Name: In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS (Video) | JoVE Journal
Uploaded: 2016-02-15T15:00:00.000+00:00
Duration: 9 min 48 s
Description: Learn in situ characterization of hydrated proteins using SALVI and time-of-flight secondary ion mass spectrometry. This method enables molecular mapping of biomolecules at solid-liquid interfaces for biointerface research.

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5 minutes

We are grateful to the Pacific Northwest National Laboratory (PNNL) Chemical Imaging Initiative-Laboratory Directed Research and Development (CII-LDRD) and Materials Synthesis and Simulation across Scales (MS3) Initiative LDRD fund for support. Instrumental access was provided through a W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) Science Themed Proposal. EMSL is a national scientific user facility sponsored by the Office of Biological and Environmental Research (BER) at PNNL. The authors thank Mr. Xiao Sui, Mr. Yuanzhao Ding, and Ms. Juan Yao for proof reading the manuscript and providing useful feedback. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.

1. Cleaning and Sterilization of the SALVI Microchannel
<ol>
	<li>Sterilization of the Microchannel in SALVI
	<ol>
		<li>Draw 2 ml of 70% ethanol aqueous solution into a syringe, connect the syringe with the inlet end of SALVI, and slowly inject 1 ml of the liquid in 10 min. Remove the syringe at the end of injection. Next, connect the inlet and outlet of SALVI using a polyetheretherketone (PEEK) union. Alternately, use a syringe pump to conduct the same procedure. For example, set the flow speed at 100 &#956;l/min.</li>
		<li>Keep the SALVI microchannel filled with 70% ethanol solution at room temperature for 4 hr.</li>
	</ol>
	</li>
	<li>Introduce Deionized (DI) Water into SALVI
	<ol>
		<li>Draw 2 ml of sterilized DI water into a syringe, connect the syringe with the inlet of SALVI, and slowly inject 1 ml of the liquid in 10 min. Remove the syringe, and connect the inlet and outlet of SALVI using a PEEK union. Alternately, use a syringe pump as mentioned in step 1.1.1.</li>
	</ol>
	</li>
</ol>
2. Immobilization of the Protein Film in SALVI
<ol>
	<li>Immobilize the Protein Film in SALVI
	<ol>
		<li>Draw 2 ml of fibronectin (10 &#956;g/ml in phosphate buffered saline, PBS) solution into a syringe, connect the syringe with the inlet of SALVI, slowly inject 1 ml of the liquid in 10 min, remove the syringe, and connect the inlet and outlet of SALVI using a PEEK union.</li>
		<li>Incubate the SALVI filled with the fibronectin solution in a clean culture dish, keep the dish in the hood at room temperature for 12 hr.</li>
		<li>Draw 2 ml of sterilized DI water into a syringe and connect the syringe with the inlet of SALVI. Slowly inject 1 ml of water in 10 min, remove the syringe, and connect the inlet and outlet of SALVI. 
		Note: If equipped with a light microscope, check the SALVI under the microscope to ensure that the SiN membrane is intact before ToF-SIMS analysis. Now the SALVI device is ready for ToF-SIMS analysis.</li>
	</ol>
	</li>
</ol>
3. Install SALVI into the ToF-SIMS Loadlock Chamber
Note: Gloves should be worn at all times when handling the SALVI device and installing it onto the ToF-SIMS stage to avoid potential contaminations during surface analysis.
<ol>
	<li>Mount SALVI onto the Stage
	<ol>
		<li>Lay the ToF-SIMS stage on a flat and clean surface. Put the SALVI device onto the stage with the silicon wafer and SiN membrane facing upward. Fix the SALVI using four metal clamping pieces and screw the metal pieces holding the corner of the device into the metal plate by four screws.</li>
		<li>Gently roll up the PFTE tubing connected to the microfluidic channel. Use four metal pieces and four screws to hold the stiff part down. Make sure that the device is positioned in the open space on the SIMS stage so that it does not interfere with the primary ion beam during analysis. Additional image and illustration of the SALVI fabrication and installation are available in previous publications.8,9,11</li>
	</ol>
	</li>
	<li>Mount the Faraday Cup onto the Stage
	<ol>
		<li>Fix the Faraday cup on the stage by a screw.</li>
	</ol>
	</li>
	<li>Load the Stage into the ToF-SIMS Loadlock Chamber
	<ol>
		<li>Open the ToF-SIMS loadlock, hold and set the stage horizontally onto the loading platform, and close the loadlock door.</li>
	</ol>
	</li>
</ol>
4. ToF-SIMS Data Acquisition
<ol>
