Name: integrated cell manipulation platform coupled with the single-probe for mass spectrometry analysis of drugs and metabolites in single suspension cells
Uploaded: 2019-06-21T15:00:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells at JoVE

The authors thank Naga Rama Kothapalli for her work in developing sample preparation for both suspension cells and cell lysate experiments. Additionally, the authors thank the NIH (R01GM116116 and R21CA204706) for funding.

1. Glass cell-selection probe fabrication
<ol>
	<li>Convert single-bore glass tubing into a tapered probe with a sharp tip.
	<ol>
		<li>Place a single-bore glass tube (ID: 0.3 mm, OD: 1.1. mm) into the clamps of a vertical pipette holder, centering the glass with respect to the heating coil and tighten to secure the tube in place. The heating coil is comprised of an 18-gauge nickel-chromium resistance wire (~60 mm in length) coiled around a metal rod (diameter = 3.90 mm) 2.5 times.</li>
		<li>Set the glass tubing with temperature program 19.5 (manufacturer&#8217;s unit). This parameter can be modified for a particular instrument.</li>
		<li>Set the solenoid plunger at 4 (manufacturer&#8217;s unit). This parameter can be modified for a particular instrument.</li>
		<li>Trigger the solenoid to pull the glass tubing. This step creates two probes fused at the tip.</li>
		<li>Use tweezers to cut ~1 mm away from the tip of each probe, creating an orifice of ~10 &#181;m in diameter at the probe tip.</li>
	</ol>
	</li>
	<li>Bend the glass probe for easy coupling to the ICMP/single-probe SCMS setup.
	<ol>
		<li>Set a pulled glass probe into the microforge, positioning the tip ~3 mm above the platinum heating wire.</li>
		<li>Turn the heat on the platinum wire to 30% of the maximum temperature.</li>
		<li>Bend the probe ~45&#176; from the original position (Figure 2).</li>
	</ol>
	</li>
</ol>
2. Integrated cell manipulation platform assembly
<ol>
	<li>Place the inverted microscope, microinjector, and two cell manipulation systems on a motorized table for easy coupling with the mass spectrometer.
	<ol>
		<li>Modify one of the cell manipulation systems to accommodate a single-probe by replacing the end with an arm clamp.</li>
		<li>Use a plastic syringe with a needle to fill the microinjector with mineral oil. Avoid bubbles in the tubing as this will affect suctioning.</li>
		<li>Replace the stage insert of the inverted microscope with the heated plate. Set the heated plate at 37 &#176;C prior to analysis.</li>
	</ol>
	</li>
	<li>Set up the glass cell-selection device.
	<ol>
		<li>Insert the glass cell-selection probe inside the metal holder of the microinjector by placing the long (non-bent) side into the capillary holder and tightening the screw to secure the probe in place. Position the probe tip&#8217;s angle parallel to the heated plate. 
		CAUTION: The glass probe is very sharp and fragile, and it breaks easily. Protect your eyes and be extra cautious while inserting the probe into the microinjector.</li>
		<li>Secure the metal holder of the microinjector into the cell manipulation system. Position the probe tip near the middle of the inverted microscope light.</li>
	</ol>
	</li>
</ol>
3. Create an extended ion transfer tube for the mass spectrometer inlet
<ol>
	<li>Use a metal cutter to cut a piece of stainless-steel tubing (OD: 0.0625 (1/16) in, ID: 0.021 in) ~250 mm in length.</li>
	<li>Measure 135 mm from the end and place a metal feral so ~135 mm will be exposed to the atmosphere and ~115 mm will be inside the mass spectrometer. Secure the feral using two wrenches to tighten it.</li>
</ol>
4. Couple the ICMP with a single-probe setup
<ol>
	<li>Secure the glass slide containing the single-probe into the arm clamp of the cell manipulation system. 
	NOTE: Single-probes are fabricated according to a previously-published protocol48 with two minor changes in the current study: the nano-ESI emitter is made longer for easy coupling to the mass spectrometer, and the single-probes are glued to the glass side on the right-hand side to avoid interfering with the spatial movement of the glass cell-selection device (Figure 2).</li>
	<li>Connect the solvent-providing capillary to a conductive union by placing the capillary into the sleeve (1/16 x .005 in) of the plastic ferrule and finger-tightening the fitting.
