0-8158 )
  6. Model 1701 and 1702 small RN syringes, 10 μl (26s gauge) and 25 μl (22s gauge), 2” needle point style 2 (Hamilton, catalog numbers: 80030 and 80230 )
  7. Unlined aluminum open-top seals, 20 mm, 1,000/case (DWK Life Sciences, WHEATON, catalog number: 224178-05 )
  8. 20 mm stopper, straight plug, ultra-pure (DWK Life Sciences, WHEATON, catalog number: W224100-405 )
  9. 200 μl Pipet Tips (VWR, catalog number: 53508-810 )
  10. Liquid nitrogen
  11. Purified ferredoxin (Fd) in 50 mM Ammonium Acetate buffer at pH 6.8 in H2O (stock concentration 150 μM) from Pf
    Note: pH adjusted with 1 N HCl.
  12. Purified NADH-dependent ferredoxin-NADP+ oxidoreductase (Nfn) in 20 mM Tris, 150 mM NaCl buffer at pH 8 in H2O (stock concentration 16.5 mg/ml) from the organism Pyrococcus furiosus (Pf)
    Note: pH adjusted with 1 N HCl.
  13. Sodium dithionite (Merck, catalog number: 1065051000 )
  14. Nicotinamide adenine dinucleotide (NAD+, Cayman Chemical, catalog number: 16077 , 500 mg)
  15. Nicotinamide adenine dinucleotide (NADH, Cayman Chemical, catalog number: 16078 , 500 mg)
  16. Nicotinamide adenine dinucleotide phosphate (NADP+, Cayman Chemical, catalog number: 10004675 , 50 mg)
  17. Nicotinamide adenine dinucleotide phosphate (NADPH, Cayman Chemical, catalog number: 9000743 , 25 mg)
  18. 99.5% formic acid (Fisher Scientific, catalog number: A117-50 )
  19. Pepsin from porcine gastric mucosa (Sigma-Aldrich, catalog number: P6887-1G )
  20. Sodium acetate trihydrate (Fisher Scientific, catalog number: S607-500 )
  21. Ammonium acetate, ≥ 99% (Sigma-Aldrich, catalog number: 09689-250G )
  22. 37% hydrochloric acid (= 12.1 M) (Merck, catalog number: HX0603-3 )
  23. Sodium hydroxide (Fisher Scientific, catalog number: BP359-500 )
  24. Deuterium oxide (Sigma-Aldrich, catalog number: 151882-100G )
  25. Tris base (Merck, catalog number: 648311-5KG )
  26. Sodium chloride (Fisher Scientific, catalog number: BP358-212 )
  27. HPLC grade water (Fisher Scientific, catalog number: W5-4 )
  28. HPLC grade acetonitrile (Fisher Scientific, catalog number: A998-4 )
  29. Nanopure water (purified in-house using a Millipore Q-Gard 2)
  30. Tris base, NaCl buffer (pH 7.0) in deuterium oxide (see Recipes)
  31. Tris base, NaCl buffer (pH 7.0) in H2O (see Recipes)
  32. Tris base, NaCl, sodium dithionite buffer (pH 7.0) in H2O (see Recipes)

Equipment

  1. HPLC stack for separation of peptides generated via pepsin digestion (e.g., 1290 Infinity series HPLC stack manufactured by Agilent Technologies) (Agilent Technologies, model: 1290 Infinity Series )
  2. LC/MS Q-TOF system for sample analysis/data acquisition (e.g., 6538 UHD Accurate-Mass Q-TOF LC/MS manufactured by Agilent Technologies) (Agilent Technologies, model: 6538 UHD Accurate-Mass Q-TOF LC/MS )
  3. Glove box capable of maintaining anaerobic conditions under positive inert gas pressure (e.g., MBraun, model: UNIlab Plus Glove Box Workstation )
  4. Nitrogen tank
  5. 20 mm Kebby standard crimper for aluminum seals (Medical Laboratory Supply, catalog number: 2001-00-C01A )
  6. Fisher Scientific isotemp 110 water bath (Fisher Scientific, model: FisherbrandTM IsotempTM, catalog number: S63077Q )
    Note: This product has been discontinued.
  7. Milli-Q purification system (Merck, catalog number: QGARD00D2 )
  8. Pipettes (10 μl and 100 μl) (Eppendorf, catalog numbers: 022478886 and 022478924 )

Software

  1. Microsoft Excel 2016 on Windows 7
  2. UCSF Chimera v. 1.11.2
  3. MassHunter Workstation Software Qualitative Analysis v. B.06.00 (Agilent Technologies)
  4. HDExaminer v. 1.3.0 beta 6 (Sierra Analytics, Inc.)
  5. MassHunter Workstation Software LC/MS Data Acquisition for 6200 series TOF/6500 series Q-TOF v. B.05.01 (Agilent Technologies)
  6. Peptide Analysis Worksheet Freeware Edition (PAWs, ProteoMetrics–freeware edition)
  7. SearchGUI v. 3.2.18 (Compomics)
  8. Peptide Shaker v. 1.16.9 (Compomics)

Procedure

  1. Reaction preparation
    1. Before preparation of stock solutions and reaction mixtures, determine the number of time points. The number of time points dictates the volume of the reaction mixtures. For every time point, 10 μl must be available to add to the quench solution, plus an additional 10 μl for the 24 h/fully deuterated time point. For instance, in the HDX experiment of Nfn, five time points were measured (1 min, 3 min, 15 min, 60 min, 3 h, and 24 h) (Berry et al., 2018). An additional 10 μl of reaction solution is included to prevent withdrawing the full reaction solution, thus bringing the total volume to 70 μl.
      Note: Time points are chosen to provide data on fast, medium, and slow exchanging residues. Data spanning multiple time scales facilitates a deeper interpretation of the results. While short time points can be very informative on protein dynamics, care should be taken to allow time to withdraw sample from the reaction chamber and quench in a reproducible manner.
    2. Prepare stock solutions and reaction mixtures under anaerobic conditions, using an MBraun glove box fed by a nitrogen tank set to 40 PSI. The anaerobic atmosphere inside the glove box is maintained by keeping a positive nitrogen pressure. The exact gas flow will depend on the specific set up, however, the gas sensor monitoring conditions inside the glove box should register 0 ppm O2 and H2O and a positive pressure of 2 mbar.
    3. Prepare two anaerobic aliquots of purified Nfn (stock concentration 16.5 mg/ml) in an MBraun glove box by adding 30 μl of Nfn into one Verex vial, and 10 μl of Nfn into another Verex vial. Purified Fd is then added to the 10 μl aliquot of Nfn in a 1:1 molar ratio. Using a double vial system, cap the Verex vials, and place inside a clear glass serum vial that contains 1 ml of degassed Tris-HCl buffer with sodium dithionite, and seal using a crimper. Remove sealed vials from the glove box, wrap in Parafilm, and store at 4 °C prior to reaction initiation.
      Note: Only 1 ml of reductant containing buffer is added to the glass serum vial. Any more volume causes the Verex vial to slightly float, making it difficult to withdraw sample at the designated time points.
    4. Make deuterated buffers with 1 mM of each pyridine nucleotide in the following combinations: NAD+, NADH, NADP+, NADPH, NADH + NADP+, and NADPH + NAD+. Also prepare an additional stock of the same buffer without pyridine nucleotides to test the Nfn:Fd complex.
      Note: A 5 ml stock of deuterated buffer was made and subsequently aliquoted out with each nucleotide combination to make a 500 µl stock of each condition to use to set up reaction mixtures. For a condition run in triplicate, 500 µl should be sufficient volume to set up reaction mixtures. These volumes can/should change depending on the number of conditions and time points being tested.
    5. Prepare reaction vials by first degassing the deuterated buffers using four-10 min cycles on a Schlenk line. The Schlenk line functions by alternating between a vacuum to remove the oxygen, and nitrogen to place the solutions under positive pressure. After degassing, transfer reaction solutions to the glove box.
    6. Place 63 μl of each reaction buffer into three Verex vials, cap, place into clear glass serum vials, and seal using a crimper. This volume of buffer accommodates the volume for all time points while also leaving room for the addition of the protein. Seal the reaction vials, remove from the glove box, and store at room temperature prior to reaction initiation.
    7. Prepare a quench solution for each time point, and keep the solutions on ice before and during the reaction. Prepare solutions in Costar microcentrifuge tubes by mixing 1% formic acid (45 μl) and porcine pepsin (15 μl of 1 mg/ml in 50 mM sodium acetate pH 4.5 (H2O; pH set with 0.1 N HCl); final concentration: 0.2 mg/ml). This final concentration of pepsin gives sufficient coverage of the target proteins (~100%) in the limited digestion time. Prepare a separate quench solution with 10 μl of reaction buffer to test the pH of the solution. The target pH for HDX quenching is pH 2.5.
      Notes:
      1. Quench solutions do not need to be prepared anaerobically because the reaction is over at this point.
      2. Pepsin digestion of the protein localizes the location of exchange onto certain regions of the protein while under low pH quench conditions.

