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Sep 2020

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Simultaneous Imaging of Single Protein Size, Charge, and Binding Using A Protein Oscillation Approach
利用蛋白质振荡方法同时成像单个蛋白质的大小、电荷和结合状态   

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Abstract

Electrophoresis and Western blot are important tools in protein research for detection and identification of proteins. These traditional techniques separate the proteins based on size and charge differences and identify the proteins by antibody binding. Over the past decade, the emergence of single-molecule techniques has shown great potential in improving the resolution of the traditional protein analysis methods to the single-molecule level. However, such single-molecule techniques measure either size or charge, and it is challenging to measure both at the same time. Recently, we have developed a single-molecule approach to address this problem. We tether the single proteins to a surface with a polymer linker and drive them into oscillation with an electric field. By tracking the electromechanical response of the proteins to the field using an optical imaging method, the size and charge can be obtained simultaneously. Binding of antibodies or ions to the tethered protein also changes the size and charge, which allows us to probe the interactions. This protocol includes fabrication of protein oscillators, configuration of the optical detection system, and analysis of the oscillation signal for quantification of protein size and charge. We wish this protocol will enable researchers to perform comprehensive single-protein analysis on a single platform.

Keywords: Single-molecule imaging (单分子成像), Single protein analysis (单一蛋白分析), Size (大小), Charge (电荷), Mobility (迁移率), Binding kinetics (结合动力学)

Background

Proteins play essential roles in many biological processes and serve as drug targets and biomarkers. Analysis of proteins rely on technologies including electrophoresis, Western Blot, and mass spectrometry, which separate and identify proteins based on the size and charge of protein molecules. Although powerful in detecting proteins, these technologies are not sensitive to single molecules, which is required for elucidating molecular heterogeneity and precision diagnosis. Recently, several single-molecule technologies have been developed to measure the size or charge of single biomolecules. Examples include interferometric scattering microscopy (iSCAT) (Young et al., 2018) and plasmonic scattering microscopy (PSM) (Zhang et al., 2020), which quantify the size by measuring the scattered light from the molecules. Single-molecule electrometry (Ruggeri et al., 2017) and anti-Brownian electrokinetic (ABEL) trap (Wang et al., 2012) measure the charge of single molecules by monitoring single molecule motion in a potential trap. However, simultaneously measuring the size and charge on a single platform remains challenging.


Recently, we have developed a single-molecule technique to solve this problem (Ma et al., 2020). We tether proteins to an indium tin oxide (ITO) surface by polyethylene glycol (PEG) linkers and drive them into oscillation in vertical direction by applying an alternating electric field (Figure 1A). To track the protein oscillation, we place the ITO on an inverted optical microscope and generate evanescent field on the ITO surface. The oscillating proteins scatter the evanescent field, which is collected by the microscope and detected by a CMOS camera (Figure 1B). Using this method, the oscillation can be tracked with nanometer precision. By analyzing the oscillation and electromechanical response of the proteins to the field, the size and charge of individual proteins can be determined. The current protocol describes the assembly of protein oscillators and the optical detection system. Applications including protein size, charge, and binding measurements will also be presented. This protocol is also applicable to other single-molecule detection platforms, such as PSM and iSCAT, to expand the capability in simultaneous charge measurements.



Figure 1. Detection principle and schematic of the detection system. A. The protein is tethered to the ITO surface by a PEG tether. An electric field is applied to the surface which drives the protein into oscillation. An evanescent field is generated on the surface to probe the oscillation of the protein. B. The detection system is based on an inverted microscope. A p-polarized incident light is directed onto the ITO surface via a high-numeric aperture objective with an incident angle slightly lower than the total internal reflection angle. The electric field is applied by a three-electrode electrochemical system, where the WE, RE, and CE represent working electrode, reference electrode, and counter electrode, respectively.


Materials and Reagents

  1. ITO coated cover slips, 22 × 22 mm, thickness #1, 70-100 Ohms resistivity (SPI Supplies, catalog number: 06470-AB)

  2. Silver wire (Alfa Aesar, catalog number: 11468-G9)

  3. Platinum wire (Alfa Aesar, catalog number: 10958-CB)

  4. Reusable silicone well (SARSTEDT, flexiPERM®, catalog number: 94.6032.039)

  5. Streptavidin (VWR, catalog number: 97062-810)

  6. (3-glycidyloxypropyl) trimethoxylsilane (Sigma-Aldrich, catalog number: 440167)

  7. Bovine serum albumin (Sigma-Aldrich, catalog number: 05470)

  8. Goat IgG (MW = 150 kDa) (Abcam, catalog number: ab76907)

  9. Rabbit anti-goat IgG (MW = 150 kDa) (Invitrogen, catalog number: A16138)

  10. Biotin-PEG10k-NHS (Nanocs, catalog number: PG2-BNNS-10k)

  11. Polystyrene (PS) nanoparticles, diameters of 50, 100, 150 and 200 nm (Bangs Laboratories, catalog numbers: PS2002, PS2004, PS2006, and PS2008)

  12. (Optional) Sodium azide purification kit (Nanopartz, catalog number: PPZ-KIT-10-MAG)

  13. 1× phosphate buffered saline (Corning, catalog number: 21-040-CV)

  14. Immersion oil type A (Cargille, catalog number: 16482)

  15. Isopropanol (Sigma-Aldrich, catalog number: 109827)

  16. Acetone (VWR, BDH®, BDH2002)

  17. Ethanol (Koptec’s Pure Ethanol 200 Proof, Decon Labs, catalog number: V1001)

  18. Ammonium Hydroxide (Mallinckrodt Chemicals, catalog number: 3256)

  19. Hydrogen peroxide (VWR, catalog number: BDH7814-3)

  20. H2O2/NH3·H2O/H2O (1:3:5) mixture (see Recipes)

  21. 0.1 mg/ml BSA blocking solution (see Recipes)