	<li>Turn on the vacuum pump and ensure suitable vacuum (e.g., on the order of 10-6 mbar or Torr) is reached in the loadlock chamber.</li>
	<li>Move the SALVI into the main chamber once the vacuum is stabilized after about 30 min. 
	Note: The vacuum should be lower than 5 x 10-6 mbar vacuum in the main chamber during data acquisition. Otherwise, higher vacuum is indicative of a leak in the SALVI system.</li>
	<li>Adjust the primary ion beam and analyzer of ToF-SIMS with a spatial resolution of approximately 400 nm according to manufacturer recommendations. The current of primary beam is 1,200 pA while the cycle time is 30 &#956;sec under DC mode. 
	Note: This process largely follows manufacturer&#39;s recommendations.</li>
	<li>Find the microfluidic channel using the optical microscope. Use the optical image center as a reference and align it to be consistent with the secondary ion image center.</li>
	<li>Before each measurement, use a 1 KeV O2+ beam (~40 nA) to scan on the SiN window with a 500 x 500 &#181;m2 area for ~15 sec to remove surface contamination. Also, use an electron flood gun to compensate surface charging during all measurements. Use the automatic function of the flood gun in the default mode for this step.</li>
	<li>Select positive or negative mode before data acquisition. Use the 25 keV Bi3+ beam as the primary ion beam in all measurements. 
	Note: The steps are similar for positive and negative data acquisitions. For the interest of space, the positive mode is described in details here.
	<ol>
		<li>Depth Profiling
		<ol>
			<li>Scan the Bi3+ beam on a round area with a diameter of ~2 &#181;m with 32 pixels by 32 pixels resolution.</li>
			<li>To minimize the time required to punch-through the SiN membrane, use a long pulse width (i.e., 180 nsec, beam current ~1.0 pA at a cycle time of 100 &#956;sec) Bi3+ beam. The intensities of secondary ions representative from the liquid surface increase when punch-through is reached. The punch-through time varies from 300 sec to 500 sec, depending on the batch of the SiN membrane. 
			Note: This difference seems to be related to the batch of SiN membranes acquired according to our experience. However, the punch-through time does not really affect the data analysis.</li>
			<li>After the SiN membrane punch-through, keep the same current for about 200 sec to obtain sufficient data for 2D image reconstructions.</li>
			<li>Reduce the pulse width to 80 nsec for data collection to acquire spectra with better mass resolution. Continue this acquisition for about another 200 sec. The spectrum data shown in Figure 1 are obtained from this region.</li>
		</ol>
		</li>
		<li>2D Imaging and Mass Spectra
		<ol>
			<li>Collect the 2D imaging and mass spectra data at the same time when measuring the depth profiling data. The ToF-SIMS instrument saves all the information of each spot during the experiment. The data are shown in different windows (FPanel - Spectra, FPanel - Profiles and FPanel - Images separately) of the digital control software. Check data in each panel judiciously during data acquisition.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. ToF-SIMS Data Analysis
Note: Data analysis is initially performed using the IonToF ToF-SIMS instrumental software.
<ol>
	<li>Open the analysis software of SIMS (Measurement Explorer) and then click the &#34;profiles&#39;, &#39;spectra&#39; and &#39;image&#39; buttons, respectively, to process depth profile, m/z spectrum, and image data.</li>
	<li>Open the data files that have the extension &#34;.ita&#34;.</li>
	<li>Select peaks of interest, type the species name in &#34;ion&#34; box, and label them with different colors in the spectra. Use the mouse scroll wheel to drag along the x-axis. Use Ctrl and scroll wheel to adjust peak heights and widths. Letter &#34;L&#34; switches between Linear and Log modes along the y-axis.</li>
	<li>Conduct data reconstruction before mass calibration. Otherwise, it is difficult to choose the spacing between peaks in the mass calibration step.
	<ol>
		<li>Select the depth profiling data of interest and reconstruct the spectra according to the depth profile temporal series.</li>
		<li>Click the reconstruction button. In the &#34;Start Reconstruction&#34; window, type in a scan range of interest. Click &#34;OK&#34; to reconstruct the depth profile data.</li>
	</ol>
	</li>
