Name: Sample Preparation for Single Cell Mass Spectrometry Metabolomics Studies: Combined Cell Washing, Quenching, Drying, and Storage (Video) | JoVE Journal
Uploaded: 2025-09-16T14:00:00.000+00:00
Duration: 8 min 7 s
Description: Learn sample preparation protocols for single-cell mass spectrometry metabolomics using liquid nitrogen quenching and cold ammonium formate washing. These methods preserve cellular metabolite integrity for accurate analysis.

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5 minutes

This study was supported by the National Science Foundation (2305182), National Institutes of Health (1R01AI177469), and Chan Zuckerberg Initiative. Figure 1 was created in BioRender (https://BioRender.com/yqn5isk).

Carry out every step inside a certified Class II biosafety laboratory and sterile conditions. In this study, the HCT-116 cell line was used as a model system, and cells were cultured in complete culture medium. The regents and the equipment used are listed in the Table of Materials.
1. Cell culture
<ol>
	<li>Prepare complete medium. Supplement McCoy&#39;s 5A medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Warm it to 37 &#176;C.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified incubator with 5% CO2. Monitor confluency daily and passage when cultures reach ~80% confluency.</li>
	<li>Passage the cells (every ~2 days).
	<ol>
		<li>Aspirate the medium and rinse once with 5 ml of 1x PBS.</li>
		<li>Add 2 mL of 0.25% trypsin-EDTA to the culture dish and incubate at 37 &#176;C for 3 min to facilitate cell detachment.</li>
		<li>Neutralize trypsin by adding 8 mL of pre-warmed complete medium and gently pipetting to disperse cells.</li>
		<li>Transfer 1 mL of the suspension into 9 mL of fresh complete medium.</li>
		<li>Count the cells. 
		NOTE: Cells were counted using a TC20 Automated Cell Counter according to the manufacturer&#39;s instructions. Trypan blue or other staining agents were not used.</li>
	</ol>
	</li>
	<li>Seed cells on the coverslips.
	<ol>
		<li>Dilute the cells to a final concentration of ~1 &#215; 106 cells/mL.</li>
		<li>Place the individual 18 mm glass coverslips into wells of a 12-well plate. 
		NOTE: Glass coverslips need to be autoclaved before being used for cell culture.</li>
		<li>Add 2 mL of the complete medium and 200 &#181;L of the cell suspension (~2 &#215; 105 cells/well).</li>
		<li>Incubate the cells overnight to allow cell attachment. 
		NOTE: To ensure cells are in normal growing status, routinely check cells&#39; morphology and their growing status (using a microscope) as well as examine potential mycoplasma contamination (using a mycoplasma detection kit).</li>
	</ol>
	</li>
</ol>
2. Cell washing with ammonium formate (AF) solution
NOTE: Prepare fresh AF solution each time.
<ol>
	<li>Prepare the AF wash buffer. Weigh 0.45 g of ammonium formate and dissolve in 50 mL of LC/MS-grade water. The final concentration of ammonium formate is 0.14 M (0.9% w/w). 
	NOTE: Keep the solution ice-cold (0-4 &#176;C) until use.</li>
	<li>Add 2 mL of cold AF solution to each well of the 12-well plate.</li>
	<li>Rinse the coverslips. Using a pair of sterile forceps to gently lift each coverslip containing adherent cells from its well and then briefly (for 2-3 s) place it in a separate well pre-filled with 2 mL of cold 0.9% ammonium formate (AF) solution. Repeat the rinsing process. 
	NOTE: To avoid disturbing the cells during the washing step, perform cell rinsing very gently.</li>
</ol>
3. Cell quenching by liquid nitrogen (LN2)
<ol>
	<li>Place each AF-rinsed coverslip (cells facing up) into the Petri dish inside the aluminum foil container. 