  22. 100 times diluted PBS buffer (see Recipes)

Equipment

  1. Inverted optical microscope (Olympus, model: IX-81)

  2. 60× NA 1.49 oil immersion TIRF objective (Olympus, model: APON60XOTIRF)

  3. Antivibration optical table (Newport, model: RS2000)

  4. XYZ translation stage (Thorlabs, model: PT3)

  5. Superluminescent light emitting diode (SLED) (Superlum, model: SLD-260-HP-TOW-PD-670)

  6. SLED current driver (Superlum, model: PILOT4-AC)

  7. CMOS camera (Hamamatsu, model: ORCA-Flash 4.0)

  8. Function generator (Agilent, model: 33521A)

  9. Potentiostat (Pine Instrument Company, model: AFCBP1)

  10. USB data acquisition card (National Instruments, model: NI USB-6251)

  11. Vacuum pump (TOPSFLO, model: TM30A-B6-P9504 (V6004))

  12. Plastic tubing, 0.02” ID × 0.06” OD (Saint-Gobain, TYGON®, model: AAD04103)

  13. Syringe with Luer-LokTM tip, 20 ml (Fisher Scientific, catalog number: 22-124-967)

  14. Perfusion manifold, 4 to 1 ports (Warner Instruments, model: MM-4)

  15. Syringe 1-way stopcock (B. Braun Medical Inc., catalog number: 455980)

  16. Dispensing needles (Weller, catalog number: KDS2312P)

  17. Cover glass staining jars (Fisher Scientific, catalog number: 02-912-636)

  18. Ultrasonic cleaner (Fisher Scientific, model: FS20D)

  19. Tweezers (Electron Microscopy Sciences, catalog number: 72750-D)

  20. Computer (Processor: Intel Xeon E-2274G; RAM: 64 GB; Hard drive: Samsung SSD 970 PRO 1TB)

Software

  1. HCImage Live (Hamamatsu)

  2. MATLAB (MathWorks)

  3. Fiji (https://imagej.net/Fiji)

  4. Origin 2019 (OriginLab)

  5. Office 365 (Microsoft)

Procedure

  1. Cleaning and silanization of ITO surface

    1. Put the ITO chips in a staining jar and sonicate the chips sequentially in acetone, ethanol, and DI water each for 20 min to remove surface contaminates.

    2. Incubate the clean ITO chips in another jar filled with a mixture of H2O2/NH3·H2O/H2O (1:3:5) at room temperature for 1 h to increase the surface density of OH.

    3. Rinse the ITO chips with DI water and dry with N2.

    4. Fill a pre-dried staining jar with 1% (3-glycidyloxypropyl) trimethoxylsilane in isopropanol, place the dried ITO chips in the jar and incubate at room temperature overnight to functionalize the chips with terminal epoxy groups (Figures 2A and 2B).

    5. Rinse the ITO chips with isopropanol and water, each for 3 times, and dry with N2. Mount a silicone well on the ITO side of the chip before fabricating protein oscillators.


  2. Preparation of protein-PEG complex

    1. Dilute the protein with PBS to reach a final concentration of 100 nM with a volume of 100 μl.

      Note: The protein sample should not contain any NHS-reactive additives such as BSA and azide. If azide-free protein sample is unavailable, sodium azide purification kits can be used to remove the azide (example: Nanopartz, catalog number: PPZ-KIT-10-MAG).

    2. Prepare 10 nM NHS-PEG10k-biotin in 100 μl PBS.

      Note: NHS degrades fast in water, always use fresh solution for the reaction.

    3. Mix the 100 μl protein solution and the 100 μl NHS-PEG10k-biotin solution and incubate the mixture at 4 °C overnight (Figures 2D-2E).

      Note: The 10/1 molar ratio of protein/PEG prevents multiple PEG molecules linking to the same protein molecule.


  3. Assembly of protein oscillators

    1. Add 200 μl 0.1 mg/ml streptavidin (dissolved in PBS) to the ITO chip in the sample well and incubate for 2 h to allow immobilization of streptavidin (Figure 2C).

    2. Wash the chip with PBS for 3 times.

    3. Add 0.1 mg/ml BSA solution to ITO and incubate for 20 min to block non-specific binding sites.

    4. Wash the chip with PBS for 3 times.

    5. Add 200 μl protein-PEG complex solution to the chip and incubate for 2 h to allow the terminal biotin in the PEG couple to the streptavidin on the ITO surface (Figure 2F).

    6. Slowly remove 100 μl solution from the well, and then gently add 400 μl of 100 times diluted PBS to the well. Do not generate fast flow in the solution because it may break the tethered protein.

    7. Slowly remove 400 μl solution from the well. Repeat Steps C6-C7 for ~10 times to reduce the amount of free protein molecules and unbound protein-PEG complexes remaining in the solution.

      Note: The surface should always be covered with at least 100 μl solution, drying out the surface will lead to irreversible adhesion of the tethered proteins to the surface. Also, avoid generating air bubbles on the surface when pipetting.



      Figure 2. Fabrication of protein oscillators. A-C. Immobilization of streptavidin on ITO surface. D-F. Assembly of protein-PEG complex and protein oscillator.


  4. Measuring the electromechanical response of protein oscillators

    1. Mount the ITO chip on the microscope with half drop of immersion oil placed between the objective and the chip. Set the camera exposure time to 1 ms. Set the camera frame rate to 800 fps at 2048 × 256 pixels. Turn off the camera cooling fan to reduce mechanical noise.