	<ol>
		<li>Connect the other side of the conductive union to a capillary (ID: 40 &#181;m, OD: 150 &#181;m), which is connected to a syringe containing the sampling solvent, by placing the capillary into the sleeve (1/32 x .007 in) and tightening the fitting. Use acetonitrile with 0.1% formic acid as the sampling solvent in these experiments. 
		NOTE: The sampling solvent is flexible, but it should primarily contain acetonitrile (or acetonitrile with formic acid for better ionization) for a rapid microscale cell lysis.</li>
		<li>Secure the syringe into the syringe pump on the mass spectrometer.</li>
		<li>Place the ionization voltage cord onto a copper wire attached to the conductive union.</li>
	</ol>
	</li>
	<li>Position the nano-ESI emitter ~1 mm to the orifice of the extended ion transfer tube.
	<ol>
		<li>Use the cell manipulation system to control the spatial movements of the Single-probe and position the nano-ESI emitter centrally in front of the extended ion transfer tubing.</li>
	</ol>
	</li>
</ol>
5. Suspended cell sample preparation
<ol>
	<li>The day before analysis (~18-24 h), seed out cells for testing in a cell culture flask (T25). K562 human myeloid leukemia cells are used as models in this study.
	<ol>
		<li>Heat 1x phosphate buffered saline (PBS) and Roswell Park Memorial Institute (RPMI) medium supplemented with 10% synthetic fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 &#176;C for 30 min.</li>
		<li>Seed ~1 x 106 cells in a total volume of 10 mL by combining cells with warm medium. In general, use a 10-mL pipette to place 8 mL of RPMI medium into a cell culture flask. Then, use a 2 mL pipette to put 2 mL of confluent K562 cells the medium for ~1 x 106 cells.</li>
		<li>Incubate the cells at 37 &#176;C and 5% CO2 until analysis.</li>
	</ol>
	</li>
	<li>Prepare cells for analysis.
	<ol>
		<li>Pipette cells from the cell culture flask into a 15-mL centrifuge tube.</li>
		<li>Spin cells down at 400 x g and 37 &#176;C for 5 min and discard the supernatant.</li>
		<li>Resuspend cells in 4 mL of RPMI medium containing the drug compound at the desired treatment concentration. 
		NOTE: For analysis of control cells, resuspend the cells in 4 mL of RPMI medium and skip to Step 6.</li>
		<li>Incubate the cells for the duration of the treatment time at 37 &#176;C and 5% CO2.</li>
		<li>Spin cells down at 400 x g and 37 &#176;C for 5 min. Aspirate the supernatant.</li>
		<li>Cells are resuspended in 10 mL of PBS, and centrifuge at 400 x g and 37 &#176;C for 5 min. After spinning, discard the supernatant. Repeat this step 3 times to minimize detection of drug from extracellular constituents.</li>
		<li>Resuspend cells in 4 mL of PBS for analysis.</li>
	</ol>
	</li>
</ol>
6. Perform SCMS measurements using the ICMP/single-probe setup
<ol>
	<li>Customize parameters for the mass spectrometer for the experiment.
	<ol>
		<li>Under the Scan Mode heading of the instrument software, select Define Scan. Use a resolution of 60,000 m/&#8710;m at m/z 400, 1 microscan, 100 ms maximum injection time, and automatic gain control (AGC) on. A mass range (m/z) of 100-1000 was utilized for the experiments. Parameters can be modified based on the instrument model.</li>
		<li>Under Syringe Pump, select a flow rate of 150 nL/min. Flow rate needs to be optimized for each experiment.</li>
		<li>Select NSI Source and apply a voltage of ~4.5 kV. This parameter also needs to be optimized for each experiment.</li>
	</ol>
	</li>
	<li>Turn on the inverted microscope (with 40x magnification selected for both the top plate and bottom lens) and connect it to the USB-port of a laptop to capture live-video feeds. Turn on the heated plate and set it to 37 &#176;C.</li>
	<li>On the computer, go to the Acquire Data tab, and select Continuously under Acquire Time.</li>
	<li>Prepare sample for analysis.
	<ol>
		<li>Pipette 2-3 mL of sample into the lid of a small Petri dish (35 mm x 12 mm).</li>
		<li>6.4.2 Position the sample in the center of the light from the inverted microscope on top of the heated plate.</li>
	</ol>
	</li>
	<li>Prepare the glass cell-selection probe for analysis. Use the cell manipulation system to move the probe so its tip is focused under the inverted microscope in the same plane as the cells.</li>