  2. Hydrogen Deuterium exchange reaction
    1. Begin the HDX reaction by adding protein via a 1:10 (v/v) dilution into the deuterated buffer using gastight Hamilton syringes (this results in a final Nfn concentration of 1.6 mg/ml). This ensures that D2O is the dominant solvent species. A workflow depicting the HDX reaction steps is shown in Figure 1.
      Note: The reaction for Nfn is performed at 60 °C. Regulation of this temperature was through a Fisher Scientific isotemp 110 water bath. This temperature was chosen to simulate the high temperature conditions where the organism, Pyrococcus furiosus lives.
    2. At specific time points (for this reaction: 1 min, 3 min, 15 min, 60 min, 3 h, and 24 h), remove 10 μl of the reaction mixture using a gas tight, Hamilton syringe, and add to the quench solution (pH 2.5, 0 °C; Concentration of Nfn in quench solution: ~0.23 mg/ml).
      Notes:
      1. This halts the exchange reaction, so any damage to the protein caused by low pH, temperature, or oxygen exposure will not impact data interpretation.
      2. Pepsin digestion is allowed to proceed for 2 min before flash freezing the solution in liquid nitrogen and storing at -80 °C.


        Figure 1. HDX-MS sample prep and work flow. Samples are kept anaerobic using a double vial system in which all reaction components are added to a small screw cap vial, which are sealed anaerobically in a slightly larger clear glass crimp vial. Protein is injected into the reaction mixture using gas tight Hamilton syringes to initiate the reaction. At designated time points, 10 μl of sample is extracted from the reaction mixture and added to an aerobic quench solution, containing formic acid and porcine pepsin. The acid not only locks in any deuterium that has exchanged onto the protein, but also serves to partially denature the protein complex, making it more accessible to pepsin digestion. Additional unfolding occurs upon exposure to oxygen. After 2 min of digestion, samples are flash frozen in liquid N2 and stored at -80 °C until data acquisition on Agilent 6538 Q-TOF. After acquisition, data is processed to display deuterium uptake over time in the form of uptake curves. Deuterium incorporation can be localized onto the structure of a protein by mapping the deuterium incorporation onto a 3D structure of the protein (if one is available).

  3. Data acquisition and processing
    1. Conduct liquid chromatography using an Onyx monolithic C18 column with the following gradient of A (H2O, 0.1% formic acid) and B (ACN, 0.1% formic acid) solvents over 10 min: 0.0-1.0 min, 5% B; 1.0-9.0 min, 5-45% B; 9.1-9.8 min, 95% B; 9.8-9.9 min, 5% B. Set the flow rate to 500 μl/min. Inject 10 μl, resulting in the injection of approximately 2.3 μg of protein onto the column. Keep the solvents cool (~0 °C) by storing the LC bottles on ice before and during data acquisition. Additionally, set the column compartment temperature to 1 °C and the auto-sampler temperature to 4 °C to help maintain a low temperature in the samples prior to injection on the mass spectrometer.
      Notes:
      1. Low temperatures in the HPLC stack helps minimize the amount of back exchange during chromatographic separation of peptides.
      2. The HPLC stack used in this protocol does not allow lower temperatures in the auto-sampler and lower temperatures in the column compartment are not stable, which can result in variations in the data.
    2. For data acquisition on an Agilent 6538 Q-TOF operating with MassHunter Workstation Software, scans are collected at 2 Hz over a scan range 50-1,700 m/z in positive mode. Set electrospray settings to the following: nebulizer gas at 3.7 bar, drying gas at 8.0 L/min, drying temperature at 350 °C, capillary voltage at 3.5 kV, and the acquisition mode was set to MS (seg).
      Note: Agilent Technologies supplies detailed information on its software for new users. For more information about different software options including Masshunter, please refer to Agilent Technologies’ website (Agilent Software: Software & Informatics).
    3. Identify compounds in the mass spectra by molecular feature in Agilent MassHunter Qualitative Analysis using the following settings: Extraction algorithm target data type, Small molecules; Peak filters, ≥ 500 counts; Ion species, +H and +Na; Isotope grouping, peak spacing tolerance 0.0025 m/z + 7.0 ppm, isotope model: peptides; Charge state assignment limited to a maximum of 5.
      Note: This is also a good chance to review the data, and look in the mass spectra for signs that deuterium exchange took place (this can also be done during data acquisition). Find an isotope envelope of a peptide in the non-deuterated sample. Then look at the same retention time in the deuterated samples to see if the isotope distribution has shifted. If it has, the experiment worked, if not, it is crucial to determine why the exchange either didn’t work, or why back exchange occurred causing a loss of deuterium ions. An example isotope comparison can be seen in Figure 2A.


      Figure 2. Isotope distribution and uptake curve of HDX-MS data. A. Example of the shift in isotope distribution for a peptide from Nfn-L. As the time for exchange increases (0-180 min) the centroid shifts to the right due to the addition of deuterium, as indicated by the dashed red line. B. Uptake curves showing the deuterium incorporation for four tested conditions. This data is for residues 124-144 of Nfn-L, the amino acid sequence located above the uptake curve.

    4. Create compound lists for non-deuterated digests of the protein sample, and export as a compound summary CSV (.csv) and import into Microsoft Excel 2016.
    5. Use the compound summary lists in a two-step process to identify peptides produced by pepsin digestion.
      Notes:
      1. The first step identifies unique peptides detected with mass spectrometry. These are masses that only have one match with the sequence of Nfn, while also falling in the mass error tolerance.
      2. The second step determines the identity of masses containing multiple peptide matches within the error tolerance by collecting fragmentation data to sequence the peptide.
    6. Place the protein sequence into PAWs to create a theoretical cleavage map using pepsin cleavage sites and an error of 100 ppm to account for the non-specific nature of pepsin cleavage.
      Note: Pepsin cleavage sites are after the following residues: L, W, I, A, F, Y, N, T, C, V, S, Q, G, E, D, R, M, K, H, and P. Cleavage sites were chosen based on outputs provided by the program PeptideShaker.
    7. PAWs identifies unique peptides for each of the subunits present in the protein complex of interest. Unique peptides are those who only have one match with the sequence of Nfn within the set error tolerance (100 ppm). Add each unique peptide to an Excel spreadsheet along with the corresponding sequence, charge, and retention time.
      Notes:
      1. Exclude any masses with multiple matches within the error tolerance.
      2. Video 1 shows how to set up PAWs for identification of unique peptides.
    8. Save the peptide list as a comma delimited (.csv) file.

      Video 1. PAWs set-up. Walkthrough of how to set up protein analysis worksheet (PAWs) for the identification of unique peptides. Walkthrough includes how to load a sequence, develop theoretical cleavage map, and input mass lists for peptide identification.