    2. Park the incident angle above the total internal reflection angle, where all the incident light is reflected. Increase the incident power by tuning the SLED current until the camera is saturated (maximum image intensity ~65,535).

    3. Slowly lower the incident angle until it is slightly below the total internal reflection angle, and half of the light is reflected to the camera (image intensity ~30,000).

    4. Set up the electrochemical system and gravity sample delivery system as shown in Figures 3A and 3B.

    5. Double-check if the electrodes are correctly connected. Short circuit or open circuit can damage the ITO surface.

    6. Find a large impurity on the surface and roughly adjust the focus until the pattern of the impurity turns to V-shape (Figure 3C) from parabolic shape (Figure 3D). Move the stage and find a clean region with no impurities (Figure 4A).



      Figure 3. Experimental setup for measuring protein oscillation. A. A picture showing the whole setup. B. A zoom-in of the sample stage (the red square in A). The ITO chip is placed on the objective. A self-adhesion silicone well is sticked to the chip for holding solution. The ITO surface, an Ag/AgCl wire, and a Pt coil serve as the working electrode (WE), reference electrode (RE), and counter electrode (CE), respectively. The sample inlet directs the sample to the surface within the field of view to minimize diffusion. The outlet tubing (connected to a vacuum pump) is fixed near the upper edge of the well and continuously removing excess solution from the well. C. An image showing a large impurity that is properly focused (inverted V shape). D. An image showing an impurity that is out of focus (parabolic patterns).


    7. Apply a sinusoidal potential of 6 V at 80 Hz to the ITO surface, and record the images for 1 s (Video 1).


      Video 1. An image sequence recorded at 800 fps for 1 s.  Any applied potential between 0 V to 10 V should generate only minor changes to the raw images that are not observable without image processing. The top video shows the raw image sequence recorded by the camera, and the bottom video shows differential images obtained by subtracting the first frame from each frame in the raw image sequence (the background is removed). The applied potential in the video is 8 V at 80 Hz. The sensor surface is modified with IgG oscillators.

    8. Extract the oscillation signal at 80 Hz using a custom MATLAB fast Fourier transform (FFT) filter. The program can be found in the supporting information in Ma et al. (2020) .

    9. Examine the FFT image to see if there are sufficient amount of protein molecules oscillating in the field of view and whether they are correctly focused. If there are not enough proteins, move the stage to another region. If the focus is incorrect, slightly tune the focus knob and repeat Steps D7-D9.

    10. Apply sinusoidal potentials from 0 V to 10 V with 1 V or 2V interval at 80 Hz. Record 1 s video at 800 fps for each potential. If needed, record the applied potential and current simultaneously using data acquisition card.


  5. Measuring binding induced size and charge changes using Anti-IgG and IgG oscillators as an example

    1. Add 20 ml of 100 times diluted PBS buffer and 2 ml of 130 nM anti-IgG in 100 times diluted PBS buffer to two different syringes in the sample delivery system (Figure 3A).

    2. Turn on the buffer channel to flow buffer over the ITO surface modified with IgG oscillators (the flow rate is about 300 μl/min if the valve is fully turned on).

    3. Apply a potential with 9 V amplitude at 80 Hz to the ITO (which fully stretches the PEG). Start recording images and if needed, use data acquisition card to record the potential and current.

    4. After flowing the buffer for 20 s to establish a baseline, turn off the buffer syringe and turn on the anti-IgG channel to start the association process. Flow anti-IgG for 30 s.

    5. Switch back to the buffer channel and flow buffer for 30 s for dissociation measurement.

    6. For charge detection, use a lower potential to oscillate the protein (~5 V) so that the PEG is not completely stretched. Do not apply high potential to fully stretch the PEG because charge change can be either positive or negative, which will either increase or decrease the oscillation amplitude.


  6. Size calibration using PS nanoparticles

    1. Dilute each PS nanoparticle stock solution (50, 100, 150, 200 nanometers) with PBS for 1000 times.

    2. Add 300 μl PBS to the sample well mounted on a bare ITO surface, and then inject 50 μl of diluted 50 nm nanoparticle solution to allow nanoparticles binding to the surface. Upon injection, start recording images at 400 fps (512 × 2048 pixels) for 1 min.

    3. Repeat Step F2 to measure the other sizes. Use a new ITO chip for each measurement.

    4. Note that the optimal nanoparticle concentration should lead to ~5 binding events per image. Particle concentration may vary, adjust the concentration if it is not appropriate.

Data analysis

  1. Constructing a size calibration curve

    1. Use Fiji software to obtain a differential image sequence showing nanoparticle binding by subtracting the first frame from each frame in the sequence (remove the background) (Video 2).

    2. Select 10 × 10 pixels regions of interest (ROIs) on the center of the particle images (red squares in Video 2) and calculate the mean intensity (I) within each ROI.


      Video 2. Nanoparticle of different sizes binding to the surface.  Each video is cropped from the original video showing a single particle (50, 100, 150, and 200 nm) binding to the surface. For better visualization, background noise is reduced by averaging every 11 neighboring frames using the moving average function in Fiji software (Select “Plugins>Stowers>Jay_Unruh>Detrend Tools>stack divide moving average jru v2”).


    3. Measure ~100 individual nanoparticles for each size (D).

    4. Plot log(I) vs. log(D) and find the slope of the curve, which should be around 2.


  2. Single protein analysis – temporal fast Fourier transform

    1. Perform fast Fourier transform (FFT) on the image sequence recorded in Procedure D (Figure 4A) using a custom MATLAB program (the same program mentioned in Procedure D8).

    2. Use the 81st image in the FFT image sequence, which shows the signal at 80 Hz, for single protein analysis (Figure 4B).