	<li>Select an individual cell for analysis.
	<ol>
		<li>Use the cell manipulation system to move the cell-selection probe tip to a targeted cell. This process is monitored using the inverted microscope. 
		NOTE: If the tip of the cell-selection probe cannot be focused in the same plane as the cells, it is possible that the bent part of the probe is not appropriately angled. Adjust the position of the cell-selection probe until both probe tips can be focused along with cells under the microscope.</li>
		<li>Gently turn the handle of the microinjector to adjust the position of the mineral oil inside the tubing. A gentle suction is provided by the microinjector to secure the targeted cell to the cell-selection probe tip. 
		NOTE: If the cell cannot be captured by the cell-selection probe through the suction force, check the cell-selection probe to ensure it is fully-inserted into the capillary holder. In addition, inspect the mineral oil levels in the microinjector and tubing, and expel air if there is any.</li>
		<li>Use the cell manipulation system to move the cell at the cell-selection probe tip to the single-probe tip, using a digital microscope focused on the single-probe tip to monitor this process. When touching, a small acetonitrile droplet at the single-probe tip induces a rapid lyses of the cell, and then cell lysate is immediately ionized for on-line MS analysis. 
		NOTE: Because the selected cell is secured to the cell-selection probe tip through a gentle suction, this cell can be potentially detached during its transfer to the Single-probe tip. Therefore, if ion signals of typical cellular lipids (see representative results below) are not observed within 5 s, it is possible that the cell became unattached, and the selection of a different cell is needed.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The integrated cell manipulation and analysis platform is constructed to expand the versatility of the single-probe MS method, allowing for on-line, rapid analysis of non-adherent cells in a near-native environment. A major advantage of the technique is that minimal sample preparation is required, so the cells are analyzed in conditions that mimic their standard state. Particularly, individual cells of interest can be visually identified and selected, minimizing the influence of matrix effect on MS ionization efficiency while maintaining cells in their natural environment, so the results are more representative cells&#8217; native status (Figure S3). This technique can be potentially used to study patient cells suspended in biofluids in future studies. Another advantage of this technique is the flexible selection of the sampling solvent. It is important to include acetonitrile as the main sampling solvent so that microscale lysis can occur rapidly. Potentially, internal standards (e.g., isotopically-labeled drug compounds) can be added into the sampling solvent for quantification of molecules of interest (e.g., drug molecules) from individual cells, including those can play a key role in revolutionizing personalizing drug treatments in the future54.
Although this integrated system can be conveniently used to analyze broad ranges of cells, a limitation of the method is that neither the single-probe nor cell-selection probe is commercially-available; dictating the need for optimization of many parameters (e.g., flow rate, voltage, length between the nano-ESI emitter and ion transfer tubing, etc.) prior to each experiment. In addition, due to the smallness of the Single-probe and cell-selection probe, environmental perturbation (e.g., air flow) may result in difficulties establishing a junction between the two probes. A short-term solution is the bending of the cell-selection probe close to the end to minimize the length of tapering. Future work includes the development of a housing to enclose the critical parts of the setup to minimize environmental effects. Due to the limited amount of cellular contents and short acquisition time (~2-3 s) from a cell, MS/MS analysis can be only conducted for relatively abundant species. Other factors influencing the detection sensitivity include the suppressed ionization efficiency due to the introduction of matrix along with the cell and potential ion loss through the extended ion transfer tubing.