  4. Peptide fragmentation data acquisition and processing
    1. Several peptides can have the same m/z, therefore in order to successfully identify these peptides fragmentation data was collected on the Agilent 6538 Q-TOF instrument. Collision energies for fragmentation were set as a gradient over the data acquisition scan range (50-1,700 m/z) ranging from 4 V to 68 V.
      Note: The HPLC and MS settings that were used are the same as discussed previously in Procedure C, Steps 1 and 2 with the exception of the data acquisition mode, which is set to auto MS/MS (seg).
    2. Open the data files in the Masshunter Qualitative Analysis software to identify fragmentation features using the command ‘Find by Auto MS/MS’. Then export the data files as a Mascot Generic File (.mgf).
    3. Use SearchGUI to identify peptides based on the fragmentation data.
      1. To set up SearchGUI, start by defining the protein sequence. For this experiment, the sequence for the Nfn small and large subunits from Pyrococcus furiosus is used. SearchGUI will ask to make decoy sequences, which will help identify false positives.
      2. Next, set the search parameters for SearchGUI. For this experiment use the following settings: Digestion: Enzyme; Enzyme: Pepsin A; Specificity: Semi-Specific; Max Missed Cleavages: 5; Fragment Ion Types: b and y; Precursor m/z Tolerance: 20 ppm; Fragment m/z Tolerance: 20 ppm; Precursor Charge: 1-5; Isotopes: 0-1.
      3. Choose both the output folder, as well as the search engines. For this experiment, X!Tandem, MyriMatch and MS Amanda are chosen for the peptide search.
      4. Select PeptideShaker for post processing. PeptideShaker provides an easy to follow output of the fragment ion identifications, and provides the sequence, m/z, and retention time of the identified peptides.
      5. Combine the peptides identified from the fragmentation data with the unique peptides identified in PAWs. The combined list of identified peptides is saved as a .csv file, and used as the input file for deuterium incorporation calculations.
        Note: Video 2 shows how to set up SearchGUI and Peptide Shaker for the identification of peptides from fragmentation data.

        Video 2. SearchGUI/PeptideShaker set-up. Walkthrough of how to set up SearchGUI to identify peptides based on fragmentation data collected using MS/MS. The search results from SearchGUI will be opened in PeptideShaker. PeptideShaker provides a user friendly interface for the examination of identified peptides, which shows the protein coverage and number of peptides identified.

  5. Calculation of deuterium uptake using HDExaminer
    1. Deuterium incorporation is calculated by measuring the shift in the centroid of the isotope distribution for a given peptide. While this can be done manually, it is extremely time consuming. Several programs are available that will calculate the deuterium incorporation for HDX data. HDExaminer is a commercially available program by Sierra Analytics Inc. that accomplishes this.
      Note: Sierra Analytics has several tutorial videos to show the features of HDExaminer and how to set up the program (HDExaminer: The Basics).
    2. To set up HDExaminer, first add the sequence for the protein of interest and add a list of tested conditions on the ‘proteins’ page. This allows the program to calculate the deuterium incorporation in each condition.
    3. Next, add the list of peptides under the ‘peptides’ page, and select the column which has the charge, sequence, and retention time of each peptide. Then add the peptides to the peptide pool.
    4. Finally, on the ‘analysis’ page add the data files for each condition. All conditions will have the non-deuterated files, which are added first. Next add the fully deuterated data files (24 h time point), followed by the partially deuterated data files (1 min, 3 min, 15 min, 1 h, 3 h). To begin calculations automatically select the ‘auto-calculate’ box in the bottom right-hand corner of the screen, otherwise calculations will have to be initiated manually. A progress bar in the bottom right-hand corner indicates when calculations are underway.
      Note: Alternatively, the maximum number of deuterium can be calculated from the number of exchangeable amide hydrogens within the peptide sequence.
    5. Once the calculations are complete, manually verify the calculations.
      1. Under the peptide tab of HDExaminer, each peptide has a dropdown menu showing the different conditions. Each condition has an additional dropdown menu showing the different charge states of the peptide based on the peptide pool.
      2. If HDExaminer successfully identifies the location of the peptide within the data files, clicking on a charge state shows the isotope distribution of the peptide in each data file on the left-hand side. The right-hand side shows the chromatogram for each data file and two red arrows indicate the retention time window containing the peptide isotope pattern.
      3. To verify the data, examine the retention time window for the peptides, they should be the same, or within a few seconds of one another for each data file. The search window can be adjusted by moving either of the red arrows. If the partially deuterated or fully deuterated data files are changed, the deuterium incorporation for only those files will be re-calculated. However, if the non-deuterated files search retention time is changed, the deuterium incorporation for all data files for that peptide will be re-calculated.
      4. Manually verifying the data ensures the same retention time search window is used for all data files for a peptide. This gives the most accurate measurement of the deuterium incorporation and helps minimize the error between replicates.
    6. After manual verification, export the peptide pool results for each condition, this provides the peptide identification, search retention time and charge. This also provides the start and end retention time, amount of deuterium incorporated (#D), percent deuterium incorporated (%D), score and confidence for each time point and replicate.
    7. The data provided in the peptide pool results can be used in a variety of ways.
      1. The easiest means of visualizing the data are uptake curves which show the #D incorporated into a peptide over time. Generate a curve for each peptide in order to compare the deuterium incorporation over time for each condition tested. Figure 2B displays an example of an uptake curve that shows a comparison of four conditions.
      2. An additional way to analyze the data is to calculate the %D incorporated and measure the percent difference to determine the relative change in accessibility to exchange between conditions. Calculate the %D by dividing the #D for a time point by the #D from the fully deuterated sample and multiply by 100. Then calculate the percent difference by subtracting the %D for two conditions. This allows comparisons in the deuterium incorporation between peptides of different length.
      3. Map the percent difference onto the structure of the protein (if available) to show the areas that are impacted by the different conditions. Mapping the exchange data onto the structure can also reveal the presence of allostery, as well as which regions of the protein are communicating with one another to enact the allosteric mechanism.
        Note: For a detailed explanation on mapping exchange data onto a protein structure, see Data analysis.

Data analysis

  1. Use MassHunter Qualitative Analysis to verify the data during data acquisition by examining the deuterated samples for evidence of exchange in the isotope distribution. This is done by comparing the deuterated samples to the non-deuterated samples. Figure 2A shows an example of this by displaying the isotope distribution for the non-deuterated, 1 min, 15 min, and 3 h time points. This allows for a comparison in the shift in the isotope distribution as more deuterium is incorporated between the tested conditions.
  2. After calculation of the deuterium incorporation, use Microsoft Excel or a similar spreadsheet program to generate uptake curves to compare deuterium incorporation over time in the tested conditions within a peptide. Figure 2B shows an example uptake curve.
  3. To compare deuterium incorporation between multiple peptides and/or conditions, convert the #D to %D and calculate the difference between two peptides. Additionally, the percent difference can be mapped onto a 3D structure of a protein using UCSF Chimera as shown in Figure 3 (Pettersen et al., 2004).


    Figure 3. HDX-MS data mapped onto a protein structure. This structural heat map shows the influence of NADPH and NAD+ binding on exchange of Nfn. H/D exchange data collected under two different conditions was mapped onto the protein structure using UCSF Chimera (see Notes). The heat map was calculated from the difference between exchange in as-purified and NADPH + NAD+ bound reactions. Color code was set to red (100% difference, increased exchange), blue (-100% difference, protection), and gray (0% difference, no change). In this example, the NADPH + NAD+ bound condition incorporates less deuterium than the as-purified protein.