	<li>Perform a mass calibration. Press &#34;F3&#34; to open the &#34;mass calibration&#34; window. Choose peaks for calibration based on the specific sample.21 In this work, use the following peaks CH3+ (m/z 15), H3O+ (m/z 19), C2H5+ (m/z 29), H5O2+ (m/z 37), C3H7+ (m/z 43), H7O3+ (m/z 55), H9O4+ (m/z 71), H11O5+ (m/z 99) and H13O6+ (m/z 109) for the positive mode. Use the following peaks CH- (m/z 13), O- (m/z 16), OH- (m/z 17), C2H- (m/z 25), C3H- (m/z 37), and C4H- (m/z 49) for the negative mode.
	<ol>
		<li>Select a peak, make sure that the downward arrow is pointed at the peak in the left bottom corner of the window. Click that peak and type the peak name in the &#34;mass calibration&#34; window, click &#34;Add&#34;, and the ion of interest is included in the mass calibration.</li>
		<li>Finally, click the &#34;Recalibrate&#34; button. Check if the areas of peaks are in the right places according to their mass to charge ratios. In the &#34;Spectrum&#34; menu, select and click &#34;Apply Mass Calibration&#34; to &#34;All Spectra&#34; or &#34;Selected spectra&#34;, depending on the specific analysis being conducted.</li>
	</ol>
	</li>
	<li>As peak selection is necessary for sample analysis, determine characteristic peaks of the sample according to literature or other previous findings. Compare the intensity of peaks of interest in the m/z spectra. If necessary, add new peaks or delete useless peaks in the peak list.
	<ol>
		<li>Add peaks. Click on a peak, find the red lines at the left bottom window. Hold the &#34;Ctrl&#34; key, scroll mouse horizontally to define peak width, which adds a peak to the peak list automatically. Another way to add a peak is to select a peak in the main spectrum window, and then to click the &#34;add peak&#34; button above the window. If there are many peaks to add, in the &#34;Spectrum&#34; menu, select &#34;search peaks&#34;, check related specifications in the newly opened window, then add peaks as needed.</li>
		<li>To export a peak list, in the &#34;Peak List&#34; menu, select &#34;Save&#8230;&#34; the peak list in the &#34;itmil&#34; format. Use the Ctrl and left button to drag along the spectrum. In the spectra window, select a peak of interest and drag the two dashed lines to desirable positions.</li>
		<li>To import a peak list, in the &#34;Mass interval List&#34; menu, choose &#34;Import&#8230;&#34;, locate an existing peak list file with the file extension &#34;.ITPL&#34;. Click &#34;Open&#34;.</li>
		<li>To use an &#34;itmil&#34; peak list, in the &#34;Mass Interval List&#34; menu, click &#34;Open&#8230;&#34;, find the file, then click &#34;Open&#34;.</li>
	</ol>
	</li>
	<li>Apply a peak list to further analyze data. At the left-bottom of &#34;Spectra&#34; window, there is a window called &#34;Mass Interval List&#34;. The imported peak list is shown up here.
	<ol>
		<li>Right-click the peak list file to be used, apply it to the &#34;active spectrum&#34; or &#34;all spectra&#34;.</li>
	</ol>
	</li>
	<li>View the data profiles. In the &#34;Profile&#34; window, Use letter &#34;M&#34; to fit to window (full range) and letter &#34;N&#34; to fit to the y-axis. Use Ctrl and scroll wheel to adjust the profile width. Or use &#34;/&#34; and &#34;*&#34; on the key board to change the y-axis range and height.</li>
	<li>Export data for further plotting using other graphic software after the initial analysis.
	<ol>
		<li>To export a depth profile, in the &#34;Profile&#34; window, click on the &#34;File&#34; menu, then select &#34;Export&#34;, and then select &#34;Save as&#34; a &#34;.txt&#34; file.</li>
		<li>To export an m/z spectrum file, in the &#34;Spectra&#34; window, click on the &#34;File&#34; menu, then select &#34;Export&#34;, and then select &#34;Save as&#34; a &#34;.txt&#34; file.</li>
		<li>To export an image file, in the &#34;Image&#34; window, print screen and save as the image file.</li>
	</ol>
	</li>
</ol>
6. ToF-SIMS Data Plotting and Presentation
Note: Use a graphic tool to plot the data after the SIMS data are processed using the instrumental software.
<ol>
	<li>Use a graphic tool to import the data.</li>
	<li>Make a plot using the m/z as the x-axis and peak intensity as the y-axis to show the reconstructed spectrum. An example is given in Figure 1.</li>
	<li>Make a plot using the sputter time as the x-axis and peak intensity as the y-axis to show the reconstructed depth profile temporal series. An example is given in Figure 2.</li>
	<li>Combine reconstructed 2D images of different m/z and form a matrix to show the ion mapping. An example is given in Figure 2.</li>
</ol>