Human biology, especially disease biology, is increasingly understood to be the result of activities on the level of individual cells, but the traditional analytical methods, such as liquid chromatography mass spectrometry (LCMS), are generally used to analyze samples prepared from populations of cells, whereas the acquired molecular information cannot accurately represent the chemical processes on the individual-cell level. These standard, traditional methods are unable to discern the effects of cellular heterogeneity on an analytical measurement, and the process of destroying and mixing the cells to prepare the lysate potentially leads to the alteration or loss of cellular components1,2. These limitations of traditional methods are especially important in the analysis of patient cells, in which the obtained samples can contain a complex mixture of many different cell types. To overcome these deficiencies, single cell molecular analysis methods, including single cell mass spectrometry (SCMS) methods, are increasingly being developed and applied to bioanalysis, especially of cellular metabolites and low molecular weight biomolecules3,4.
The first SCMS techniques developed used vacuum-based techniques to perform the analyses under non-ambient conditions2,5,6,7,8,9,10,11. Non-ambient SCMS techniques are capable of analyzing cellular lipids and metabolites, but require sample pretreatment under artificial conditions, and therefore are not suitable for real-time analysis. The sample preparation process for non-ambient analysis includes the addition of matrix components, and this preparation can alter cellular components from their natural environment12. Therefore, ambient mass spectrometry (MS) techniques, which do not require a vacuum for the sampling environment, are utilized to analyze cells in a near-native environment. Not having a vacuum environment allows for versatility in the experimental design; cameras can be added to monitor the cellular process and softer ionization techniques can be combined with separation techniques to receive better information from each single-cell experiment4,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42.
The single-probe SCMS method is an ambient technique that analyzes live, mammalian cancer cell lines in a near-native environment21,43,44,45,46. In addition, the single-probe device has been used for other mass spectrometry applications, including analysis of extracellular molecules in multicellular spheroids and MS imaging of tissues47,48,49,50,51,52. However, since cell immobilization on substrates is required for this method, suspension cells cannot be directly analyzed using this technique3,53. Therefore, the single-probe SCMS system could not be directly used to sample non-adherent single cells, such as non-adherent cell lines or suspension cells isolated from a patient&#8217;s blood or other bodily fluids54. In this work, an integrated cell manipulation platform (ICMP) is coupled with the single-probe SCMS technique to analyze live, suspension cells on-line with minimal sample preparation (Figure 1)46. The ICMP consists of an inverted microscope to monitor cell selection, a glass cell-selection probe, a microinjector to capture individual floating cells, a heated plate to maintain cellular temperature, two cell manipulation systems to control spatial movements of both the glass cell-selection probe and single-probe, and a digital microscope to observe cell transfer from the cell-selection probe tip to the single-probe tip. The fabrication of the single-probe is detailed in previous publications and will not be addressed here21,48. The ICMP/single-probe system is coupled to a high resolution mass spectrometer. This integrated setup allows for the sampling of identified single cells from complex biological samples with minimal effects from matrix molecules.