	NOTE: Wear cryogenic-rated gloves, safety goggles, and a lab coat. Perform all LN2 handling in a well-ventilated area. Aluminum foil can be used to fold a container for LN2 quenching.</li>
	<li>Slowly pour 10-20 mL of liquid nitrogen over the coverslips, ensuring complete coverage.</li>
	<li>Immediately tilt the Petri dish with tweezers to decant any remaining liquid nitrogen. 
	NOTE: Complete this action within a few seconds to prevent large ice-crystal formation from residual wash moisture.</li>
</ol>
4. Cell freeze-drying in vacuum
<ol>
	<li>Freeze-dry immediately after LN2 quenching to prevent thawing. 
	NOTE: Remove the rotor of the SpeedVac to create a flat platform that accommodates a 100 mm Petri dish.</li>
	<li>Using cryogenic gloves and insulated tweezers, place the Petri dish containing the LN2 chilled coverslips onto the vacuum concentrator chamber.</li>
	<li>Process using its standard vacuum settings and continue for 5-7 min until visible LN2 has evaporated and coverslips appear dry. Dried coverslips should not contain any residual LN2 and ice crystals.</li>
</ol>
5. Cell storage in a -80 &#176;C freezer
<ol>
	<li>Wrap the container with a paper towel and transfer it to a &#8722;80 &#176;C freezer for 48 h.</li>
	<li>After storage, immediately transfer the coverslips containing the Petri dish into a room-temperature desiccator for ~10 min. Ensure condensed frost or water on coverslips completely disappears before taking them out of the desiccator. 
	NOTE: Rapidly transferring the coverslips to the desiccator minimizes condensation; even minor surface wetting can dissolve dried metabolites.</li>
	<li>After AF washing, divide the samples into two groups (Group 1 and Group 2). Apply the following processing protocols. 
	Group 1: LN2 quench &#8594; freeze-dry &#8594; SCMS analysis (no storage). 
	Group 2: LN2 quench &#8594; freeze-dry &#8594; &#8722;80 &#176;C storage 48 h &#8594; SCMS analysis.</li>
	<li>Proceed with SCMS analysis as described below. 
	NOTE: More cell groups can be prepared using different conditions. Group 1 and Group 2 are used to illustrate the general protocols.</li>
</ol>
6. Single-probe fabrication and SCMS setup
NOTE: The Single-probe is fabricated in accordance with established procedures21,24,60,61, and only brief protocols are provided here.
<ol>
	<li>Pull the dual-bore quartz needle using a laser puller and generate a ~10 &#181;m tip. Assemble a Single-probe by inserting the fused silica capillary and nano-ESI emitter into the dual-bore quartz needle. Secure all components to a glass slide with quick-setting epoxy.</li>
	<li>Mount the Single-probe on the XYZ stage. Affix the Single-probe to a motorized XYZ manipulator positioned beneath a digital microscope.</li>
	<li>Couple the setup to a mass spectrometer for SCMS analysis.</li>
	<li>Use acetonitrile (&#8805;99.9%) containing 0.1 % formic acid as an extraction solvent at a flow rate of 150 nL/min delivered by a syringe pump.</li>
	<li>Set the MS parameters. 
	Positive ion mode: m/z 200-1500, +2.9 kV, mass resolution 120 k (at m/z 200). 
	Negative ion mode: m/z 70-900, &#8722;2.1 kV, mass resolution 120 k (at m/z 200). 
	NOTE: Because negative ions are generally detected in the m/z range lower than that of positive ions, different m/z ranges can be used for these two ion modes. Additional MS settings include 100 ms maximum injection time, one microscan, standard automatic gain control (AGC), full scan mode (for MS analysis), higher-energy collisional dissociation (HCD) (for MS/MS analysis) with 10-35 Normalized Collision Energy (NCE), and 320 &#176;C for the ion transfer tube temperature. A total ion chromatogram (TIC) was generated at each scan/time point to monitor signal stability and single-cell detection.</li>
</ol>
7. Single-probe SCMS experiments
<ol>
	<li>Place the two coverslips (Group 1 and Group 2) together on the motorized XYZ stage of the Single-probe SCMS setup21,24,61.</li>
	<li>To minimize batch effects, select the cells randomly under the microscope.</li>
	<li>Acquire positive and negative ion mode mass spectra. Analyze multiple (e.g., ~30) cells per group with the same Single-probe, solvent flow rate, and ionization voltage. 