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<li>Deng, J., Ren, T. C., Zhu, J. Y., Mao, Z. W., Gao, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adsorption+of+plasma+proteins+and+fibronectin+on+poly%28hydroxylethyl+methacrylate%29+brushes+of+different+thickness+and+their+relationship+with+adhesion+and+migration+of+vascular+smooth+muscle+cells%5BTitle%5D)+AND+%22Regen+Biomater%22%5BJournal%5D)">Adsorption of plasma proteins and fibronectin on poly(hydroxylethyl methacrylate) brushes of different thickness and their relationship with adhesion and migration of vascular smooth muscle cells.</a> Regen Biomater. , 17-25 (2014).</li>
</ol>

The authors have nothing to disclose.

SALVI is a microfluidic interface that allows dynamic liquid surface and liquid-solid interface analysis by vacuum based instruments, such as ToF-SIMS and scanning electron microscopy (SEM). Due to the usage of small apertures to expose liquid directly in vacuum, SALVI is suitable for many finely focused spectroscopy and imaging techniques without any modifications;22 the portability and versatility of microfluidics make it a true multimodal imaging platform. Distinct features and significance of SALVI compare...

Hydration is crucial to the structure,1 conformation,2 and biological activity3 of proteins. Proteins without water molecules surrounding them would not have viable biological activities. Specifically, water molecules interact with the surface and internal structure of proteins, and different hydration states of proteins make such interactions distinct.4 The interaction of proteins with solid surfaces is a fundamental phenomenon with implications in nanotechnology, biomaterials and tissue engineering processes. Studies have long indicated that conformational changes may occur as a protein encounters a surface. ToF-SIMS has been envisioned as the technique that has the potential to study the protein-solid interface.5-7 It is important to understand the hydration of proteins on solid surfaces, which potentially provides a fundamental understanding of the mechanism of their structure, conformation, and biological activity.
However, major surface analytical techniques are mostly vacuum-based and direct applications for volatile liquid studies are difficult due to the rapid evaporation of volatile liquid under the vacuum environment. We developed a vacuum compatible microfluidic interface, System for Analysis at the Liquid Vacuum Interface (SALVI), to enable direct observations of liquid surfaces and liquid-solid interactions using time-of-flight secondary ion mass spectrometry (ToF-SIMS).8-11 The unique aspects include the following: 1) the detection window is an aperture of 2-3 &#956;m in diameter allowing direct imaging of the liquid surface, 2) surface tension is used to hold the liquid within the aperture, and 3) SALVI is portable among multiple analytical platforms.11,12
SALVI is composed of a silicon nitride (SiN) membrane as the detection area and a microchannel made of polydimethylsiloxane (PDMS). It is fabricated in the clean room, and the fabrication and key design factors have been detailed in previous papers and patents.8-12 The applications of ToF-SIMS as an analytical tool were demonstrated using a variety of aqueous solutions and complex liquid mixtures, some of which contained nanoparticles.13-17 Specifically, SALVI liquid ToF-SIMS allows dynamic probing of the liquid-solid interface of live biological systems (i.e., biofilms), single cells, and solid-electrolyte interface, opening new opportunities for in situ condensed phase studies including liquids using ToF-SIMS. However, the current design does not permit gas-liquid interactions yet. This is a direction for future development. SALVI has been used to study the hydrated protein film in this work for the first time.
Fibronectin is a commonly used protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds,18 which is well-known for its ability to bind cells.19,20 It was chosen as a model system to illustrate that the hydrated protein film could be dynamically probed using the SALVI liquid ToF-SIMS approach. The protein solution was introduced into the microchannel. After incubating for 12 hr, a hydrated protein film formed on the back side of the SiN membrane. Deionized (DI) water was used to rinse off the channel after protein introduction. Information was collected from hydrated fibronectin protein molecules in the SALVI microchannel using dynamic ToF-SIMS. DI water was also studied as a control to compare with results obtained from hydrated fibronectin thin film. Distinct differences were observed between the hydrated protein film and DI water. This work demonstrates that protein adsorption on surface in the liquid environment can be studied using the novel SALVI and liquid ToF-SIMS approach. The video protocol is intended to provide technical guidance for people who are interested in utilizing this new analytical tool for diverse applications of SALVI with ToF-SIMS and reduce unnecessary mistakes in liquid handling as well as ToF-SIMS data acquisition and analysis.

