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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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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 compared to other existing techniques are summarized in several recent reviews.22-25 As the developer of the SALVI technology, our group has been actively applying it to a variety of studies involving dynamic liquid surfaces and liquid-solid interfaces. Some examples of new applications include mammalian cell membranes, bacteria biofilm attachment,15,16 or nanoparticle formation as a result of aqueous surface oxidations. In this paper, we present initial liquid ToF-SIMS results of the hydrated protein thin film adsorbed on the SiN surface.
It is quite critical to carefully handle the highlighted steps in the protocol. For example, when drawing liquid into the syringe in steps 1 and 2, make sure that there are not any bubbles inside the syringe. Because once the bubbles are injected into SALVI, they may induce leakage in vacuum.9 Thus, if any bubbles are observed, it is prudent to get rid of them by setting the syringe upright and gently pushing the bubbles out. In addition, it is very important to make the SiN window in SALVI flat for optimized ToF-SIMS analysis, as described in step 3. This levelness of the detection area directly affects the data quality according to the design of ToF-SIMS.26 It is necessary to visually inspect the device after securing everything onto the SIMS stage. If the SiN window is not horizontal, adjust the screws and change the height of the SALVI device to ensure that everything is at the same plane as much as possible. These delicate points need experience and carefulness, which determine the success and reliability of the whole experiment.
As shown in Figure 1b, the peaks with m/z 19, 37, 55, 73, 91 and 109 have high counts. This indicates that there are a good number of water clusters (H3O+, H5O2+, H7O3+, H9O4+, H11O5+ and H13O6+) in the positive secondary ions. The other water clusters (data not shown) with higher m/z values also have relatively strong peaks compared with their neighboring peaks. These large amounts of water clusters are emitted from the DI water surface after the SiN punch-through. Water cluster signals in the positive mode have not been observable in the previous work; and this was attributed to the low counts of secondary ions and non-optimized ToF-SIMS conditions in the past.8 With the continuous effort in optimizing the SALVI and ToF-SIMS acquisition conditions such as the primary ion beam (i.e., Bi+ vs. Bi3+), pulse width and current conditions during depth profiling, it is now possible to observe up to 44 water clusters in the positive mode using the conditions presented in this paper. Results shown here are promising to further investigate the role of water in dynamics of various biological molecules and biological systems.
Here as an example to illustrate the advantages of SALVI and ToF-SIMS in studying biological molecular dynamics, the hydration of fibronectin protein was presented. The peaks of m/z 30, 44, 61, 74, 81, 83, and 98 belong to the amino acid fragments according to the previous report,27,28 which are much stronger than those of DI water shown in Figure 1b. This difference in the increased intensity of the amino acid fragments in the fibronectin sample suggests that these secondary ions are indeed from the proteins themselves. In addition, the peaks of water clusters in Figure 1a are much stronger than those of DI water in Figure 1b, which implies that the properties of water molecules in the protein adsorption surface and interfacial solution could be very different from those in pure water.
Figure 2a shows representative depth profiling temporal series of two different samples: fibronectin and DI water in the SALVI microchannel. The depth profiling data were collected as the SiN window was being bombarded by the Bi3+ primary ion beam with the long pulse width. As seen in Figure 2a, the SiN window is punched through at around 300 sec. Before the SiN window in SALVI is punched through, the intensities of selected secondary ions are pretty low. Once the SiN window is punched through, the intensities of the secondary ions immediately increase as the liquid surface starts being bombarded by the primary ion beam. For example, the intensities of H+ and H3O+ show a large increase, indicating the observation of the water surface.8,9 Although the intensities are unstable right after the punch-through or at the interface, they may reach relatively stable values after a period of time (e.g., 60 sec for fibronectin and 220 sec for DI water, respectively, in Figure 2a). The difference in the long pulse width drilling does not really affect the analysis results illustrated here when reconstructing m/z spectra (e.g., Figure 1), because the effective m/z spectra are acquired after the SiN punch-through. However, a researcher may choose to use the same conditions for all the samples for consistency and ease in choosing data sections during data analysis. In order to obtain better mass resolution, the pulse width was subsequently reduced and the intensities were decreased accordingly in the latter portion of the depth profiling experiment. The depth profile time series were continuously collected for another 200 sec to provide ample data for m/z spectral analysis. The segments of time that m/z spectra were reconstructed are highlighted in green shaded areas.
Fibronectin has been widely used for surface adsorption prior to cell culturing; many studies have shown fibronectin is responsible for cell adhesion and cell-biomaterial interactions.19,20,29 A recent study characterized the thickness of fibronectin adsorbed on poly(hydroxylethyl methacrylate) brushes using ellipsometry. The fibronectin thin film was determined to be 3-12 nm thick.30 Such thickness is reasonable for the depth profiling analysis in an aqueous solution.8,9 With the development of SALVI, direct analysis of the hydrated protein adsorbed on the solid surface is now possible. This approach potentially can have wide applications in studying the interactions of proteins with solid surfaces dynamically.
Figure 2b shows the selected 2D images of different secondary ions from fibronectin and DI water samples, respectively. 2D images are obtained from the depth profiling temporal data in green shaded areas in Figure 2a, which correspond to the liquid surface after the SiN punch-through as described earlier. The images of selected amino acid fragment secondary ions CH4N+, C5H7O+ and C4H4NO2+ (i.e., m/z 30, 83 and 98) for the fibronectin thin film were much brighter than those for DI water. This indicates that the amino acid fragment secondary ions are observed from the fibronectin molecules inside the 2 &#956;m diameter aperture. Furthermore, the images of selected peaks (e.g., m/z 1, 19, 37, and 55) corresponding to several water clusters of fibronectin are remarkably brighter than those of DI water. This result demonstrates that the hydration of fibronectin makes the water molecules surrounding the biomolecule distinct from the water molecules in the DI water.
In summary, this work has provided striking evidence on protein hydration induced water molecule property alterations. Such results could provide an improved fundamental understanding on the structure, conformation, and biological activity of proteins adsorbed on a solid surface in the liquid microenvironment.