  4. Generate a structure-based heat map of the HDX data in UCSF Chimera (Pettersen et al., 2004).
    1. Create a txt file that lists the residue number and the %D that correlates to that residue in the following format:
      Attribute: nad_nadph-as_purified_nfn_3hrs_percent_deuterium
      match mode: 1-to-1
      recipient: residues         

      :1.A 20
      :2.A 20
      :3.A 45
      :4.A 45
      :5.A 45

      The header names the attribute that will be displayed onto the model and defines that the residues are what will be colored according to the attribute (rather than atoms). The ‘:1.A’ defines the residue number and chain name. A tab separates the value from the residue identifier. These columns can easily be generated in Excel or a similar spreadsheet program and either saved as a txt file or copy/pasted into a text file. If there is no data for a residue, leave empty space next to the residue identifier or completely remove the line.
    2. Open the pdb protein structure file in Chimera. Go to Tools > Structure Analysis > Define Attribute. Select the .txt file generated in step a.
      Note: An error will open in a pop up window if data is listed for residues that are not resolved in the structure file. This error can be ignored.
    3. Once the attribute is defined, the Render/Select by Attribute window opens. Use this window to determine which colors will represent which %D values.
      1. Residues that are listed in the txt file but do not have data associated with them will be colored according to the highest %D color unless an additional color is added above this value.
      2. To add additional color bars use Ctrl + left click in the histogram plot. Ctrl + left click on existing color bars to remove them.
      3. Move color bars either by the mouse or by entering a value in the ‘Value’ box.
    4. Click ‘Apply’ to color the protein structure according to the data file.
    5. Figure 3 shows an example of a colored structure.
    Note: Video 3 shows how to map HDX-MS data onto a 3D structure of a protein.

    Video 3. UCSF Chimera protein mapping. Walkthrough showing how to format data to map onto a 3D protein structure in UCSF Chimera. Walkthrough also shows the correct menus to select to map data onto the structure, as well as how to set up a color gradient and specify what colors to use.

Notes

  1. The recommended alternative column for HPLC separation of peptides is the same as the one used in the protocol described here, however the internal diameter is 1 mm larger. This will influence the flow rate of solvent through the column, which can affect the elution of peptides from the column. It is highly recommended to test different flow rates and gradients to optimize separation of peptides prior to performing the exchange reaction.
  2. The measured pH of the deuterated buffers must be corrected to pD by adding the correction factor (0.4 pH units) to the measured pH. For this experiment the tris base buffer prepared in D2O had a measured pH of 7.0, therefore the pD of the buffer is 7.4 (Krezel and Bal, 2004).
  3. Before beginning the exchange reaction, check that the quench solution with formic acid, pepsin, and reaction buffer are at pH 2.5 so back exchange does not occur during protein digestion.
  4. Place HPLC solvents on ice, set the column compartment to 0 °C, and the auto-sampler to 4 °C to help reduce back exchange of the deuterated amides.
  5. HDX-MS can be performed under a wide range of temperatures. While exchange rate does have a dependence on temperature, pH can affect it much more (Walters et al., 2012). However, it should be noted that a comparative analysis between HDX-MS reactions run at different temperatures is difficult because of the change in exchange rate as the temperature is varied. HDX-MS is most commonly run at 25 °C (room temperature), however, some cases are reported to run as low as 0 °C and as high as 60 °C.
  6. When planning an HDX-MS experiment, it is highly recommended that the starting concentration of the protein of interest be approximately 10 mg/ml, this ensures that approximately 1.5 μg of protein is injected onto the instrument.
    1. Lower amounts of protein injected onto the instrument will cause peptides to be detected at low intensity, which in turn can prevent accurate deuterium incorporation calculations.
    2. A 1:10 dilution of protein into D2O buffer is encouraged, it is possible to do less if a particular protein is not easy to obtain aliquots with a high concentration. It is then recommended to do some pilot experiments to ensure that deuterium incorporation is satisfactory, and the extra water present in the reaction is not causing back exchange of the deuterium ions.

Recipes

  1. Tris base buffer in deuterium oxide (shelf life ~one week)
    100 mM Tris-HCl
    150 mM NaCl
    Adjust pH to 7.0 with 1 N HCl
  2. Tris base buffer in H2O
    100 mM Tris-HCl
    150 mM NaCl
    Adjust pH to 7.0 with 1 N HCl
  3. Tris base buffer w/sodium dithionite
    100 mM Tris-HCl
    150 mM NaCl
    2 mM sodium dithionite
    Adjust pH to 7.0 with 1 N HCl

Acknowledgments

This work was supported as part of the Biological and Electron Transfer and Catalysis (BETCy) EFRC, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science (DE-SC0012518). The Mass Spectrometry Facility at MSU was supported in part by the Murdock Charitable Trust and an NIH IDEA program grant P20GM103474. The work detailed in this protocol was adapted from work described in recent publications (Lubner et al., 2017; Berry et al., 2018). The authors declare that they do not have any conflicts of interest.

References

  1. Berry, L., Poudel, S., Tokmina-Lukaszewska, M., Colman, D. R., Nguyen, D. M. N., Schut, G. J., Adams, M. W. W., Peters, J. W., Boyd, E. S. and Bothner, B. (2018). H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle. Biochim Biophys Acta 1862(1): 9-17.
  2. Demmer, J. K., Rupprecht, F. A., Eisinger, M. L., Ermler, U. and Langer, J. D. (2016). Ligand binding and conformational dynamics in a flavin-based electron-bifurcating enzyme complex revealed by Hydrogen-Deuterium Exchange Mass Spectrometry. FEBS Lett 590(24): 4472-4479.
  3. Krezel, A. and Bal, W. (2004). A formula for correlating pKa values determined in D2O and H2O. J Inorg Biochem 98: 161-166.
  4. Lubner, C. E., Jennings, D. P., Mulder, D. W., Schut, G. J., Zadvornyy, O. A., Hoben, J. P., Tokmina-Lukaszewska, M., Berry, L., Nguyen, D. M., Lipscomb, G. L., Bothner, B., Jones, A. K., Miller, A. F., King, P. W., Adams, M. W. W. and Peters, J. W. (2017). Mechanistic insights into energy conservation by flavin-based electron bifurcation. Nat Chem Biol 13(6): 655-659.
  5. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. and Ferrin, T. E. (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25(13): 1605-1612.
  6. Walters, B. T., Ricciuti, A., Mayne, L. and Englander, S. W. (2012). Minimizing back exchange in the hydrogen exchange-mass spectrometry experiment. J Am Soc Mass Spectrom 23(12): 2132-2139.

简介

本文详述的方案描述了一种在氧敏感蛋白质上进行氢氘交换偶联质谱(HDX-MS)的方法。 HDX-MS是研究蛋白质结构 - 功能关系的强大工具。将这种技术应用于厌氧蛋白质可以深入了解执行氧敏化学的蛋白质的机制。使用HDX-MS研究厌氧蛋白时的一个问题是,有许多部分需要不断运动进出厌氧室。这会影响密封,增加氧气暴露的可能性。暴露于氧气使得与这些蛋白质结合的辅因子(一个常见的例子是FeS簇)不再与负责配位FeS簇的氨基酸残基相互作用,造成簇的丢失和蛋白质的不可逆失活。为了解决这个问题,开发了一种双瓶系统,可以厌氧地制备溶液和反应混合物,但也可以将这些溶液转移到有氧环境中,同时屏蔽溶液中的氧气。此外,运动不受限制,就像厌氧室一样,确保更一致的数据,并在反应过程中减少错误。