	NOTE: Use the same Single-probe, solvent flow rate, and ionization voltages for every experiment to ensure data comparability. Calibrate the Orbitrap daily before acquisition. Both MS and MS/MS analyses can be performed at the single-cell level. Collect MS data to obtain accurate m/z values for studies of metabolite profiles and tentative labels in non-targeted analysis. Conduct MS/MS analysis of selected ions for molecular identification in targeted analysis. Data were acquired using a compatible data acquisition and interpretation software.</li>
</ol>
8. Data analysis
<ol>
	<li>Preprocess the raw SCMS data using a custom R script. Preprocessing steps include background and noise removal and ion intensity normalization to TIC11,12,23.</li>
	<li>Deisotope the SCMS data using the Python package ms_deisotope.</li>
	<li>Perform peak alignment using an in-house Python script60. Use a bin width of 0.01 m/z for binning. Use a mass tolerance of 0.01 m/z or 5 ppm for peak alignment.</li>
	<li>Perform statistical data analysis using MetaboAnalyst 6.062. Conduct a t-test and generate a heat map (for relative metabolite levels). Select Use top 100 in T-test/ANOVA to generate the top 100 metabolites, ranked by p-values from low to high, with a significant difference (p &#60; 0.05) from the comparison. Plot a heatmap using these top 100 metabolites.</li>
	<li>Search accurate m/z values and MS/MS spectra against the Human Metabolome Database (HMDB63). Use a mass tolerance of 10 ppm in MS database searching. 
	NOTE: Other databases, such as LipidMaps64, can be used for database searching.</li>
</ol>

The authors have no conflicts of interest to disclose.

A critical step in this protocol is the immediate quenching of live cells using LN2 following washing with AF, which substantially reduces enzymatic activity and halts ongoing metabolic processes. Additionally, washing cells with AF prior to quenching enhanced ion intensity and reduced matrix effects, supporting its inclusion as a preparatory step to improve SCMS sensitivity. In the current studies, cells in different groups (i.e., Group 1 and Group 2) were cultured using the same initial batch to minimize their biological variance. 48-h storage time was chosen because cells with additional growing time (Group 1) can still maintain suitable cell densities (e.g., to avoid cell aggregates) for comparative SCMS analysis with stored cells (Group 2). However, caution is warranted when relying on cold storage, even for 48 h, as significant metabolic changes were still observed in comparisons between freshly dried and stored samples (Group 1 vs. Group 2). Although in actual studies, cell and tissue samples are commonly stored for longer periods (e.g., for weeks and months) in a -80 &#176;C freezer, it is suggested to shorten the storage time to minimize changes in metabolites.
Troubleshooting should therefore focus on minimizing the time between drying and SCMS analysis, ensuring that defrosting conditions avoid condensation or partial rehydration. Residual water content and water condensation during sample transitions can partially rehydrate cells and enable enzymatic and chemical reactions such as hydrolysis, oxidation, and proteolysis that degrade metabolites65, leading to artificial changes in metabolite composition. Thoroughly dried samples effectively arrest these processes and preserve molecular integrity66. Ensuring a moisture-free desiccation environment and rapid transition to SCMS is key to avoiding these pitfalls.
Despite the success of this method in preserving metabolite profiles, limitations remain. The method requires access to a vacuum drying system and a -80 &#176;C freezer, and it is not fully effective for long-term storage. Furthermore, while rapid room-temperature drying preserves metabolites temporarily, it is not a substitute for LN2 quenching. Storage-related alterations may still occur due to incomplete water removal and the risk of rehydration during sample handling. These limitations highlight the need for method refinement if longer storage or higher throughput is desired.