  3. Single protein analysis – background removal

    1. Perform k-space FFT to the temporal FFT image obtained above and convert the image to k-space (Figure 4C) using Fiji (Select “Process>FFT>FFT”).

    2. Apply a band-block filter (Figure 4D) which filters out the low-frequency background patterns while keeping the signal. Use the rectangle selection tool to select the low-frequency region, and then use “Edit>Clear” to remove the selected region.

    3. Use “Process>FFT>Inverse FFT” to transform the image back to time domain (Figure 4E).


  4. Oscillation amplitude vs. applied potential

    1. Select ROIs (10 × 10 pixels) on the bright spots which are tethered proteins (Figure 4F). Measure the mean intensity of each ROI and subtract background intensity (the mean intensity of the whole image).



      Figure 4. Image processing. A. Raw image sequence obtained in Procedure D. The calibration bar shows the image intensity in gray scale. B. Temporal FFT image of IgG molecules obtained by performing FFT on the image sequence. Applied potential: 8 V at 80 Hz; FFT integration time: 1 s. C. k-space FFT is performed on the temporal FFT image. D. A band-block filter (black region) is applied to partially remove the background. E. Image after background removal. F. ROIs of 10 × 10 pixels are selected for single protein size and charge analysis.


    2. Measure the mean intensity (with background subtraction) of each tethered protein at different applied potentials.

    3. Plot intensity (y-axis) vs. applied potentials (x-axis). The intensity increases linearly with applied potential at first and reaches a plateau at high potentials (often > 6 V) because the tether is fully stretched (Figure 5).

    4. Convert the intensity to oscillation amplitude in nanometers using the equation:

      where ∆z0 is the oscillation amplitude, I is the evanescent wave scattering intensity of a protein, ∆I(∆z0 ) is the protein oscillation intensity at z0, and d is the decay constant of evanescent field. I is determined from the oscillation plateau regime when ∆z0 is 63 nm (the length of the PEG) and ∆I(∆z0=63 nm) is the maximum FFT image intensity of the protein.


  5. Single protein size analysis

    1. Find the ∆I(∆z0=63 nm) value for each individual protein using the intensity vs. applied potential plots (Figure 5). Determine I for each protein using equation [1].



      Figure 5. Calculating the size and charge. The plot shows the oscillation response (intensity) vs. the applied potential of a single IgG molecule. At low potentials, the oscillation increases with the potential in a roughly linear manner, because the PEG is stretched by the electric field. At high potentials, the PEG is fully stretched, and the oscillation (intensity) reaches the maximum value. The protein size is determined by the intensity at the plateau (∆I(∆z0=63 nm)), and the charge is determined by the potential (U) or field when the PEG is fully stretched. The positions for ∆I(∆z0=63 nm) and U are marked on the plot.


    2. Convert I to diameter Dapp using the size calibration curve. Here, Dapp is the effective diameter of the protein-PEG complex.

    3. Calculate the diameter of protein using , where DPEG is the diameter of PEG coil measured by dynamic light scattering. DPEG is ~4.5 nm for PEG10k in 100 times diluted PBS according to our dynamic light scattering measurement.


  6. Single protein charge analysis

    1. Find the potential U on the intensity vs. applied potential plot where the oscillation amplitude reaches the plateau (Figure 5).

    2. Convert the potential U to electric field E0 using the equation E= 3.53×106 U/m.

      Note: This relationship is determined on our system, and we recommend the readers to calibrate it before experiment in consideration of system differences. Please refer to Ma et al. (2020) for details.

    3. Determine the charge q using , where kPEG is the entropic spring constant of the PEG tether. For PEG10k, kPEG~3.62×10-4 N/m.

    4. Determine the mobility μ of each protein by μ=q/(3πηD), where η is the solution viscosity.

Recipes

  1. H2O2/NH3·H2O/H2O (1:3:5) mixture

    To make 45 ml:

    5 ml H2O2

    15 ml NH3·H2O

    25 ml DI water

  2. 0.1 mg/ml BSA blocking solution

    Add 1 mg of BSA to 10 ml PBS buffer (pH 7.4)

  3. 100 times diluted PBS buffer

    To make 40 ml:

    400 μl PBS

    39.6 ml H2O

Acknowledgments

We thank financial support from National Institute of Health (R44GM126720). This protocol is based on our work published in Nature Communication ( Ma et al., 2020 ).

Competing interests

A US patent application (16/584,120) has been filed by Arizona Board of Regents on behalf of Arizona State University based on the method described in this protocol and published on 03/26/2020.

References

  1. Ma, G., Wan, Z., Yang, Y., Zhang, P., Wang, S. and Tao, N. (2020). Optical imaging of single-protein size, charge, mobility, and binding. Nat Commun 11(1): 4768.
  2. Ruggeri, F., Zosel, F., Mutter, N., Rozycka, M., Wojtas, M., Ozyhar, A., Schuler, B. and Krishnan, M. (2017). Single-molecule electrometry. Nat Nanotechnol 12(5): 488-495.
  3. Wang, Q., Goldsmith, R. H., Jiang, Y., Bockenhauer, S. D. and Moerner, W. (2012). Probing single biomolecules in solution using the anti-Brownian electrokinetic (ABEL) trap. Accounts Chem Res 45(11): 1955-1964.
  4. Young, G., Hundt, N., Cole, D., Fineberg, A., Andrecka, J., Tyler, A., Olerinyova, A., Ansari, A., Marklund, E. G., Collier, M. P., Chandler, S. A., Tkachenko, O., Allen, J., Crispin, M., Billington, N., Takagi, Y., Sellers, J. R., Eichmann, C., Selenko, P., Frey, L., Riek, R., Galpin, M. R., Struwe, W. B., Benesch, J. L. P. and Kukura, P. (2018). Quantitative mass imaging of single biological macromolecules. Science 360(6387): 423-427.
  5. Zhang, P., Ma, G., Dong, W., Wan, Z., Wang, S. and Tao, N. (2020). Plasmonic scattering imaging of single proteins and binding kinetics. Nat Methods 17(10): 1010-1017.