【背景】许多氧敏感蛋白质是生物在缺氧环境中繁殖所必需的。这些蛋白质中的一些通过被称为基于黄素的电子分叉(FBEB)(Lubner等,2017)的方法向厌氧微生物提供替代的能量供应。 FBEB产生还原型铁氧还蛋白,可被氧化产生能量。能够降低铁氧还蛋白的蛋白质引起了极大的兴趣,并且一直是最近使用HDX-MS研究的重点(Demmer et al。,2016; Lubner 等人), 2017; Berry等人,2018)。 HDX-MS是研究蛋白质稳定性,动力学和配体结合的强大技术,提供关于结构和功能之间关系的信息。 HDX-MS利用酰胺氢的内在特性与溶液中的氢交换,以追踪蛋白质/蛋白质复合物结构和动力学的变化。通过用重水(D 2 O)代替单同位素水(H 2 O)制备缓冲液,蛋白质上的酰胺氢将与溶液中的氘交换。给定氨基酸的交换速率受二级结构中氢键的稳定性以及单一蛋白质或蛋白质复合物内的三级和四级相互作用的影响。使用质谱法,通过测量氘化和非氘化样品之间的同位素分布的变化来确定氘掺入。 HDX-MS已被广泛应用于大量蛋白质和蛋白质复合物。为了用HDX-MS成功研究这些蛋白质,建立一种在台面上执行该反应的手段是必要的,以避免繁忙的进出厌氧室的耗费时间和繁重的工作。问题是如何在有氧环境中保持蛋白质样品长时间厌氧的同时操作样品。为了解决这个问题,将反应混合物和蛋白质储备溶液置于双瓶系统中,其允许添加和除去样品,同时保持严格的厌氧条件。设置背后的逻辑是创建一个气闸。将小瓶置于带有螺帽瓶的氮气的正压下,置于含有还原剂的较大卷曲小瓶内。有了这个双重屏障系统,小容量的空气可以被困在外瓶中,不会接触样品。

关键字:HDX-MS, 质谱法, 厌氧蛋白, H/D交换, 蛋白质动力学, 蛋白-蛋白相互作用, 蛋白-配体相互作用

材料和试剂

  1. Verex TM小瓶试剂盒,9mm,螺旋盖,聚丙烯,300μl+ PTFE /硅胶帽,蓝色,1,000 /包(Phenomenex,目录号:AR0-9991-13)
  2. 具有20毫米卷曲顶部涂饰的透明玻璃血清瓶,10毫升,100 /盒(DWK Life Sciences,WHEATON,目录号:225278)
  3. Curwood Parafilm M TM实验室包装膜(Bemis,目录号:PM996)
  4. Costar Microcentrifuge Tubes 0.65 ml,500 /袋(Corning,目录号:3208)
  5. Onyx Monolithic C18色谱柱,100 x 2 mm(Phenomenex,目录号:CH0-8467)
    备选色谱柱(详见说明):Onyx Monolithic C18色谱柱,100 x 3 mm(Phenomenex,目录号:CH0-8158)

  6. 模型1701和1702小型注射器,10μl(26s规格)和25μl(22s规格),2“针形式2(汉密尔顿,目录号:80030和80230)
  7. 无衬铝开顶密封,20 mm,1,000 /箱(DWK Life Sciences,WHEATON,产品目录号:224178-05)
  8. 20毫米塞子,直塞,超纯(DWK Life Sciences,WHEATON,目录号:W224100-405)
  9. 200μl移液吸头(VWR,目录号:53508-810)
  10. 液氮
  11. 纯化的铁氧化还原蛋白(Fd)在50mM乙酸铵缓冲液(pH6.8)中于H 2 O(储存浓度150μM)中来自Pf
    注意:用1N HCl调节pH值。
  12. 在20mM Tris,150mM NaCl缓冲液(pH8)中在H 2 O(储备浓度16.5mg / ml)中纯化NADH依赖性铁氧还蛋白-NADP +氧化还原酶(Nfn)来自生物体 Pyrococcus furiosus ( Pf )
    注意:用1N HCl调节pH值。
  13. 连二亚硫酸钠(Merck,目录号:1065051000)
  14. 烟酰胺腺嘌呤二核苷酸(NAD +,Cayman Chemical,目录号:16077,500mg)
  15. 烟酰胺腺嘌呤二核苷酸(NADH,Cayman Chemical,目录号:16078,500mg)
  16. 烟酰胺腺嘌呤二核苷酸磷酸(NADP +,Cayman Chemical,目录号:10004675,50mg)
  17. 烟酰胺腺嘌呤二核苷酸磷酸(NADPH,Cayman Chemical,目录号:9000743,25mg)
  18. 99.5%甲酸(Fisher Scientific,目录号:A117-50)
  19. 来自猪胃粘膜的胃蛋白酶(Sigma-Aldrich,目录号:P6887-1G)
  20. 醋酸钠三水合物(Fisher Scientific,目录号:S607-500)
  21. 乙酸铵,≥99%(Sigma-Aldrich,目录号:09689-250G)
  22. 37%盐酸(= 12.1M)(Merck,目录号:HX0603-3)
  23. 氢氧化钠(Fisher Scientific,目录号:BP359-500)
  24. 氧化氘(Sigma-Aldrich,目录号:151882-100G)
  25. Tris碱(Merck,目录号:648311-5KG)
  26. 氯化钠(Fisher Scientific,目录号:BP358-212)
  27. HPLC级水(Fisher Scientific,目录号:W5-4)
  28. HPLC级乙腈(Fisher Scientific,目录号:A998-4)
  29. Nanopure水(使用Millipore Q-Gard 2内部纯化)
  30. Tris碱,在氧化氘中的NaCl缓冲液(pH7.0)(见食谱)
  31. Tris碱,NaCl缓冲液(pH7.0)在H 2 O(参见配方)中
  32. Tris碱,NaCl,连二亚硫酸钠缓冲液(pH 7.0)在H 2 O中的溶液(参见食谱)。

设备

  1. 用于分离通过胃蛋白酶消化产生的肽的HPLC叠层(例如,Agilent Technologies制造的1290 Infinity系列HPLC叠层)(Agilent Technologies,型号:1290 Infinity系列)
  2. 用于样品分析/数据采集的LC / MS Q-TOF系统(Agilent Technologies制造的6538UHD Accurate-Mass Q-TOF LC / MS)(Agilent Technologies,型号:6538UHD Accurate-Mass Q-TOF LC / MS)
  3. 手套箱能够在正的惰性气体压力下保持厌氧条件(例如,MBraun,型号:UNIlab Plus手套箱工作站)
  4. 氮气罐
  5. 20毫米用于铝密封的Kebby标准卷曲机(医学实验室供应,目录号:2001-00-C01A)
  6. Fisher Scientific isotemp 110水浴(Fisher Scientific,型号:Fisherbrand TM TM Isotemp TM,目录号:S63077Q)
    注意:此产品已停产。
  7. Milli-Q纯化系统(Merck,目录号:QGARD00D2)
  8. 移液器(10μl和100μl)(Eppendorf,目录号:022478886和022478924)

软件

  1. 在Windows 7上的Microsoft Excel 2016
  2. UCSF Chimera v。1.11.2
  3. MassHunter工作站软件定性分析v.06.00(安捷伦科技)
  4. HDExaminer v。1.3.0 beta 6(Sierra Analytics,Inc.)
  5. MassHunter工作站软件用于6200系列TOF / 6500系列Q-TOF v。B.05.01(Agilent Technologies)的LC / MS数据采集
  6. 肽分析工作表免费版(PAWs,ProteoMetrics免费版)
  7. SearchGUI v。3.2.18(Compomics)
  8. Peptide Shaker v。1.16.9(Compomics)