Protocols reported in this work offer a simplified and effective approach for preserving the single-cell metabolome for ambient SCMS. Unlike methods requiring elaborate cryoprotectants or in situ freezing, our workflow is readily compatible with existing ambient ionization techniques and allows for flexible sampling and analysis timelines. The integration of cell washing, quenching, drying, and short-term storage without substantial loss of metabolomic fidelity offers a practical solution for labs lacking on-demand access to mass spectrometers. This approach holds strong potential for advancing research in single-cell systems biology, cancer metabolism, drug response heterogeneity, and metabolic phenotyping of rare cell populations. The broad applicability of these protocols, particularly for ambient ionization-based SCMS platforms, makes them an important contribution to the field of single-cell analytical techniques. In future studies, these protocols can be adapted and refined to accommodate a wider variety of cell types. With additional sample preparation steps (e.g., matrix application), the processed cells can be potentially analyzed by other SCMS techniques (e.g., MALDI and SIMS), offering higher spatial resolution and throughput for analysis of larger numbers of cells (e.g., highly confluent cells). The results also suggest that these quenching-drying-storage protocols can be potentially used to prepare cells for shipping (e.g., in dry ice containers), allowing for collaborative SCMS studies at different locations.

Cell-to-cell heterogeneity has emerged as a fundamental principle of biological systems, especially in the contexts of disease progression, drug resistance, and cellular differentiation1,2. In multicellular organisms, even genetically identical cells in the same microenvironment can exhibit distinct phenotypes, arising from differences at the transcriptomic3, proteomic4, and, crucially, metabolomic levels5. While single-cell genomics and transcriptomics have gained substantial traction in the past decade, single-cell metabolomics remains comparatively underdeveloped due to the inherent complexity and rapid dynamics of metabolites6. Metabolites are highly sensitive to environmental cues, have diverse chemical structures and polarities7, and exist at low concentrations in small cell volumes8. These properties make their reliable measurement from individual cells an analytical challenge. As metabolomics offers the closest snapshot of a cell&#39;s real-time functional state, advancing methods to robustly study the metabolome at single-cell resolution is essential for a deeper understanding of cellular physiology and pathology7.
Traditional bulk metabolomics techniques average signals across large cell populations, masking cellular heterogeneity and potentially obscuring rare but significant metabolic signatures. In contrast, single-cell metabolomics reveals unique biochemical profiles at the individual cell level, making it invaluable for studying processes like stem cell differentiation, drug responses, and disease mechanisms6,9,10,11,12,13. Mass spectrometry (MS)-based methods, such as matrix-assisted laser desorption-ionization (MALDI) MS and secondary ion (SI) MS, have advanced the field but face challenges like complex preparation, vacuum requirements, and destructive ionization that limit live-cell analysis9,14. Particularly, recent developments in instrumentation resulted in cutting-edge techniques, including transmission-mode MALDI coupled with laser-induced postionization (t-MALDI-215) and Orbitrap secondary ion mass spectrometry (OrbiSIMS)16,17, enabled label-free, high-resolution 3D metabolic imaging at the single-cell and subcellular level, offering exceptional mass accuracy and spatial resolution.
Ambient ionization MS addresses these issues. The representative ambient SCMS techniques include live single-cell video mass spectrometry (Video-MS)18, DESI19, and nano-DESI20. Among them, the single-probe SCMS is one of the techniques that stands out for its ability to directly sample and ionize live cells in real time using a dual-capillary system21,22,23,24. This technique minimizes sample disturbance while enabling high-resolution, in situ metabolic profiling and has been successfully applied to studies of drug uptake and influence on cell metabolism22,25,26,27,28,29,30, environmental responses31,32, and metabolic heterogeneity33,34,35,36 among individual cells.