简介

[摘要]电泳和蛋白质印迹是蛋白质研究中用于检测和鉴定蛋白质的重要工具。这些传统技术根据大小和电荷差异分离蛋白质,并通过抗体结合鉴定蛋白质。Ø版本在过去的十年中,单分子技术的出现显示了将传统蛋白质分析方法的分辨率提高到单分子水平的巨大潜力。然而,这样的单分子技术测量大小或电荷,并且同时测量这两者是挑战性的。最近,我们已经开发出一种单分子方法来解决这个问题。我们使用聚合物接头将单个蛋白质束缚在表面上,并通过电场驱动它们振荡。通过使用光学成像方法跟踪蛋白质对电场的机电响应,可以同时获得大小和电荷。抗体或离子与拴系蛋白的结合也会改变大小和电荷,这使我们能够探查相互作用。该协议包括蛋白质振荡器的制造,光学检测系统的配置以及用于定量蛋白质大小和电荷的振荡信号的分析。我们希望该协议将使研究人员能够在单个平台上执行全面的单蛋白分析。



[背景]蛋白质在许多生物学过程的发挥重要作用,并作为药物靶标和生物标志物。蛋白质分析依赖于电泳,蛋白质印迹和质谱等技术,这些技术根据蛋白质分子的大小和电荷来分离和鉴定蛋白质。尽管这些技术在检测蛋白质方面功能强大,但它们对阐明分子异质性和精确诊断所必需的单个分子不敏感。最近,已经开发了几种单分子技术来测量单个生物分子的大小或电荷。实例包括:i nterferometric散射显微镜(iSCAT )(杨氏等人。,2018)和等离子体激元散射显微镜(PSM) (张等人。,2020) ,其通过从分子测量散射光量化的大小。单分子电测定法(Ruggeri等,2017)和反布朗电动(ABEL)陷阱(Wang等,2012)通过监测电势陷阱中的单分子运动来测量单分子的电荷。然而,在单个平台上同时测量尺寸和电荷仍然具有挑战性。

最近,我们已经开发出一种单分子技术来解决这个问题(Ma等,2020)。我们通过聚乙二醇(PEG)接头将蛋白质束缚在氧化铟锡(ITO)表面,并通过施加交变电场使它们在垂直方向上振荡(图1A)。为了跟踪蛋白质的振荡,我们将ITO放在倒置的光学显微镜上,并在ITO表面上产生van逝场。振荡的蛋白质会散射渐逝场,渐逝场由显微镜收集并由CMOS相机检测(图1B)。使用这种方法,可以以纳米精度跟踪振荡。通过分析蛋白质对磁场的振荡和机电响应,可以确定单个蛋白质的大小和电荷。当前的协议描述了蛋白质振荡器和光学检测系统的组装。还将介绍包括蛋白质大小,电荷和结合力测量在内的应用。该协议还适用于其他单分子检测平台,例如PSM和iSCAT ,以扩展同时进行电荷测量的能力。



图1.检测原理和检测系统原理图。A.蛋白质通过PEG系链拴在ITO表面。电场被施加到表面上,从而驱动蛋白质振荡。在表面上会产生一个渐逝场,以探测蛋白质的振荡。B.检测系统基于倒置显微镜。甲p偏振光入射的光经由与入射角的高数值孔径物镜引导到ITO表面比所述全内反射角度略低。电场由三电极电化学系统施加,其中WE,RE和CE分别代表工作电极,参比电极和对电极。

关键字:单分子成像, 单一蛋白分析, 大小, 电荷, 迁移率, 结合动力学



材料和试剂


1. ITO Ç oated Ç过小号唇,22 × 22毫米,吨hickness#1,70-100欧姆- [R esistivity (SPI小号upplies ,目录号:06470-AB )     

2.银线(Alfa Aesar ,目录号:11468-G9 )     

3.铂金线(Alfa Aesar ,目录号:10958-CB)     

4.可重复使用的小号ilicone阱(SARSTEDT,flexiPERM ® ,目录号:94.6032.039)     

5.链霉亲和素(VWR,目录号:97062-810 )     

6. (3-缩水甘油基羟丙基)三甲氧基硅烷(Sigma-Aldrich,目录号:440167)     

7.牛血清白蛋白(Sigma-Aldrich,目录号:05470)     

8.山羊IgG(MW = 150 kDa )(Abcam ,目录号:ab76907)     

9.兔抗山羊IgG(MW = 150 kDa )(Invitrogen,目录号:A16138)     

10.生物素-PEG10k-NHS (N anocs ,目录号:PG2-BNNS-10k) 

11.聚苯乙烯(PS)的纳米颗粒,d的iameters 50,100,150和200纳米(邦斯实验室,目录号:PS2002,PS2 004,PS2006,PS2008和) 

12. (可选)叠氮化钠纯化试剂盒(Nanopartz ,目录号:PPZ-KIT-10-MAG) 

13. 1 ×磷酸盐缓冲盐水(Corning,目录号:21-040-CV) 

14. A型浸油(Cargille ,目录号:16482) 

15.异丙醇(Sigma-Aldrich,目录号:109827) 

16.丙酮(VWR,BDH ® ,BDH2002) 

17.乙醇(Koptec的纯乙醇200证明,去污实验室,目录号:V1001) 