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

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 mass spectra of the hydrated fibronectin film and DI water, respectively. The peaks of interest including water clusters (i.e., m/z 19, 37, 55, 73, 91 and 109) and amino acid fragments (i.e., m/z 30, 44, 61, 74, 81, 83, and 98) are indicated by the red arrows. In addition to the characteristic peaks of amino acid fragments, which have been widely reported using dry protein samples, the peaks of water clusters are observed in the mass spectra of hydrated fibronectin film in DI water. Moreover, the intensities of these water cluster peaks are very different from those in the mass spectra of DI water. This result provides the direct evidence of the property of water molecules surrounding and inside the hydrated protein molecules that play a role in their adsorption on a surface.
Figure 2a shows the depth profile time series of the hydrated fibronectin film and DI water. As the sample is under the SiN membrane, the intensities of selected second ions are negligible before the SiN membrane is punched through (i.e., 280 sec for fibronectin and 340 sec for DI water). Once the SiN membrane is punched through, the intensities of these secondary ions increase significantly. When using the long pulse width, the mass resolution is relatively low. Therefore, the short pulse width process is used to obtain data with better mass resolution for image and spectrum reconstructions as highlighted in Figure 2a. After a period of time (i.e., 60 sec for fibronectin and 220 sec for DI water), the reduced pulse width is used to obtain m/z spectra with better mass resolution. The final m/z spectra were reconstructed from 200 sec measurements highlighted in green shaded areas in the depth profile temporal series. Figure 2b shows the 2D images from the hydrated fibronectin film and DI water, which correspond to the green shaded area in Figure 2a. These 2D images represent the counts of selected secondary ions from the sample: the brighter the image, the higher the secondary ion count. Such intuitive images give a clear comparison of selected secondary ions between different samples, which is helpful in data analysis. According to our experiences, the difference in the SiN punch-through time varies from batch to batch of the SiN membranes. However, it does not affect the results of the dynamic ToF-SIMS analysis.
<img alt="Figure 1" src="/files/ftp_upload/53708/53708fig1.jpg" /> 
Figure 1: Comparisons of the ToF-SIMS positive spectra of the hydrated fibronectin film and DI water in the SALVI microchannel. (a) The reconstructed m/z spectrum of fibronectin. (b) The DI water spectrum.
<img alt="Figure 2" src="/files/ftp_upload/53708/53708fig2.jpg" /> 
Figure 2: Depth profiles and 2D images of hydrated fibronectin and DI water. (a) The depth profiling time series of fibronectin and DI water in the positive mode. (b) 2D images reconstructed corresponding to the green shaded 200 sec time segments along the depth profile time series in (a). Several characteristic mass to charge ratios of relevance to water clusters (e.g., m/z 1, 19, 37, 55) are shown in addition to known amino acid fragments (e.g., m/z 30, 83, 98). <a href="https://www.jove.com/files/ftp_upload/53708/53708fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS at JoVE.com

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

8.2K Views.  Pacific Northwest National Laboratory. 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.

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

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

Cancer Research

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the time-profile volume and enthalpy changes of light induced conformational changes in proteins on microsecond to millisecond time-scales that are not accessible using traditional stop-flow instruments. In addition, since overall changes in volume and/or enthalpy are probed, these techniques can be applied to proteins and other biomacromolecules that lack a fluorophore and or a chromophore label. To monitor dynamics and energetics of structural changes associated with Ca2+ binding to calcium transducers, such neuronal calcium sensors, a caged calcium compound, DM-nitrophen, is employed to photo-trigger a fast (&#964; &#60; 20 &#956;sec) increase in free calcium concentration and the associated volume and enthalpy changes are probed using photothermal beam deflection technique.

Here we report an application of the photothermal beam deflection technique in combination with a caged calcium compound, DM-nitrophen, to monitor microsecond and millisecond dynamics and energetics of structural changes associated with the calcium association to a neuronal calcium sensor, Downstream Regulatory Element Antagonist Modulator.

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

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

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

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

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.

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Biochemistry

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

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Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)