While many ambient SCMS methods enable live cell analysis, they typically suffer from low throughput due to the manual handling required for individual cell selection23,37,38. Since cell metabolism is highly dynamic, extended sample preparation can alter metabolite profiles. To address this, researchers apply quenching techniques immediately after isolating cells39,40. Quenching halts metabolic activity either by rapid cooling41,42,43,44 or enzyme denaturation43,45,46,47, preserving the cells&#39; biochemical state at a specific moment. This step is vital for ensuring accurate and temporally relevant metabolomic data. An effective quenching protocol must rapidly and thoroughly stop intracellular metabolic activity. Various methods have been evaluated, including cold isotonic saline48, chilled acetonitrile46, cold methanol44,47,48,49, ice-cold phosphate buffer solution (PBS)43,50, LN243,44,51, and even hot air treatments52. While many of these were originally developed for bulk cell metabolomics, some, like cold methanol and acetonitrile, have been adapted for single-cell techniques such as Pico-ESI-MS39 and MALDI-MS46. Despite their effectiveness, each method has drawbacks: organic solvents may cause metabolite leakage and damage to cell membranes48,53, and nonvolatile salt solutions can interfere with MS by causing ion suppression54, reducing sensitivity, and compromising data accuracy47,55.
LN2 snap freezing is commonly used in biological research56 because it rapidly halts cellular metabolism without leaving behind nonvolatile salts, making it a good fit for SCMS. However, it can damage cell membranes, which is problematic for SCMS57,58. To reduce this damage, some studies used a method involving fast filtration, cold NaCl washing, and LN2 freezing, which helps retain fast-turnover metabolites53,59. Still, this method isn&#39;t ideal for SCMS due to difficulties in isolating individual cells and potential matrix effects from salt residues. Storage at -80 &#176;C is another common preservation method, but its effects on single-cell metabolite profiles remain unclear. We have previously reported studies to address these limitations by combining multiple key steps: cells are first washed with a volatile, MS-compatible AF solution, then quenched with LN2, vacuum freeze-dried, and stored at low temperature (-80 &#176;C)60. The current approach minimizes cell damage and metabolite degradation, enabling more accurate SCMS measurements. This work is focused on experimental details, aiming to promote the adoption of this cell preparation method for robust SCMS techniques.

<table><tbody><tr><td>-80 &amp;deg;C freezer</td><td>Eppendorf New Brunswick</td><td>U700</td><td>Sample preparation </td></tr><tr><td>Acetontrile</td><td>ThermoFisher Scientific</td><td>240744</td><td>SCMS experiment setup</td></tr><tr><td>Ammonium formate </td><td>ThermoFisher Scientific</td><td>401152500</td><td>Sample preparation </td></tr><tr><td>Coverslip 18 mm</td><td>VMR micro cover glass</td><td>48380046</td><td>Cell culture </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>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>Formic acid</td><td>Ward&#039;s Science </td><td>470301-120 500mL</td><td>SCMS experiment setup</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</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>SCMS experiment setup</td></tr><tr><td>Glass coverslips, 18mm</td><td>VWR </td><td>48380046</td><td>Cell culture</td></tr><tr><td>HyClone Synthetic serum (FBS)</td><td>Hyclone</td><td>SH3006603</td><td>Cell culture</td></tr><tr><td>Incubator </td><td>HeraCell (Heraeus)</td><td></td><td>Cell culture </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>McCoy&#039;s 5A </td><td>Gibco </td><td>2537128</td><td>Cell culture </td></tr><tr><td>Microunion</td><td>IDEX Health &amp;amp; Science LLC</td><td>M-539</td><td>SCMS experiment setup</td></tr><tr><td>Modified FAIMS pro interface </td><td>Thermo Fisher </td><td>98100-20046</td><td>Analysis </td></tr><tr><td>MycoAlert PLUS Mycoplasma Detection