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&#38;F Co.</td>
			<td>Supereyes T004</td>
			<td>Analysis</td>
		</tr>
		<tr>
			<td>Disposable micropipette, 1-5 &#181;L</td>
			<td>Rochester Scientific</td>
			<td>5065</td>
			<td>Cell-selection probe fabrication</td>
		</tr>
		<tr>
			<td>Dual bore quartz tubing, 1.120&#34;x0.005&#34;x12&#34;</td>
			<td>Friedrich &#38; 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 &#181;m, OD: 100 &#181;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 &#181;m, OD: 150 &#181;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 &#38; 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 &#38; Science LLC</td>
			<td>M-539</td>
			<td>Conductive union</td>
		</tr>
		<tr>
			<td>PEEK tubing, 1/32x0.005x 5ft</td>
			<td>IDEX Health &#38; Science LLC</td>
			<td>1576</td>
			<td>Conductive union setup</td>
		</tr>
		<tr>
			<td>PEEK tubing, 1/32x0.007x 5ft</td>
			<td>IDEX Health &#38; 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&#34; OD</td>
			<td>IDEX Health &#38; Science LLC</td>
			<td>VHP-200-01x</td>
			<td>Ion transfer tube fabrication</td>
		</tr>
		<tr>
			<td>Stainless steel tubing, 1/32x 205 &#181;m x30 cm</td>
			<td>IDEX Health &#38; Science LLC</td>
			<td>U-1128</td>
			<td>Ion transfer tube fabrication</td>
		</tr>
		<tr>
			<td>Syringe, 250 &#181;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&#34; 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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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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

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 ...

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein&#39;s structure by MS, specific chemical reactions are performed in solution that encode a protein&#39;s structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as &#946;2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry analysis.

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

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 ...