<table><tbody><tr><td>Acetontrile</td><td>Millipore Co.</td><td>AX0145-1</td><td>Sampling solvent</td></tr><tr><td>CellTram Vario</td><td>Eppendorf</td><td>6221</td><td>ICMP</td></tr><tr><td>Copper wire</td><td>stores.ebay.com/jewelerheaven</td><td>Dead soft, round, 20 guage, 25 ft</td><td>Conductive union setup</td></tr><tr><td>Digital stereomicroscope</td><td>Shenzhen D&amp;amp;F Co.</td><td>Supereyes T004</td><td>Analysis</td></tr><tr><td>Disposable micropipette, 1-5 &amp;micro;L</td><td>Rochester Scientific</td><td>5065</td><td>Cell-selection probe fabrication</td></tr><tr><td>Dual bore quartz tubing, 1.120&quot;x0.005&quot;x12&quot;</td><td>Friedrich &amp;amp; Dimmock, Inc.</td><td>MBT-005-020-2Q</td><td>Single-probe fabrication</td></tr><tr><td>Epoxy resin</td><td>Devcon</td><td>Part No. 20945</td><td>Single-probe fabrication</td></tr><tr><td>Eppendorf cell manipulation system</td><td>Eppendorf</td><td>Transferman NK517800397-U.R.</td><td>ICMP</td></tr><tr><td>External nut</td><td>VALCO*CHEMINERT</td><td>EN1</td><td>Ion transfer tube fabrication</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>399388-500ML</td><td>Sampling solvent</td></tr><tr><td>Fused silica capillary, ID: 40 &amp;micro;m, OD: 100 &amp;micro;m</td><td>Polymicro Technologies</td><td>TSP040105</td><td>Single-probe fabrication, conductive union setup</td></tr><tr><td>Fused silica capillary, ID: 50 &amp;micro;m, OD: 150 &amp;micro;m</td><td>Polymicro Technologies</td><td>1068150015</td><td>Conductive union setup</td></tr><tr><td>HyClone Synthetic fetal bovine serum (FBS)</td><td>Fischer Sci</td><td>SH3006603</td><td>Cell culture</td></tr><tr><td>Inline MicroFilter</td><td>IDEX Health &amp;amp; Science LLC</td><td>M-520</td><td>Conductive union setup</td></tr><tr><td>Laser puller</td><td>Sutter Instrument Co.</td><td>Model P-2000</td><td>Single-probe fabrication</td></tr><tr><td>LED UV lamp</td><td>Foshan Liang Ya Dental Equipment</td><td>LY-C240</td><td>Single-probe fabrication</td></tr><tr><td>LTQ Orbitrap mass spectrometer</td><td>Thermo Scientific</td><td>LTQ Orbitrap XL</td><td>Analysis</td></tr><tr><td>Microforge</td><td>Narishige, Co.</td><td>MF-9</td><td>Cell-selection probe fabrication</td></tr><tr><td>Microunion</td><td>IDEX Health &amp;amp; Science LLC</td><td>M-539</td><td>Conductive union</td></tr><tr><td>PEEK tubing, 1/32x0.005x 5ft</td><td>IDEX Health &amp;amp; Science LLC</td><td>1576</td><td>Conductive union setup</td></tr><tr><td>PEEK tubing, 1/32x0.007x 5ft</td><td>IDEX Health &amp;amp; Science LLC</td><td>1577</td><td>Conductive union setup</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco/Life Technologies</td><td>15140-122</td><td>Cell culture</td></tr><tr><td>Petri dish, 35x10 mm</td><td>VWR</td><td>25382-334</td><td>Sample preparation</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>VWR</td><td>0780-50L</td><td>Cell culture</td></tr><tr><td>Platinum wire</td><td>Narishige, Co.</td><td>Model PT-A</td><td>Microforge</td></tr><tr><td>Power supply</td><td>Nikon</td><td>PSM-2120</td><td>ICMP</td></tr><tr><td>RPMI, 1X with Corning glutagro</td><td>Corning</td><td>10-104-CV</td><td>Cell culture</td></tr><tr><td>Single-bore tubes</td><td>Boralex</td><td>5065</td><td>Cell-selection probe fabrication</td></tr><tr><td>Stainless steel ferrules, for 1/16&quot; OD</td><td>IDEX Health &amp;amp; Science LLC</td><td>VHP-200-01x</td><td>Ion transfer tube fabrication</td></tr><tr><td>Stainless steel tubing, 1/32x 205 &amp;micro;m x30 cm</td><td>IDEX Health &amp;amp; Science LLC</td><td>U-1128</td><td>Ion transfer tube fabrication</td></tr><tr><td>Syringe, 250 &amp;micro;L</td><td>Hamilton</td><td>1725LTN250UL</td><td>Sampling syringe</td></tr><tr><td>T25 flask</td><td>CellStar</td><td>690160</td><td>Cell culture</td></tr><tr><td>Thermo LTQ XL ion source interface flange</td><td>New Objective</td><td>PB5500</td><td>Analysis</td></tr><tr><td>ThermoPlate</td><td>TokaiHit</td><td>55R30N</td><td>ICMP</td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12605-010</td><td>Cell culture</td></tr><tr><td>Tube cutter, for 1/16&quot; stainless steel</td><td>SUPELCO</td><td>58692-U</td><td>Ion transfer tube fabrication</td></tr><tr><td>USB digital photography microscope</td><td>dx.com</td><td>SO2 25~500X</td><td>Analysis</td></tr><tr><td>UV curing resin</td><td>Prime Dental</td><td>Item No. 006.030</td><td>Single-probe fabrication</td></tr><tr><td>Vertical pipette puller</td><td>David Kopf Instruments</td><td>Model 720</td><td>Cell-selection probe fabrication</td></tr><tr><td>Voltage housing</td><td>PicoChip</td><td>PCH-A00120</td><td>ICMP/MS interface</td></tr><tr><td>Wire cutter</td><td>Craftsman</td><td>4 1/2 in end nipper</td><td>Conductive union setup</td></tr></tbody></table>