Kit</td><td>Lonza</td><td>LT07-703</td><td>Cell culture</td></tr><tr><td>Optical board </td><td>Thorlabs Inc., Newton</td><td></td><td>Analysis </td></tr><tr><td>Optima LC/MS-grade water (Fisher Chemical, USA)</td><td>ThermoFisher Scientific</td><td>W6500</td><td>Sample preparation </td></tr><tr><td>Orbitrap Exploris 240 mass spectrometer </td><td>ThermoFisher Scientific</td><td></td><td>SCMS experiment setup</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Cell culture</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>VWR</td><td>0780-50L</td><td>Cell culture</td></tr><tr><td>SpeedVac Concentrator (Savant)</td><td>ThermoFisher Scientific</td><td> SPD111 V</td><td>Sample preparation </td></tr><tr><td>Syringe Pump (SKE10)</td><td>Chemyx Inc.</td><td>80561</td><td>SCMS experiment setup</td></tr><tr><td>Syringe, 250 &amp;micro;L</td><td>Hamilton</td><td>1725LTN250UL</td><td>SCMS experiment setup</td></tr><tr><td>TC20 Automated Cell Counter</td><td>Bio-Rad Laboratories </td><td>1450102EDU</td><td>Cell count</td></tr><tr><td>Tissue Culture Dish, 100mm</td><td>Fisher scientific </td><td>FB012924</td><td>Cell culture </td></tr><tr><td>Trypsin-EDTA</td><td>ThermoFisher Scientific</td><td>25200-072</td><td>Cell culture</td></tr><tr><td>USB digital microscope (&quot;Scorpio&quot; 500 Zoom)</td><td>Chinavasion, Wholesale Ltd.</td><td></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>Xcalibur software</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>XYZ-stage (CONEX- MFACC)</td><td>Newport Corp.</td><td></td><td>Analysis </td></tr></tbody></table>

sample preparation for single cell mass spectrometry metabolomics studies: combined cell washing, quenching, drying, and storage

Sample Preparation for Single Cell Mass Spectrometry Metabolomics Studies: Combined Cell Washing, Quenching, Drying, and Storage

Single-cell mass spectrometry (SCMS) has become an indispensable tool for studying cellular metabolism. Owing to advancements in modern mass spectrometry (MS) techniques and demand in studies of cell heterogeneity in fundamental biological sciences and human diseases, a variety of different SCMS techniques have been developed and applied in laboratory research. As metabolites can accurately reflect cell status, SCMS metabolomics analysis of live cells is regarded as a powerful tool to provide molecular information about cells. However, a major challenge in SCMS analysis of live cells is preserving the endogenous metabolite profiles during sample preparation, transport, and measurement. Cellular metabolites undergo rapid turnover and are highly sensitive to environmental changes, making them susceptible to degradation or transformation prior to analysis. To address this limitation, we present a robust and reproducible cell preparation protocol designed to preserve cellular metabolite integrity for SCMS. The protocol integrates cell washing with a volatile ammonium formate (AF) solution, rapid quenching with liquid nitrogen (LN2), vacuum freeze-drying, and storage at -80 &#176;C. This approach minimizes cell membrane damage while effectively halting metabolic activity. The results indicate that rapid cell quenching is vital; however, limiting storage time at -80 &#176;C is necessary to preserve cell metabolites. The proposed protocol can potentially be used for other existing SCMS techniques for broad applications.

This report describes a methodology that integrates cell washing, rapid cell quenching, vacuum drying, and storage at -80 °C (Figure 1). Specifically, this approach is designed to preserve the metabolomic state of cells prior to SCMS analysis. The effectiveness of this protocol was tested by preparing cells under two different conditions: freshly quenched and dried cells (Group 1, serving as the control), and quenched, dried, and -80 °C stored cells (Group 2). Each group was analyzed using the Single-probe SCMS method in both positive and negative ion modes. Principal Component Analysis (PCA) and heat map visualization were used to evaluate the differences in metabolomic profiles across groups.