程序

  1. 反应制备
    1. 在制备储备溶液和反应混合物之前,确定时间点的数量。时间点的数量决定了反应混合物的体积。对于每个时间点,10μL必须可用于添加淬火溶液,再加上10μL的24小时/完全氘化时间点。例如,在Nfn的HDX实验中,测量了五个时间点(1分钟,3分钟,15分钟,60分钟,3小时和24小时)(Berry等人,2018年) 。包括额外的10μl反应溶液以防止全部反应溶液的排出,从而使总体积达到70μl。
      注:选择时间点以提供快速,中等和慢速交换残基的数据。跨多个时间尺度的数据有助于更深入地解释结果。尽管短时间点对于蛋白质动力学可能非常有用,但应注意留出时间从反应室取出样品并以可重现的方式进行淬灭。
    2. 在厌氧条件下制备储备溶液和反应混合物,使用由设置为40PSI的氮气罐供料的MBraun手套箱。保持氮气正压,维持手套箱内的无氧环境。确切的气体流量取决于具体的设置,但手套箱内的气体传感器监测条件应记录0 ppm O 2和H 2 O,
      2毫巴的压力
    3. 在MBraun手套箱中,通过将30μlNfn加入一个Verex小瓶中,并将10μlNfn加入另一个Verex小瓶中,准备两个纯化的Nfn(储备浓度16.5mg / ml)的厌氧等分试样。然后将纯化的Fd以1:1的摩尔比加入到10μl的Nfn等分试样中。使用双瓶系统,盖上Verex小瓶,并置于透明玻璃血清小瓶内,其中含有1ml连二亚硫酸钠的脱气Tris-HCl缓冲液,并使用卷曲机密封。从手套箱中取出密封的小瓶,用Parafilm包裹,并在反应开始前储存在4°C。
      注意:在玻璃血清瓶中只加入1毫升含还原剂的缓冲液。任何更大的体积都会导致Verex小瓶轻微浮动,使得在指定的时间点很难取出样品。
    4. 用下列组合中的每种吡啶核苷酸1mM制备氘化缓冲液:NAD +,NADH,NADP +,NADPH,NADH + NADP + ,NADPH + NAD + 。另外准备一个不含吡啶核苷酸的相同缓冲液的另外的储备物来测试Nfn:Fd复合物。
      注意:制备5ml的氘化缓冲液储备液,随后用每种核苷酸组合物等分出来以制备每种条件的500μl储液以用于建立反应混合物。对于一式三份的情况,500μl应该具有足够的体积以建立反应混合物。这些体积可以/应该改变,具体取决于测试条件和时间点的数量。
    5. 首先在Schlenk生产线上使用四个10分钟的循环对氘化缓冲液脱气来制备反应瓶。 Schlenk生产线通过在真空中除去氧气和用氮气将溶液置于正压下进行交替运转。脱气后,将反应溶液转移到手套箱中。
    6. 将63μl的每个反应缓冲液放入三个Verex小瓶中,盖上,放入透明玻璃血清小瓶中,并使用卷曲机密封。该体积的缓冲液适用于所有时间点的体积,同时也为蛋白质的添加留出空间。密封反应瓶,从手套箱中取出,在室温下储存,然后开始反应。
    7. 为每个时间点准备一个淬灭溶液,并在反应前和反应过程中将溶液保存在冰上。通过将1%甲酸(45μl)和猪胃蛋白酶(15μl1mg / ml在50mM乙酸钠pH 4.5中(H 2 O; pH设定为0.1)在Costar微量离心管中制备溶液N HCl);终浓度:0.2mg / ml)。胃蛋白酶的最终浓度在有限的消化时间内给出足够的靶蛋白覆盖率(〜100%)。用10μl反应缓冲液制备单独的淬灭溶液以测试溶液的pH。
      HDX淬火的目标pH值为2.5 注意:
      1. 淬火解决方案不需要厌氧准备,因为此时反应已结束。
      2. 在低PH值淬火条件下,蛋白质的胃蛋白酶消化将交换位置定位到蛋白质的某些区域。

  2. 氢氘交换反应
    1. 通过使用气密Hamilton注射器将1:10(v / v)稀释液加入氘化缓冲液中(这导致最终Nfn浓度为1.6mg / ml)开始HDX反应。这确保了D 2 O是主要的溶剂物质。描绘HDX反应步骤的工作流程如图1所示。
      注:Nfn的反应在60°C下进行。通过Fisher Scientific isotemp 110水浴对此温度进行调节。选择该温度是为了模拟有机体Pyrococcus furiosus居住的高温条件。
    2. 在特定的时间点(对于该反应:1分钟,3分钟,15分钟,60分钟,3小时和24小时),使用不透气的汉密尔顿注射器移除10μl反应混合物,并添加至骤冷溶液(pH 2.5,0℃;淬灭溶液中Nfn的浓度:约0.23mg / ml)。
      注意:
      1. 这阻止了交换反应,因此低pH值,温度或氧气暴露对蛋白质造成的任何破坏都不会影响数据解释。
      2. 胃蛋白酶消化可以进行2分钟,然后在液氮中快速冷冻溶液并储存在-80℃。


        图1. HDX-MS样品制备和工作流程。使用双瓶系统使样品保持厌氧,其中将所有反应组分加入到小型螺帽小瓶中,将其在厌氧下密封在稍大的透明玻璃卷曲小瓶中。使用气密Hamilton注射器将蛋白质注入反应混合物以引发反应。在指定的时间点,从反应混合物中提取10μl样品,并将其加入到含甲酸和猪胃蛋白酶的好氧急冷溶液中。酸不仅锁定了交换到蛋白质上的任何氘,还用于部分变性蛋白质复合物,使其更容易被胃蛋白酶消化。暴露于氧气后会发生额外的展开。消化2分钟后,将样品在液体N 2中快速冷冻并储存在-80℃直到在Agilent 6538Q-TOF上获得数据。采集后,处理数据以显示吸收曲线形式的氘摄取随时间推移。通过将氘掺入蛋白质的3D结构(如果有的话),氘掺入可以定位在蛋白质的结构上。

  3. 数据采集和处理
    1. 使用Onyx单片C18柱,使用以下梯度的A(H 2 O,0.1%甲酸)和B(ACN,0.1%甲酸)溶剂在10分钟内进行液相色谱:0.0-1.0分钟,5%B; 1.0-9.0分钟,5-45%B; 9.1-9.8分钟,95%B; 9.8-9.9分钟,5%B.将流速设定为500μl/分钟。注入10μl,导致在柱上注射大约2.3μg蛋白质。在数据采集之前和数据采集过程中,将LC瓶保存在冰上保持溶剂冷却(〜0°C)。此外,将色谱柱室温设置为1°C,并将自动进样器温度设置为4°C,以帮助样品在注入质谱仪之前保持较低的温度。
      注意:
      1. 本协议中使用的HPLC堆栈不允许自动进样器中的较低温度和柱室中较低的温度不稳定,这可能导致数据的变化。
    2. 对于使用MassHunter工作站软件操作的Agilent 6538 Q-TOF上的数据采集,以正模式扫描范围为50-1,700 m / z的2 Hz采集扫描。将电喷雾设置设置为:3.7巴的雾化器气体,8.0 L / min的干燥气体,350°C的干燥温度,3.5 kV的毛细管电压,并将采集模式设置为MS(seg)。
      注意:Agilent Technologies为新用户提供其软件的详细信息。有关包括Masshunter在内的不同软件选项的更多信息,请参阅Agilent Technologies网站( 安捷伦软件:软件与信息学 )。
    3. 在Agilent MassHunter定性分析中使用以下设置通过分子特征识别质谱中的化合物:提取算法目标数据类型,小分子;峰值滤波器,≥500个计数;离子种类,+ H和+ Na;同位素分组,峰间距公差0.0025μm/ z + 7.0ppm,同位素模型:肽;充电状态分配限制为最多5个。
      注意:这也是一个很好的机会来查看数据,并在质谱中查找发生氘交换的迹象(这也可以在数据采集过程中完成)。在非氘代样品中找到肽的同位素包膜。然后观察氘代样品中的相同保留时间,以确定同位素分布是否发生了变化。如果有的话,实验工作,否则,确定为什么交换不起作用,或为什么回流交换发生造成氘离子的损失是至关重要的。图2A中可以看到示例性的同位素比较。