18.氢氧化铵(Mallinckrodt Chemicals,目录号:3256) 

19.过氧化氢(VWR,目录号:BDH7814-3) 

20. H 2 O 2 / NH 3 ·H 2 O / H 2 O(1:3:5)混合物(请参阅食谱)   

21. 0.1 mg / ml BSA封闭溶液(请参阅食谱)   

22. 100倍稀释的PBS缓冲液(请参阅食谱)   



设备


倒置光学显微镜(奥林巴斯,型号:IX-81)
60 × NA 1.49油浸TIRF物镜(奥林巴斯,型号:APON60XOTIRF )
防震光学平台(Newport,型号:RS2000)
XYZ平移台(Thorlabs ,型号:PT3)
超发光发光二极管(SLED)(Superlum ,型号:SLD-260-HP-TOW-PD-670)
SLED电流d河(Superlum ,型号:PILOT4-AC)
CMOS相机(滨松,型号:ORCA-Flash 4.0)
函数发生器(Agilent,型号:33521A)
恒电位仪(Pine仪器公司,型号:AFCBP1)
USB数据采集卡(National Instruments,型号:NI USB-6251)
V acuum泵(TOPSFLO,型号:TM30A-B6-P9504(V6004) )
塑料管,0.02 ” ID × 0.06 ” OD (圣戈班,TYGON ® ,型号:AAD04103)
带Luer-Lok TM尖端的注射器,20 m l (Fisher Scientific,目录号:22-124-967 )
灌注歧管,4至1个端口(Warner Instruments,型号:MM-4)
注射器1通旋塞阀(B.Braun Medical Inc.,目录号:455980)
点胶针头(Weller,目录号:KDS2312P )
盖上玻璃染色罐(Fisher Scientific,目录号:02-912-636 )
超声波Ç精简(Fisher Scientific公司,型号:FS20D)
镊子(电子显微镜科学,目录号:72750-D)
计算机(处理器:Intel Xeon E-2274G; RAM:64 GB;硬盘:Samsung SSD 970 PRO 1TB)


软件


HCImage Live(滨松)
MATLAB (MathWorks )
斐济(https://imagej.net/Fiji)
Origin 2019(OriginLab)
Office 365(微软)


程序


ITO表面的清洁和硅烷化
将ITO芯片放入染色罐中,然后依次在丙酮,乙醇和去离子水中分别超声处理20分钟,以去除表面污染物。
在另一个装有H 2 O 2 / NH 3 ·H 2 O / H 2 O(1:3:5)混合物的广口瓶中,将干净的ITO芯片在室温下孵育1小时,以增加OH的表面密度。
用去离子水冲洗ITO芯片,然后用N 2冲洗y y 。
用异丙醇中的1%(3-环氧丙氧基丙基)三甲氧基硅烷填充预干燥的染色罐,将干燥的ITO芯片放入罐中,并在室温下孵育过夜,以使带有末端环氧基的芯片功能化(图2A和2 B)。
用异丙醇和水冲洗ITO芯片,每次3次,然后用N 2干燥。安装一个小号ilicone在阱的ITO的侧制造蛋白质振荡器之前芯片。


Prepar的通货膨胀蛋白-PEG复合
稀释用PBS蛋白以达到一个最终的浓度100 nM的与100的体积μ升。
注意:蛋白质样品中不应含有任何NHS反应性添加剂,例如BSA和叠氮化物。如果叠氮化-free蛋白质样品是不可用的,钠叠氮化物纯化试剂盒可用于去除叠氮化物(例如:Nanopartz ,目录号:PPZ-KIT-10-MAG)。


制备10 nM的NHS-PEG10k生物素在10 0 μ升PBS。
注意:NHS在水中会快速降解,请始终使用新鲜溶液进行反应。


混合100 μ升蛋白溶液和100 μ升NHS-PEG10k-生物素溶液并在4孵育混合物℃过夜(图2D- 2 E)。
注意:蛋白质/ PEG的摩尔比为10/1,可以防止多个PEG分子连接至同一蛋白质分子。


蛋白质振荡器的组装
加入200 μ升0.1毫克/米升链霉(溶解在PBS中)至孔中的样品在ITO芯片和孵育2 ħ以允许链霉(图2C)的固定。
用PBS清洗芯片3次。
向ITO中添加0.1 mg / ml的BSA溶液,孵育20分钟以封闭非特异性结合位点。
用PBS清洗芯片3次。
加入200 μ升蛋白质-PEG复合物溶液到芯片和孵育2 ħ以允许PEG耦合到ITO表面(图2F)上的链霉亲和终端生物素。
慢慢除去100 μ升从井溶液,然后轻轻地添加400 μ升的100倍稀释的PBS到孔中。不要在溶液中产生快速流动,因为它可能会破坏拴系的蛋白质。
慢慢除去400 μ升从井中溶液。重复小号TEPS C6- Ç 7〜10倍,以减少游离的蛋白分子和未结合的蛋白-PEG复合物残留在溶液中的量。
注:表面应始终覆盖有至少100 μ升溶液,干燥的表面将导致拴系蛋白对表面的不可逆的粘附性。还请注意,移液时避免在表面上产生气泡。




