Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

NOTE: In this protocol, room temperature (RT) is 18-22 °C.

1. Carrier and reference material generation

1. Cell isolation
   1. Collect cells of interest into cell suspensions in ice-cold 1x PBS.
      NOTE: If possible, the carrier and reference material should be prepared from the cell population(s) chosen to be studied at the single-cell level. Similar numbers of cells from the different experimental conditions (such as stimulated and unstimulated immune cells) should constitute the carrier and reference. In situations in which a large enough number of these cells cannot be harvested, a suitable cell population should be selected based on biological similarity.
   2. Using a hemocytometer or other means of cell counting (such as FACS), count the number of cells in the suspension.
   3. Spin down and resuspend 22,000 cells of each cell type in 11 µL of HPLC-grade water, separately into PCR tubes (standard 250 µL PCR tubes).
      NOTE: The number of cells advised here are sufficient for the carriers and references for the number of sets that can be prepared from a 384-well plate full of single cells. The cell input in this step and reagent amounts in subsequent steps can be scaled proportionally for greater numbers of sets. The speed of spinning down is dependent on the cell culture practices of the lab. A typical spin down: 300 × g for 5 min.
   4. Transfer the samples to a -80 °C freezer for at least 30 min.
      PAUSE POINT: Samples can stay frozen for several months with no consequences for single-cell data.
2. Cell lysis
   1. Heat the PCR tube with the cells to 90 °C for 10 min (with thermal cycler lid set at 105 °C), and then let the tubes cool to 12 °C right after the heating cycle.
   2. Vortex the tube briefly, and then spin down in a bench-top PCR tube spinner at RT to collect all the liquid at the bottom of the tube.
   3. In a water bath sonicator, sonicate the tubes for 5 min, then place them on ice at RT.
3. Trypsin digestion
   1. Add 2.2 µL of the master mix (see Table 1 for the components) to each sample tube while on ice.
      CAUTION: Trypsin can cause skin, respiratory, and eye irritation. Be sure to wear personal protective equipment. Handle under a chemical fume hood.
   2. Vortex the tubes briefly to mix and spin down in a bench-top PCR tube spinner at RT to collect all liquid at the bottom of each tube.
   3. Heat the samples at 37 °C for 3 h (with the thermal cycler lid set at 52 °C).
4. TMT labeling reaction
   1. Spin down the samples after digestion in a bench-top PCR tube spinner at RT.
   2. Split each sample into two equal volumes of 6.6 µL each, so that each tube contains 11,000 cells.
   3. To the tubes meant for the carrier, add 3.3 µL of TMT label 126 that has been resuspended at a concentration of 85 mM.
   4. To the tubes meant for the reference, add 3.3 µL of TMT label 127N that has been resuspended at a concentration of 85 mM.
   5. Vortex the tubes briefly and spin down in a bench-top PCR tube spinner at RT to collect the liquid at the bottom.
   6. Leave the tubes at RT for 1 h.
5. Quenching of TMT labeling reaction
   1. Add 1.65 µL of 0.5% hydroxylamine to each tube.
      CAUTION: Hydroxylamine can cause skin irritation. Be sure to wear personal protective equipment.
   2. Vortex the tubes briefly and spin down in a bench-top PCR tube spinner at RT to collect the liquid at the bottom.
   3. Leave the tubes at RT for 30 min.
6. Combination of carrier and reference
   1. If samples that will constitute the carrier have been separately labeled, mix them in equal ratios.
   2. If samples that will constitute the reference have been separately labeled, mix them in equal ratios.
   3. Mix the carrier and reference so that the final concentration of the carrier is 100-200 cells/µL, and the reference concentration is 5-10 cells/µL.
   4. Assess the quality of the carrier and reference materials prior to combining them with single-cell sets.
      NOTE: Quality control of the carrier and reference material can be carried out by a variety of methods, but it is recommended to use the DO-MS software, available at do-ms.slavovlab.net¹⁷. Critical measurements of quality include miscleavage rate (<25%, a metric calculated as the number of confidently identified peptides with a missed cleavage divided by the number of confidently identified peptides in total) and labeling efficiency (>99%, a metric calculated as the number of labeling sites, namely the peptide N-terminus and Lysine, with a label divided by the total number of labeling sites).
      PAUSE POINT: The carrier and reference materials can be stored at -80 °C until required.