      图2. HDX-MS数据的同位素分布和摄取曲线。 :一种。来自Nfn-L的肽的同位素分布转变的例子。随着交换时间增加(0-180分钟),由于添加了氘,质心向右移动,如红色虚线所示。 B.显示四种测试条件下氘掺入的吸收曲线。该数据适用于Nfn-L的残基124-144,该氨基酸序列位于摄取曲线上方。

    4. 为蛋白质样品的非氘化摘要创建化合物列表,并将其作为复合摘要CSV(.csv)导出并导入到Microsoft Excel 2016中。

    5. 在两步过程中使用化合物总结列表来鉴定胃蛋白酶消化产生的肽。
      注意:
      1. 第一步确定用质谱法检测到的独特肽。这些质量只与Nfn的序列匹配,同时也落在质量误差容限内。
      2. 第二步是通过收集碎片数据对肽进行测序来确定包含多个肽匹配的质量在误差容限内的特性。
    6. 将蛋白质序列置于PAW中以使用胃蛋白酶切割位点产生理论切割图并且误差为100ppm以解释胃蛋白酶切割的非特异性质。
      注:胃蛋白酶切割位点在以下残基之后:L,W,I,A,F,Y,N,T,C,V,S,Q,G,E,D,R,M,K, H和P.裂解位点是根据PeptideShaker程序提供的输出进行选择的。
    7. PAW为目标蛋白复合物中存在的每个亚基鉴定独特的肽。独特的多肽是那些在设定的误差容限(100 ppm)内只与Nfn序列匹配的那些肽。
      在Excel电子表格中添加每个独特的肽以及相应的序列,电荷和保留时间。
      注意:
      1. 在容错范围内排除具有多个匹配的任何质量。
      2. 视频1显示了如何设置PAW以识别独特的多肽。
    8. 将肽列表保存为逗号分隔(.csv)文件。

      s视频1

  4. 肽碎片数据采集和处理
    1. 几种肽可具有相同的m / z,因此为了成功鉴定这些肽片段,在Agilent 6538 Q-TOF仪器上收集数据。在数据采集扫描范围(50-1,700m / z)范围内,用于碎裂的碰撞能量设置为4V至68V范围内的梯度。
      注意:除了设置为自动MS / MS(seg)的数据采集模式之外,所使用的HPLC和MS设置与先前在过程C,步骤1和2中所讨论的相同。
    2. 打开Masshunter定性分析软件中的数据文件,使用命令“通过自动MS / MS查找”来识别碎片特征。然后将数据文件导出为Mascot通用文件(.mgf)。
    3. 使用SearchGUI根据碎片数据识别肽段。
      1. 要设置SearchGUI,首先定义蛋白质序列。对于这个实验,使用来自火球菌的Nfn小亚基和大亚基的序列。 SearchGUI会要求制作诱饵序列,这有助于识别误报。
      2. 接下来,设置SearchGUI的搜索参数。对于这个实验使用以下设置:消化:酶;酶:胃蛋白酶A;特异性:半特异性;最大错过的劈理:5;片段离子类型:b和y;前体 m / z 公差:20 ppm;片段 m / z 公差:20 ppm;前体充电:1-5;同位素:0-1。
      3. 选择输出文件夹以及搜索引擎。对于这个实验,选择X!Tandem,MyriMatch和MS Amanda进行多肽搜索。
      4. 选择PeptideShaker进行后期处理。 PeptideShaker提供了一个易于遵循的片段离子鉴定输出,并提供了识别肽段的序列,m / z和保留时间。
      5. 将从碎裂数据中鉴定的肽与在PAW中鉴定的独特肽组合。已识别肽的组合列表保存为.csv文件,并用作氘合并计算的输入文件。
        注意:视频2显示了如何设置SearchGUI和Peptide Shaker来从碎片数据中鉴定肽。

        小号视频2

  5. 使用HDExaminer计算氘摄入量
    1. 通过测量给定肽的同位素分布质心的偏移来计算氘掺入。虽然这可以手动完成,但这非常耗时。有几个程序可以计算HDX数据的氘结合。 HDExaminer是由Sierra Analytics Inc.提供的商业可用程序,可以完成此项任务。
      注意:Sierra Analytics有几个教程视频来展示HDExaminer的功能以及如何设置该程序( HDExaminer:The Basics )。
    2. 要设置HDExaminer,首先添加感兴趣蛋白质的序列,并在“蛋白质”页面上添加测试条件列表。这允许程序计算每种情况下的氘掺入量。
    3. 接下来,在“肽”页面下添加肽列表,并选择具有每种肽的电荷,序列和保留时间的列。然后将肽添加到肽库。
    4. 最后,在'分析'页面上添加每个条件的数据文件。所有条件都会有非氘化文件,首先添加。接下来添加完全氘化的数据文件(24小时时间点),然后添加部分氘化的数据文件(1分钟,3分钟,15分钟,1小时,3小时)。要自动开始计算,请选择屏幕右下角的“自动计算”框,否则将不得不手动开始计算。
      。右下角的进度条表示计算何时正在进行。
      注意:或者,可以从肽序列中可交换的酰胺氢的数量计算氘的最大数量。
    5. 计算完成后,手动验证计算结果。
      1. 在HDExaminer的多肽标签下,每种肽都有一个显示不同条件的下拉菜单。每种条件都有一个额外的下拉菜单,显示基于肽库的肽的不同电荷状态。
      2. 如果HDExaminer成功识别数据文件中肽的位置,点击电荷状态将显示左侧每个数据文件中肽的同位素分布。右侧显示每个数据文件的色谱图,两个红色箭头显示含有肽同位素模式的保留时间窗口。
      3. 要验证数据,请检查肽的保留时间窗口,它们应该是相同的,或者每个数据文件在几秒钟之内。可以通过移动任一红色箭头来调整搜索窗口。如果部分氘化或完全氘化的数据文件发生变化,则仅重新计算这些文件的氘掺入量。但是,如果非氘化文件搜索保留时间发生变化,则重新计算该肽所有数据文件的氘掺入。
      4. 手动验证数据可确保为肽的所有数据文件使用相同的保留时间搜索窗口。这样可以最精确地测量氘的掺入量,并有助于最小化重复之间的误差。
    6. 手动验证后,输出每种条件下的肽库结果,这提供了肽的鉴定,检索保留时间和收费。这也提供了开始和结束保留时间,掺入氘的量(#D),掺入氘的百分比(%D),每个时间点和重复的分数和置信度。
    7. 肽库结果中提供的数据可以多种方式使用。
      1. 数据可视化的最简单方法是吸收曲线,显示随时间推移掺入肽中的#D。生成每个肽的曲线,以便比较每个测试条件随时间推移的氘掺入。图2B显示了一个吸收曲线的例子,显示了四种情况的比较。
      2. 另一种分析数据的方法是计算并入的%D并测量百分比差异以确定条件之间交换可访问性的相对变化。通过将#D从一个时间点除以完全氘代样品的#D来计算%D,然后乘以100.然后通过减去两个条件的%D来计算百分比差异。这样可以比较不同长度的肽之间的氘掺入。
      3. 将百分比差异映射到蛋白质结构(如果可用)以显示受不同条件影响的区域。将交换数据映射到结构上还可以揭示联合体的存在,以及蛋白质的哪些区域彼此通信以实现变构机制。
        注意:有关将交换数据映射到蛋白质结构的详细说明,请参阅数据分析。

数据分析

  1. 使用MassHunter定性分析在数据采集过程中通过检查氘代样品中同位素分布交换的证据来验证数据。这是通过比较氘化样品和非氘化样品来完成的。图2A通过显示非氘化,1分钟,15分钟和3小时时间点的同位素分布显示了一个例子。这允许比较同位素分布的变化,因为在测试条件之间掺入更多的氘。
  2. 计算氘掺入量后,使用Microsoft Excel或类似的电子表格程序生成吸收曲线,以比较肽内测试条件下氘掺入随时间的变化。图2B显示了一个示例摄取曲线。
  3. 为了比较多种肽和/或条件之间的氘掺入,将#D转换为%D并计算两种肽之间的差异。此外,使用UCSF嵌合体可以将差异百分比映射到蛋白质的3D结构上,如图3所示(Pettersen等人,2004年)。