PCA results in the positive ion mode (Figure 2A) revealed that Groups 1 and Group 2 had similar overall metabolite profiles. The nearly identical profiles of Group 1 and Group 2 demonstrate that -80 °C storage for 48 h does not significantly alter the metabolome if cells are properly quenched before drying. In the negative ion mode (Figure 2B), a comparable pattern is observed, although Group 2 appears slightly more distinct-potentially due to ionization differences among specific metabolite classes, for instance, phosphatidylcholines are better detected in positive mode, while fatty acids ionize more efficiently in negative mode60. Despite this variation, the consistent clustering between Groups 1 and 2 across both modes supports the conclusion that the integrated quenching-drying-storage protocol maintains metabolic integrity.
Heat map analysis further validated the PCA findings by depicting relative abundances of the top 100 metabolites across two groups. As shown in Figure 3, the top 100 metabolites display highly consistent abundance patterns between Group 1 and Group 2 in both ion modes. No major shifts or group-specific clustering are observed, although minor variations in a few metabolite clusters may reflect differences in ionization efficiency or metabolite stability. These results collectively demonstrate that the quenching-drying-storage workflow preserves cellular metabolomic profiles with high fidelity, making it well-suited for ambient SCMS applications.
<img alt="HCT-116 cell freeze-drying process; SCMS results; diagram shows LN2 quenching, storage." class="xfigimg" src="/files/ftp_upload/68995/68995fig1.jpg" data-altupdatedat="1762509930000" data-alt="Figure 1"> 
Figure 1: General workflow for single-cell mass spectrometry (SCMS) analysis investigating how LN2 quenching and 48-h storage at -80 °C affect metabolite profiles in single cells. (A) Cells were seeded, incubated, and rinsed using ammonium format (AF) solution. (B) Two cell groups were prepared-Group 1: cells were washed, quenched, and freeze-dried for immediate SCMS experiments without storage; Group 2: cells were washed, quenched, freeze-dried, and stored at -80 °C. <a href="https://www.jove.com/files/ftp_upload/68995/68995fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Principal component analysis charts, groups 1-2, variance distribution in data sets A-B, diagram." class="xfigimg" src="/files/ftp_upload/68995/68995fig2.jpg" data-altupdatedat="1762509930000" data-alt="Figure 2"> 
Figure 2: Principal Component Analysis (PCA) of SCMS data from HCT-116 cells in experimental groups in (A) positive ion mode and (B) negative ion mode. <a href="https://www.jove.com/files/ftp_upload/68995/68995fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Heatmap diagram showing gene expression levels for two groups with color scale from -4 to 4." class="xfigimg" src="/files/ftp_upload/68995/68995fig3.jpg" data-altupdatedat="1762509930000" data-alt="Figure 3"> 
Figure 3: Heat maps showing the relative levels of the top 100 metabolites detected in single HCT-116 cells prepared under various conditions, presented for (A) positive ion mode and (B) negative ion mode. <a href="https://www.jove.com/files/ftp_upload/68995/68995fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol aims to preserve cell metabolites for single-cell mass spectrometry (SCMS) by combining volatile salt solution washing, rapid liquid nitrogen quenching, freeze-drying, and &#8722;8&#8239;&#176;C storage. It demonstrates that liquid nitrogen quenching is essential for maintaining metabolite profiles, while prolonged cold storage should be minimized to prevent metabolic alterations.

Single-cell mass spectrometry (SCMS) has become an indispensable tool for studying cellular metabolism. Owing to advancements in modern mass spectrometry ...