图2.蛋白质振荡器的制造。交流电 链霉亲和素在ITO表面的固定化。DF。蛋白质-PEG复合物和蛋白质振荡器的组装。


中号EAS uring蛋白振荡器的机电响应
将ITO芯片安装在显微镜上,并在物镜和芯片之间放半滴浸油。将相机曝光时间设置为1 ms 。设置相机帧速率到800个在2048 FPS × 256像素。关闭相机冷却风扇,以减少机械噪音。
将入射角停在全内反射角上方,所有入射光都会在此反射。通过调节SLED电流直至摄像机饱和(最大图像强度〜65,535),增加入射功率。
缓慢降低入射角直到略低于全内反射的角度,并且光的一半被反射到相机(图像强度〜30 ,000)。
设置电化学系统和重力样品输送系统,如图3A和3B所示。
仔细检查电极是否正确连接。小号园艺或开路会损坏ITO表面。
在表面上找到较大的杂质,并大致调整焦点,直到杂质的图案从抛物线形状(图3D)变为V形(图3C)。移动平台,找到没有杂质的干净区域(图4A)。




图3.用于测量蛋白质振荡的实验装置。A.显示整个设置的图片。B.放大样品台(A中的红色方块)。ITO芯片放置在物镜上。将自粘硅树脂孔粘贴到芯片上以固定溶液。ITO表面,Ag / AgCl导线和Pt线圈分别用作工作电极(WE),参比电极(RE)和对电极(CE)。样品入口可将样品引导至视野内的表面,以最大程度地减少扩散。出口管(连接到真空泵)固定在井的上边缘附近,并不断从井中去除多余的溶液。C.显示适当聚焦的大杂质(倒V形)的图像。D.图像显示出焦点不清晰的杂质(抛物线图案)。


在ITO表面施加80 Hz的6 V正弦波电势,并记录图像1 s (视频1)。




视频1.以800 fps录制1秒钟的图像序列。0 V至10 V之间的任何施加电势都只会对原始图像产生微小的变化,如果不进行图像处理就无法观察到这些变化。顶部的视频显示了相机记录的原始图像序列,底部的视频显示了通过从原始图像序列中的每个帧中减去第一帧而获得的差分图像(背景被移除)。视频中施加的电势为80 V(80 Hz)。传感器表面用IgG振荡器修饰。


使用自定义的MATLAB快速傅立叶变换(FFT)滤波器提取80 Hz的振荡信号。该程序可以在Ma等人的支持信息中找到。(2020年)。
检查FFT图像,以查看视野中是否有足够数量的蛋白质分子振荡,以及它们是否正确聚焦。如果蛋白质不足,请将载物台移至另一个区域。如果重点是不正确,稍微调整调焦旋钮和重复小号TEPS d 7 d 9。
以80 Hz的频率以1 V或2V的间隔施加0 V至10 V的正弦电位。每个电位以800 fps录制1 s视频。如果需要,使用数据采集卡同时记录施加的电势和电流。


以抗IgG和IgG振荡器为例来测量结合诱导的大小和电荷变化
添加2 0米升的100倍稀释的PBS缓冲液和2M升的130 nM的抗IgG中稀释100倍PBS缓冲液中,以样品递送系统(图3A)的两个不同的注射器。
打开缓冲通道流动缓冲在ITO表面上用IgG振荡器改性(流速为约300 μ升/分钟,如果阀被完全打开)。
甲PP LY在80赫兹到ITO(其充分伸展PEG)与9 V振幅的电位。开始记录图像,如果需要,使用数据采集卡记录电位和电流。
在使缓冲液流动20秒钟以建立基线后,请关闭缓冲液注射器并打开抗IgG通道以启动关联过程。流动抗IgG 30 s。
切换回缓冲液通道并流动缓冲液30秒,以进行解离测量。
对于电荷检测,请使用较低的电位来振荡蛋白质(约5 V),以使PEG不能完全伸展。不要施加高电位来完全拉伸PEG,因为电荷变化可以是正的或负的,这会增加或减小振荡幅度。


使用PS纳米颗粒进行尺寸校准
用PBS稀释每种PS纳米颗粒储备溶液(50、100、150、200纳米)1000倍。
添加300 μ升P BS到样品井安装在裸ITO表面上,然后注入50 μ升的稀释50纳米的纳米颗粒溶液中以使纳米颗粒结合到表面上。注入后,开始以400 fps(512 × 2048像素)的速度记录图像1分钟。
重复小号TEP ˚F 2来衡量其他尺寸。每次测量均使用新的ITO芯片。
请注意,最佳纳米颗粒浓度应导致每个图像约5个结合事件。颗粒浓度可能会有所不同,如果不合适,请调整浓度。


数据分析


绘制尺寸校准曲线
使用斐济软件ö btain差分图像序列示出了纳米颗粒通过减去来自每个帧中的第一帧的序列中结合(去除背景)(视频2)。
在粒子图像的中心(视频2中的红色方块)上选择10 × 10像素的感兴趣区域(ROI),然后计算每个ROI内的平均强度(I )。




视频2.不同大小的纳米颗粒结合到表面。每个视频都是从原始视频中裁剪出来的,该视频显示了绑定到表面的单个粒子(50、100、150和200 nm)。为了更好地可视化,背景噪声通过使用在斐济软件的移动平均函数每11个相邻帧平均降低(选择“插件>斯托尔斯> Jay_Unruh >消除趋势工具>堆栈划分均线JRU V2”)。


对于每种尺寸(D ),测量约100个单独的纳米颗粒。
绘制log(I )与log(D )并找到曲线的斜率,该斜率应为2左右。


单一蛋白质分析–时间快速傅里叶变换
使用自定义MATLAB程序(过程D8中提到的同一程序),对在过程D(图4 A)中记录的图像序列执行快速傅里叶变换(FFT )。
在FFT图像序列中使用第81个图像,该图像显示80 Hz的信号,用于单个蛋白质分析(图4 B)。


单一蛋白质分析–去除背景
对上面获得的时间FFT图像执行k空间FFT ,然后使用斐济(选择“处理> FFT> FFT”)将图像转换为k空间(图4 C)。
应用带阻滤波器(图4 D),该滤波器滤出低频背景图案,同时保留信号。使用矩形选择工具选择低频区域,然后使用“编辑>清除”删除所选区域。
使用“ Process> FFT> Inverse FFT”将图像转换回时域(图4 E)。