Table 1: Reagent amount of master mix. The final concentrations of reagents needed for trypsin digestion are listed.

2. SCoPE2 sample preparation

1. Cell isolation
   1. Supplement HPLC-grade water with 25 fmol per peptide per µL of a synthetic peptide solution.
      NOTE: Waters MassPrep peptide solution is an example of what can be used. It is composed of seven peptides that do not ionize very well. This is a key aspect of this solution: these peptides do not interfere with accurate mass spectrometry quantitation of single cells. Any highly purified mixture of a few peptides unlikely to be in the samples of interest will be appropriate.
   2. Add 1 µL of this mix to each well of a 384-well plate using a liquid-dispensing robot or manual pipette.
   3. Seal the plate and spin it down in a bench-top plate spinner at RT to collect the liquid at the bottom.
      PAUSE POINT: Plates with this solution can be stored at -80 °C until required.
   4. Obtain a suspension of unfixed cells that are disaggregated.
      NOTE: How exactly the cell suspension is obtained is based on the cell type of interest. Here are broad examples:
      1. Suspension cells: spin down the cells into a pellet, and remove the cell culture medium.
      2. Adherent cells: remove the cells from the dish or flask through trypsinization or scraping. Then, spin them down to remove the cell culture medium.
   5. Wash the cell suspension twice with 1x ice-cold PBS. Leave the cells suspended in 1x PBS after the second wash.
   6. If the 384-well plate was frozen, thaw it out at RT. Spin it down to ensure all liquid is at the bottom of the wells.
   7. Use a cell sorter or other available means, such as manual handpicking, to distribute single cells into respective wells.
      NOTE: Add single cells to wells while leaving some wells empty for negative and positive controls (see discussion). When using flow cytometry, use the lowest possible flow rate for the flow cytometer. Lower flow rate is thought to be gentler for cell handling. Additionally, the nozzle size should be appropriate for use with the cells of interest. It is recommended to collaborate with a flow cytometry facility.
   8. Add positive controls of 2-5 cell equivalents lysate to selected wells pipetted manually or with liquid handling robots.
   9. Seal and spin down the plate with sorted cells in a bench-top plate spinner at RT to collect the liquid at the bottom. Freeze the plate at -80 °C as soon as possible.
      PAUSE POINT: Plates with sorted single cells can be stored at -80 °C until required.
2. Cell lysis
   1. Take the 384-well plate with the sorted single cells and put it into the thermal cycler as quickly as possible.
   2. Heat the plate to 90 °C for 10 min (with the lid temperature set to 105 °C), and then let the plate cool to 12 °C.
   3. Spin down the plate briefly in a bench-top plate spinner at RT.
   4. In a water bath sonicator, sonicate the plate for 5 min at RT, then place it on ice.
3. Trypsin digestion
   1. Prepare 100 µL of the master mix (see Table 1 for components) per 384-well plate.
   2. To each well of the 384-well plate, add 0.2 µL of the master mix prepared in step 2.3.1 using a liquid handler.
      NOTE: Use larger volumes if manual pipetting is used, making sure that the final concentrations of reagents are still the same per well.
   3. Seal the plate, vortex for 5 s, and spin down in a bench-top plate spinner at RT.
   4. Heat the 384-well plate at 37 °C (with the lid temperature set to 52 °C) for 3 h.
4. TMT labeling reaction
   1. Take the 85 mM stocks of TMT labels (128N through 135N) out of the -80 °C freezer. Warm the tubes to room temperature before opening them.
   2. Dilute the labels to 22 mM in anhydrous acetonitrile.
      CAUTION: Acetonitrile is a flammable liquid. It can irritate the skin, eyes, respiratory tract, and central nervous system. Be sure to wear personal protective equipment. Handle under a chemical fume hood.