<table><tbody><tr><td>ToF-SIMS</td><td>IONTOF</td><td>TOF.SIMS 5</td><td>Resolution: &gt;10,000 m/&amp;Delta;m for mass resolution; &gt;4,000 m/&amp;Delta;m for high spatial resolution&amp;nbsp;</td></tr><tr><td>System for Analysis at the Liquid Vacuum Interface (SALVI)</td><td>Pacific Northwest National Laboratory</td><td>N/A</td><td>SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum-based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level.</td></tr><tr><td>PEEK Union</td><td>Valco</td><td>ZU1TPK</td><td>for connecting the inlet and outlet of SALVI</td></tr><tr><td>5 Axes Sample Stage</td><td>IONTOF</td><td>N/A</td><td>Stage is self-made for mounting SALVI in ToF-SIMS</td></tr><tr><td>Barnstead Nanopure Water Purification System</td><td>Thermo Fisher Scientific</td><td>D11921</td><td>ROpure LP Reverse Osmosis filtration module (D2716)</td></tr><tr><td>Syringe</td><td>BD</td><td>309659</td><td>1 ml</td></tr><tr><td>Pipette</td><td>Thermo Fisher Scientific</td><td>21-377-821</td><td>Range: 100 to 1,000 ml</td></tr><tr><td>Pipette Tip</td><td>Neptune</td><td>2112.96.BS</td><td>1,000 &amp;micro;l</td></tr><tr><td>Centrifuge Tube</td><td>Corning</td><td>430791</td><td>15 ml</td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td>1 mg/ml</td></tr><tr><td>Ethanol</td><td>Thermo Fisher Scientific</td><td>S25310A</td><td>95% Denatured</td></tr><tr><td>Gibco PBS</td><td>Thermo Fisher Scientific</td><td>10010-023</td><td>pH 7.4</td></tr></tbody></table>

in situ characterization of hydrated proteins in water by salvi and tof-sims

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

A couple of representative results are presented to demonstrate the advantages of the proposed protocol. By using the SALVI microfluidic interface, the primary ion beam (Bi3+) can directly bombard on the hydrated fibronectin film in DI water. Thus the molecular chemical mapping of the liquid surface can be successfully acquired.
Figure 1a and 1b show the positive ToF-SIMS m...