    图3. HDX-MS数据映射到蛋白质结构上。这种结构热图显示了NADPH和NAD +结合对Nfn交换的影响。在两种不同条件下收集的H / D交换数据使用UCSF嵌合体(参见注释)映射到蛋白质结构上。从纯化的交换和NADPH + NAD +结合的反应之间的差异计算热图。颜色代码被设置为红色(100%差异,增加交换),蓝色(-100%差异,保护)和灰色(0%差异,不变化)。在这个例子中,NADPH + NAD +结合条件比纯化的蛋白掺入更少的氘。

  4. 在UCSF Chimera中生成一个基于结构的HDX数据热图(Pettersen et al。,2004)。
    1. 创建一个txt文件,以下列格式列出残留物编号和与残留物相关的%D:
      属性:nad_nadph-as_purified_nfn_3hrs_percent_deuterium
      匹配模式:1对1
      收件人:残留物         

      :1.A 20
      :2.A 20
      :3.A 45
      :4.A 45
      :5.A 45

      标题命名将显示在模型上的属性,并定义残留是根据属性(而不是原子)进行着色的。 ':1.A'定义了残基编号和链名。选项卡将值与残余标识符分开。这些列可以在Excel或类似的电子表格程序中轻松生成,并保存为txt文件或复制/粘贴到文本文件中。如果没有残留物的数据,请在残留物识别符旁边留出空白处,或者彻底清除残留物。
    2. 在Chimera中打开pdb蛋白质结构文件。转到工具>结构分析>定义属性。选择步骤a中生成的.txt文件。
      注意:如果列出了在结构文件中未解析的残基的数据,则会在弹出窗口中打开错误。这个错误可以忽略。
    3. 一旦定义了属性,就会打开“按属性渲染/选择”窗口。使用此窗口可确定哪些颜色代表哪些%D值。
      1. 在txt文件中列出但没有与它们相关的数据的残留物将根据最高的%D颜色进行着色,除非在此值以上添加了其他颜色。
      2. 要添加其他颜色条,请使用Ctrl +在直方图中左键单击。 Ctrl +左键单击现有的颜色条以将其删除。

      3. 通过鼠标移动颜色条或在'数值'框中输入一个值。
    4. 点击“应用”根据数据文件着色蛋白质结构。
    5. 图3显示了一个彩色结构的例子。
    注意:视频3显示了如何将HDX-MS数据映射到蛋白质的3D结构。

    小号视频3

笔记

  1. 推荐的用于HPLC分离多肽的替代色谱柱与本文所述方案中使用的色谱柱相同,但内径要大1mm。这将影响通过色谱柱的溶剂流速,这可能会影响色谱柱中肽段的洗脱。强烈建议在进行交换反应之前测试不同的流速和梯度以优化肽的分离。
  2. 通过将校正因子(0.4个pH单位)加到测量的pH值,必须将测得的氘化缓冲液的pH校正为pD。对于该实验,在D 2 O中制备的三碱缓冲液具有7.0的测量pH,因此缓冲液的pD是7.4(Krezel和Bal,2004)。
  3. 在开始交换反应之前,检查含有甲酸,胃蛋白酶和反应缓冲液的骤冷溶液的pH为2.5,因此在蛋白质消化过程中不会发生回流交换。
  4. 将HPLC溶剂置于冰上,将柱温箱置于0°C,将自动进样器置于4°C以帮助减少氘代酰胺的回流交换。
  5. HDX-MS可以在很宽的温度范围内进行。虽然汇率确实对温度有依赖性,但pH值可能会对它造成更多影响(Walters et al。,2012)。然而,应该注意的是,由于随着温度变化的汇率变化,在不同温度下运行的HDX-MS反应之间的比较分析是困难的。 HDX-MS通常在25°C(室温)下运行,但据报道有些情况下运行温度低至0°C,高达60°C。
  6. 在规划HDX-MS实验时,强烈建议感兴趣蛋白的起始浓度大约为10 mg / ml,这可以确保大约1.5μg蛋白被注入仪器。
    1. 注入仪器的蛋白质量较低会导致肽在低强度下被检测到,这反过来可能会阻止准确计算氘的含量。
    2. 鼓励将蛋白质1:10稀释到D 2 O缓冲液中,如果特定蛋白质不容易获得高浓度的等分试样,则可以做得更少。然后推荐进行一些中试实验以确保氘的掺入是令人满意的,并且反应中存在的额外的水不会引起氘离子的交换。

食谱

  1. 氧化氘中的Tris碱缓冲液(保质期〜一周)
    100mM Tris-HCl
    150 mM NaCl
    用1N HCl调节pH至7.0
  2. 在H 2 O中的Tris碱缓冲液 100mM Tris-HCl
    150 mM NaCl
    用1N HCl调节pH至7.0
  3. 含有连二硫酸钠的Tris碱缓冲液 100mM Tris-HCl
    150 mM NaCl
    2 mM连二硫酸钠
    用1N HCl调节pH至7.0

致谢

作为由美国能源部科学办公室(DE-SC0012518)资助的能源前沿研究中心生物和电子转移与催化(BETCy)EFRC的一部分,这项工作得到了支持。 MSU的质谱仪器部分由Murdock慈善信托基金和NIH IDEA计划资助P20GM103474资助。本协议中详述的工作改编自最近出版物(Lubner等人,2017; Berry等人,2018)中描述的工作。作者声明他们没有任何利益冲突。

参考

  1. Berry,L.,Poudel,S.,Tokmina-Lukaszewska,M.,Colman,D.R.,Nguyen,D.M.N.,Schut,G.J.,Adams,M.W.,Peters,J.W.,Boyd,E.S和Bothner,B。(2018)。 H / D交换质谱和统计耦合分析揭示了铁蛋白依赖性分叉中的作用transhydrogenase catalytic cycle。
  2. Demmer,J.K.,Rupprecht,F.A.,Eisinger,M.L.,Ermler,U.和Langer,J.D。(2016)。 氢 - 氘交换显示的基于黄素的电子分叉酶复合物中的配体结合和构象动力学Mass Spectrometry。 FEBS Lett 590(24):4472-4479。
  3. Krezel,A.和Bal,W。(2004)。 关联p K a 的公式在D 2 O和H 2 O中确定的值。Inorg Biochem 98:161- 166。
  4. Lubner,CE,Jennings,DP,Mulder,DW,Schut,GJ,Zadvornyy,OA,Hoben,JP,Tokmina-Lukaszewska,M.,Berry,L.,Nguyen,DM,Lipscomb,GL,Bothner,B.,Jones ,AK,Miller,AF,King,PW,Adams,MWW和Peters,JW(2017)。 通过基于黄素的电子分叉对机械能的保护 Nat Chem Bolsol 13(6):655-659。
  5. Pettersen,E.F。,Goddard,T.D.,Huang,C.C。,Couch,G.S。,Greenblatt,D.M.,Meng,E.C。和Ferrin,T.E。(2004)。 UCSF Chimera - 用于探索性研究和分析的可视化系统。 J Comput Chem 25(13):1605-1612。
  6. Walters,B. T.,Ricciuti,A.,Mayne,L.和Englander,S.W。(2012)。 尽量减少氢气交换 - 质谱实验中的回流。 J Am Soc Mass Spectrom 23(12):2132-2139。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Berry, L., Patterson, A., Pence, N., Peters, J. W. and Bothner, B. (2018). Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins. Bio-protocol 8(6): e2769. DOI: 10.21769/BioProtoc.2769.
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