   3. Using this labeling strategy, add 0.5 µL of diluted TMT labels to each well of the 384-well plate. Use either a liquid-dispensing robot (if using a liquid handler, be sure to use associated equipment that is suitable for organic solvents) or a manual pipette.
      NOTE: See Table 2 for the labeling strategy. See Table 3 for an example plate layout.
   4. Seal the plate, vortex for 5 s, and spin down in a bench-top plate spinner at RT.
   5. Let the labeling reaction proceed at RT for 1 h.
5. Quenching of TMT labeling reaction
   1. To each well of the 384-well plate, add 0.2 µL of 0.5% hydroxylamine (diluted in HPLC-grade water) using a liquid handler.
      NOTE: Use larger volumes if manual pipetting is used, making sure that the final concentrations of reagents are still the same per well.
   2. Seal the plate, vortex for 5 s, and spin down in a bench-top plate spinner at RT.
   3. Keep the plate at RT for 30 min.
6. Preparation for LC-MS/MS analysis: combination of single cells and control wells with carrier/reference material
   1. Remove the combined carrier/reference material from the -80 °C freezer.
   2. For each set, pipette 1 µL of carrier and reference material into the first well that will be part of a single set. Pipette the complete volume of the first well to the subsequent well. Continue pipetting the complete volume from one cell to the next until all wells to be included in a single set have been combined in the final well.
      NOTE: The carrier and reference material should be at a concentration of 200- and 5-cell equivalents, respectively, but as discussed previously²¹, these amounts may vary. Each set, as outlined in the labeling strategy (see Table 2), should contain carrier, reference, and up to 12 or 14 single-cell or control samples when using TMTpro-16plex or TMTpro-18plex, respectively (see step 2.4.3).
   3. For each set, add 5 µL of 50% acetonitrile (diluted in HPLC-grade water) to the first single-cell well to be included in each set. Pipet the complete volume from the first well to the subsequent well. Continue pipetting the complete volume from one cell to the next until all wells to be included in a single set have been combined in the final well.
      NOTE: This washing step can help in sample recovery from the wells.
   4. Transfer each set into their individual autosampler glass inserts.
      NOTE: These sets can also be combined into single wells of a 384-well plate and directly placed in the autosampler for injection²⁸.
   5. Once all sets are combined and placed into glass inserts, dry the samples.
      PAUSE POINT: Dried sets can be stored at -80 °C for at least 1 week prior to running on a mass spectrometer.
   6. Prior to LC-MS/MS analysis, add 1.2 µL of 0.1% formic acid (diluted in HPLC-grade water) to each set to resuspend the labeled peptides.
      CAUTION: Formic acid is a flammable liquid. It can cause serious eye damage or skin burns. Be sure to wear personal protective equipment. Handle in a well-ventilated area.
   7. Place the autosampler inserts into glass autosampler vials and close each sample with a cap.
   8. Vortex each vial for 5 s to make sure resuspension is complete, and then spin down in a vial spinner at RT.
   9. Ensure that each sample is at the bottom of the insert, rather than splashed on the sides.
   10. Place the vials in the autosampler tray.
   11. For each set, inject 1 µL for LC-MS/MS analysis.

Table 2: Example SCoPE2 TMTpro-18plex labeling scheme. The carrier and reference are usually labeled in the first two TMT labels. Then, the next two labels are skipped due to potential isotopic contamination that would make the quantitation less accurate. The other labels left are for single cells or controls.

Table 3: Example plate layout for labeling using TMTpro-16plex. Using a 384-well plate format (or any other plate format), it is important to randomize the wells in which cells are sorted into. A different plate layout will be used for TMTpro-18plex.