We have developed protocols to preserve the integrity of cellular metabolites for single cell mass spectrometry studies. We investigated how washing, quenching and storage conditions affect cell metabolites. New sample preparation methods and mass spectrometry techniques have been recently developed to advance single-cell metabolomics studies to better understand cellular heterogeneity and disease mechanisms.Researchers need to overcome multiple key challenges, including limited sample amount, reduced detection sensitivity due to sample complexity and altered metabolites due sample preparation and analysis. We demonstrate comprehensive sample preparation techniques that can preserve cellular metabolites and cell integrity for single cell mass spectrometry metabolomic studies. Our protocols could offer a simple and effective way for preparation, storage and transportation of samples for single cell metabolomic studies using different mass spectrometric techniques.To begin, incubate the cells at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide. Monitor the confluency of the cells daily and passage them when cultures reach 80%confluency. For passaging, aspirate the medium from the dish and rinse once with five milliliters of PBS.Incubate the cells with two milliliters of 0.25%trypsin EDTA at 37 degrees Celsius for three minutes to facilitate cell detachment. Neutralize the trypsin by adding eight milliliters of prewarm complete medium and gently pipette to disperse the cells. Transfer one milliliter of the suspension into nine milliliters of fresh, complete medium for counting the cells.To seed the cells, dilute them to a final concentration of one times 10 to the power of six cells per milliliter. Place individual 18 millimeter glass cover slips into the wells of a 12 Well plate. Add two milliliters of complete medium and 200 microliters of the cell suspension into each well to achieve two times 10 to the power of five cells per well.Incubate the cells overnight to allow attachment. For washing the cells, add two milliliters of cold ammonium formate solution to each well of the 12 well plate. Then using sterile forceps, gently lift each cover slip containing adherence cells from its well and briefly place it for two to three seconds in a separate well prefilled with two milliliters of cold 0.9%ammonium formate solution.Place each ammonium formate rinsed cover slip with the cells facing upward into a petri dish inside an aluminum foil container. Slowly pour 10 to 20 milliliters of liquid nitrogen over the cover slips to ensure complete coverage. And immediately tilt the petri dish with tweezers to decant any remaining liquid nitrogen.Using cryogenic gloves and insulated tweezers, place the petri dish containing the liquid nitrogen chill cover slips into the vacuum concentrator chamber. Process the samples using the standard vacuum settings for five to seven minutes until the visible liquid nitrogen evaporates and the cover slips appear dry. Ensure that the dried cover slips do not contain any residual liquid nitrogen or ice crystals.Next, wrap the container with a paper towel and transfer it to a 80 degree Celsius freezer for 48 hours. After storage, transfer the cover slips containing the petri dish into a room temperature desiccate for 10 minutes. Ensure condensed frost or water on the cover slips completely disappears before removing them from the desiccator.Divide the samples into two groups and treat them appropriately. Now, mount the single probe onto the XYZ stage and affix it to a motorized XYZ manipulator positioned beneath a digital microscope. Couple the single probe setup to a mass spectrometer for SCMS analysis.Use acetone nitrile containing 0.1%formic acid at a purity of at least 99.9%as the extraction solvent and deliver it at a flow rate of 150 nanoliters per minute using a syringe pump. Set the mass spectrometry parameters for positive and negative ion modes. Now, place the two cover slips from group one and group two together on the motorized XYZ stage of the single probe SCMS setup and use the microscope to randomly select cells for analysis.After acquiring mass spectra, analyze approximately 30 cells per group using the same single probe, solvent flow rate and ionization voltage. Pre-processed the raw SCMS data using a custom R script and apply background and noise removal followed by ion intensity normalization to total ion current. De-isotope the SCMS data using the Python package MSD isotope.Perform peak alignment Using an in-house Python script. Apply a bin width of 0.01 master charge ratio for binning and a mass tolerance of 0.01 master charge ratio or five parts per million for peak alignment. Conduct statistical data analysis using Metabo Analyst 6.0.Perform a T-test and generate a heat map showing relative metabolite levels. Select the option used top 100 and T-test or Inova to generate the top 100 metabolites ranked by significance and plot a heat map using these top 100 metabolites with significant differences. Finally, search the accurate master charge values against the human metabolome database using a mass tolerance of 10 parts per million for database searching.Principle component analysis in the positive ion mode showed that group one and group two overlapped closely, indicating highly similar metabolite profiles. In the negative ion mode, a comparable pattern was observed, although group two appeared slightly more distinct. Heat map analysis of the top 100 metabolites displayed highly consistent abundance patterns between group one and group two in both ion modes.No major shifts or group specific clustering are observed. Although minor variations in a few metabolite clusters may reflect differences in ionization efficiency or metabolite stability.

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