振荡幅度与施加电势的关系
在被束缚的蛋白质的亮点上选择ROI(10 × 10像素)(图4 F)。测量每个ROI的平均强度,然后减去背景强度(整个图像的平均强度)。




图4.图像处理。A.在步骤D中获得的原始图像序列。校准栏以灰度显示图像强度。B.通过对图像序列执行FFT获得的IgG分子的时间FFT图像。施加的电势:80 Hz时为8 V;FFT积分时间:1 s。C. ķ在颞FFT图像执行-space FFT。D.应用带状滤镜(黑色区域)以部分去除背景。E.去除背景后的图像。F.选择10 × 10像素的ROI用于单个蛋白质大小和电荷分析。


在不同的施加电势下,测量每种束缚蛋白的平均强度(扣除背景)。
绘制强度(y轴)与施加电势(x轴)的关系。强度首先随着施加的电势线性增加,并在高电势(通常> 6 V)时达到平稳状态,因为系链已完全拉伸(图5)。
使用以下公式将强度转换为以纳米为单位的振荡幅度:


              [1]


其中,振荡振幅,I是蛋白质的e逝波散射强度,是z 0处的蛋白质振荡强度,d是van逝场的衰减常数。I是在63nm (PEG的长度)时从振荡平台状态确定的,并且是蛋白质的最大FFT图像强度。



单一蛋白质大小分析
使用强度与施加电势图(图5)找到每种蛋白质的值。确定我使用方程每种蛋白质[1] 。




图5.计算大小和费用。该图显示了振荡响应(强度)与单个IgG分子施加的电势的关系。在低电势下,振荡会随着电势以大致线性的方式增加,因为PEG被电场拉伸了。在高电位下,PEG被完全拉伸,并且振荡(强度)达到最大值。蛋白质的大小取决于平台的强度(),电荷的大小取决于PEG完全拉伸时的电势(U )或电场。和U的位置在图中标出。


使用尺寸校准曲线将I转换为直径D app 。在此,D app是蛋白质-PEG复合物的有效直径。
计算蛋白质的直径使用,其中d PEG是PEG线圈的通过动态光散射测得的直径。根据我们的动态光散射测量,在100倍稀释的PBS中,PEG10k的D PEG约为4.5 nm 。


单一蛋白质电荷分析
发现潜在ù上的I ntensity与施加在振荡幅度达到平台期电位曲线图(图5)。
转换的可能性û电场Ë 0使用情商uation 。
注意:此关系是在我们的系统上确定的,考虑到系统差异,建议读者在进行实验之前对其进行校准。请参考Ma等。(2020年)了解详情。


确定充电q使用,其中ķ PEG是PEG系绳的熵弹簧常数。对于PEG10k ,。 
确定所述移动性每种蛋白质的通过,在那里是溶液粘度。


菜谱


H 2 O 2 / NH 3 ·H 2 O / H 2 O(1:3:5)混合物
Ť ø使45毫升:


5毫升H 2 O 2


15毫升NH 3 ·H 2 O


25毫升去离子水


0.1 mg / ml BSA封闭液
向10 ml PBS缓冲液(pH 7.4 )中加入1 mg BSA。


100倍稀释的PBS缓冲液
Ť ø使40毫升:


400微升PBS


39.6毫升H 2 O


致谢


感谢国家卫生研究院(R44GM126720)的财政支持。该协议基于我们在《自然通讯》(Ma等,2020)中发表的工作。


利益争夺


亚利桑那州董事局已代表亚利桑那州立大学基于该协议中描述的方法提交了美国专利申请(16 / 584,120),该方法已于2020年3月26日发布。


参考


Ma,G.,Wan,Z.,Yang,Y.,Zhang,P.,Wang,S. and Tao,N.(2020年)。单个蛋白质大小,电荷,迁移率和结合的光学成像。Nat Commun 11(1):4768。
Ruggeri,F.,Zosel ,F.,Mutter,N.,Rozycka ,M.,Wojtas ,M.,Ozyhar ,A.,Schuler,B. and Krishnan,M.(2017)。单分子电泳。Nat Nanotechnol 12(5):488-495。
Wang,Q.,Goldsmith,RH,Jiang,Y.,Bockenhauer ,SD和Moerner,W.(2012)。使用抗布朗电动(ABEL)阱探测溶液中的单个生物分子。Accounts Chem Res 45(11):1955-1964。
Young,G.,Hundt ,N.,Cole,D.,Fineberg ,A.,Andrecka ,J.,Tyler,A.,Olerinyova ,A.,Ansari,A.,Marklund ,EG,Collier,MP,Chandler, SA,Tkachenko ,O.,Allen,J.,Crispin,M.,Billington ,N.,Takagi,Y.,Sellers,JR,Eichmann,C.,Selenko ,P.,Frey,L.,Riek ,R。 ,Galpin ,MR,Struwe ,WB,Benesch ,JLP和Kukura ,P.(2018)。单个生物大分子的定量质量成像。科学360(6387):423-427。
Zhang,P.,Ma,G.,Dong,W.,Wan,Z.,Wang,S. and Tao,N.(2020年)。等离子散射成像的单个蛋白质和结合动力学。Nat Methods 17(10):1010-1017。
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引用:Ma, G., Wan, Z. and Wang, S. (2021). Simultaneous Imaging of Single Protein Size, Charge, and Binding Using A Protein Oscillation Approach. Bio-protocol 11(5): e3934. DOI: 10.21769/BioProtoc.3934.
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