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

The overall goal of this procedure is to characterize adsorbed protein films in aqueous solutions, using the System for Analysis at the Liquid Vacuum Interface, and time-of-flight secondary ion mass spectrometry. This method can help answer key questions about biomolecules and the biointerface. For example, characterization of adsorbed proteins at a solid surface.The main advantages of this technique is that the biomolecules can be started in situ in the liquid environment, and in combination with molecular mapping. Though this method can provide insight into biomolecules at the solid-liquid interface, it can also be applied to other systems, including investigation into single cancer cells, drug interactions, and the attachment of biofilms. Into a five milliliter syringe, draw up two milliliters of a 70%ethanol solution.Connect the syringe to the inlet end of the SALVI device, and slowly inject one milliliter of the liquid over the course of 10 minutes. At the end of the injection, remove the syringe, and connect the inlet and outlet of the SALVI using a polyether-ether-ketone union. Keep the SALVI microchannel filled with the 70%ethanol solution at room temperature for four hours.Then, fill a second syringe with sterile deionized water. Connect it to the inlet of the SALVI and slowly inject the deionized water into the device over the course of 10 minutes. Remove the syringe and reconnect the inlet and the outlet of the device together.In order to immobilize a protein film, start by drawing up two milliliters of fibronectin solution into a syringe. Connect the syringe to the inlet of the SALVI device, and slowly inject one milliliter of the liquid over the course of 10 minutes. Then, remove the syringe, and again reconnect the inlet and outlet.Incubate the setup in a clean culture dish at room temperature for 12 hours. Following incubation, slowly inject one milliliter of sterile deionized water over the course of 10 minutes, and then reconnect the inlet to the outlet. To begin, lay the ToF-SIMS stage on a flat, clean surface.Put the SALVI device onto the stage with the silicon wafer and silicon nitride membrane facing upward. Fix the SALVI device to the stage using four metal clamping pieces, and screw the metal pieces holding the corner of the device into the metal plate by four screws. Next, gently roll up the PFTE tubing connected to the microfluidic channel.Use four metal pieces and four screws to hold the stiff part down. Make sure that the device is positioned in the open space on the stage, so that it does not interfere with the primary ion beam during analysis. Next, mount the Faraday cup onto the stage using a screw.Then, open the ToF-SIMS loadlock door and set the stage horizontally onto the loading platform before closing the door. Turn on the vacuum pump and let the loadlock chamber pump down to around 10 to the minus sixth millibar. Then, move the SALVI device into the main chamber after about 30 minutes, once the vacuum is stabilized.Adjust the primary ion beam, and analyzer of the ToF-SIMS, to acquire a spatial resolution of approximately 400 nanometers by following the manufacturer's recommended protocol. Next, find the microfluidic channel using the optical microscope. Use the optical image center as a reference, and align it to be consistent with the secondary ion image center.Visual demonstration of this step is critical, as it is difficult to learn, because the sample is thicker than normal solid samples, and it's difficult to adjust the settings to find the best focus of the channel. Before each measurement, use a one kiloelectronvolt oxygen beam to scan on the silicon nitride window for about 15 seconds to remove surface contamination. Also, use an electron flood gun to compensate surface charging during all measurements.Next, select the positive or negative mode before starting data acquisition. Use the 25 kiloelectronvolt bismuth three plus as the primary ion beam in all measurements. For depth profiling, scan the primary ion beam on a round area with a diameter of about two micrometers with a resolution of 32 pixels by 32 pixels.To minimize the time required to punch through the silicon nitride membrane, use a long pulse width primary ion beam. After the silicon nitride membrane punch-through, keep the same current for about 200 seconds to obtain sufficient data for two-dimensional image reconstructions. For data collection, reduce the pulse width to 80 nanoseconds in order to acquire spectra with better mass resolution.Continue this acquisition for about another 200 seconds. Using the SALVI microfluidic interface, the primary ion beam can directly bombard on the hydrated fibronectin film. A number of water clusters and amino acid fragments are indicated by the ToF-SIMS mass spec data.This provides direct evidence of the property of water molecules surrounding and inside the hydrated protein molecules. Here, one can see the depth profile time series of the hydrated fibronectin film and of deionized water. The intensities of selected second ions are negligible before the silicon nitride membrane is punched through.Once the silicon nitride membrane is punched through, the intensities of the secondary ions increase significantly. The short pulse width process is used to obtain data with better mass resolution for image and spectrum reconstructions. The 2-D images from the hydrated fibronectin film and deionized water represent the counts of selected secondary ions from the sample.The brighter the image, the higher the secondary ion counts. These intuitive images give a clear comparison of selected secondary ions between different samples, which is helpful in data analysis. While attempting this procedure, it is important to remember that a microreactor needs to be leak-free before installing in the vacuum chamber.This technique paved the way for researchers in the field of biology and biointerfaces to explore protein adsorption, biofilm attachment, surface-assembled monolayers, and many other phenomena. After watching this video, you should have a good understanding of how to study various liquid samples using SALVI and the liquid ToF-SIMS.

in-situ-characterization-hydrated-proteins-water-salvi-tof

Research

JoVE Journal

Chemistry

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

More than half of proteins are small proteins (molecular mass &lt;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a  homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

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.

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

We present a procedure for real-time imaging and elemental composition analysis of boehmite particles in deionized water by in situ liquid Scanning Electron Microscopy.

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

Here, we present a protocol to investigate the structure and dynamics of interfacial water at the atomic scale, in terms of submolecular resolution imaging, molecular manipulation, and single-bond vibrational spectroscopy.

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Learn in situ characterization of hydrated proteins using SALVI and time-of-flight secondary ion mass spectrometry. This method enables molecular mapping of biomolecules at solid-liquid interfaces for biointerface research.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

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