integrated cell manipulation platform coupled with the single-probe for mass spectrometry analysis of drugs and metabolites in single suspension cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

First, untreated K562 cells are used to establish the experimental method. In a typical SCMS experiment, obvious changes of mass spectra can be observed from transferring a cell, during the detection of cellular contents, and after finishing the measurement (Figure S1). Three common cellular lipid peaks (phosphatidylcholine, PC), including PC(34:4) (m/z 754.536), PC(36:4) (m/z 782.567), and PC(38:5) (m/z 808.583), are monitored to ensure the cell is successfully transferred and cellular contents are detected (Figure S2)21,43,46,55,56. If lipid peaks are not seen within 5 s, the mineral oil level in the microinjector is altered to reduce the suction holding the cell at the cell-selection probe tip; caution needs to be taken so that no mineral oil is pushed out from the cell-selection probe. The identity of many PC&#8217;s in the mass range of m/z 750-850 are confirmed using MS/MS on untreated cell lysate samples (Figure 3, Figure S2, Table 1)46.
K562 cells are also subjected to treatment with various drug compounds to expand the versatility of the method. K562 cells are incubated with gemcitabine (1 &#181;M) and taxol (1 &#181;M) for 1 h and OSW-1 (100 nM, 1 &#181;M) for 4 h and 2 h, respectively. Cells are then washed with PBS to minimize the detection of drug compounds from extracellular content. The contribution of matrix (e.g., ions from cell culture medium, PBS, and solvent) to mass spectra of cellular contents can be eliminated through data subtraction, due to their significantly different ion signals (Figure S3). All three drug compounds are detected using the ICMP/single-probe MS setup (Figure S4)46. These results suggest this method can be used to study intracellular lipids, drugs, and metabolites on the single-cell level from cells in solution in a near-native environment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59875/59875fig1.jpg" /> 
Figure 1. Experimental setup for single suspension cell MS experiments. (A) The integrated cell manipulation platform (ICMP) coupled with a mass spectrometer. (B) Schematic for analysis of suspended cells. (C) Experimental view of K562 cells to be selected using the cell-selection probe. Reprinted with permission from Standke et al.46. Copyright 2019 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/59875/59875fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59875/59875fig2.jpg" /> 
Figure 2. Photos of a modified single-probe and a cell-selection probe utilized for single suspension cell MS experiments. <a href="https://www.jove.com/files/ftp_upload/59875/59875fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59875/59875fig3.jpg" /> 
Figure 3. Zoomed-in mass spectrum from a single cell showing the representative species (m/z 750-850). Chemical structures are confirmed using MS/MS analysis (Figure S1). Reprinted with permission from Standke et al46. Copyright 2019 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/59875/59875fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Drug Molecule*</td>
			<td>m/z</td>
			<td>Mass Error (ppm)</td>
		</tr>
		<tr>
			<td>[Gemcitabine + H]+</td>
			<td>264.076</td>
			<td>11.32</td>
		</tr>
		<tr>
			<td>[Taxol + Na]+</td>
			<td>876.318</td>
			<td>2.74</td>
		</tr>
		<tr>
			<td>[OSW-1 + Na]+</td>
			<td>895.445</td>
			<td>0.89</td>
		</tr>
		<tr>
			<td>Cellular Lipids</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>[PC(34:4) + H]+</td>
			<td>754.535</td>
			<td>3.71</td>
		</tr>
		<tr>
			<td>[PC(34:3) + H]+</td>
			<td>756.551</td>
			<td>3.44</td>
		</tr>
		<tr>
			<td>[PC(34:2) + H]+</td>
			<td>758.569</td>
			<td>0.66</td>
		</tr>
		<tr>
			<td>[PC(36:5) + H]+</td>
			<td>780.551</td>
			<td>3.07</td>
		</tr>
		<tr>
			<td>[PC(36:4) + H]+</td>
			<td>782.568</td>
			<td>2.17</td>
		</tr>
		<tr>
			<td>[PC(36:3) + H]+</td>
			<td>784.585</td>
			<td>0.64</td>
		</tr>
		<tr>
			<td>[PC(38:7) + H]+</td>
			<td>804.551</td>
			<td>4.1</td>
		</tr>
		<tr>
			<td>[PC(38:6) + H]+</td>
			<td>806.567</td>
			<td>2.48</td>
		</tr>
		<tr>
			<td>[PC(38:5) + H]+</td>
			<td>808.583</td>
			<td>2.72</td>
		</tr>
		<tr>
			<td>[PC(38:4) + H]+</td>
			<td>810.601</td>
			<td>0</td>
		</tr>
		<tr>
			<td>[PC(40:7) + H]+</td>
			<td>832.583</td>
			<td>3.12</td>
		</tr>
	</tbody>
</table>
Table 1. Identified cellular components using the ICMP/Single-probe setup. The detection of all drug compounds were confirmed by comparing the MS/MS results with standard compound.

Watch this Scientific Journal Video about Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells at JoVE

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

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Research

JoVE Journal

Chemistry

5.7K Views.  University of Oklahoma. An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Video: Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Bioengineering

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

Cancer Research

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Biochemistry

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

A protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM is a convenient method for multiplexed targeted proteomics analysis of small numbers of cells, including single cells. It capitalizes on using excessive exogenous protein as a carrier and high-specificity LC-SRM for targeted quantification.

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Biology

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...