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

SCoPE2 can quantify thousands of proteins from thousands of single mammalian cells without antibodies. This protocol can help uncover proteome heterogeneity that would otherwise be invisible to bulk analyses. Single cell proteomics, using the SCoPE2 approach, was designed to be accessible to researchers with a range of resources, from just a pipette, to liquid handling robotics.

SCoPE2 is broadly applicable to many research areas, including cancer and immunology, by providing insight into how proteins are differential in abundance between single cells from the same population. To begin, start the cell lysis procedure by placing a 384-well plate with sorted single cells into the thermal cycler, heating it for 10 minutes at 90 degrees Celsius followed by cooling at 12 degrees Celsius. Spin down the plate briefly in a benchtop plate spinner at 18 to 22 degrees Celsius.

Sonicate the plate for five minutes in a water bath sonicator at 18 to 22 degrees Celsius. Then, place it on ice. To proceed with trypsin digestion, prepare 100 microliters of the master mix and add 0.2 microliters of the master mix to each well of the 384-well plate using a liquid handler.

Seal the plate, vortex for five seconds, and spin down in a benchtop plate spinner at 18 to 22 degrees Celsius. Heat the 384-well plate for three hours at 37 degrees Celsius with the lid temperature set to 52 degrees Celsius. To set a tandem mass tagging reaction, take the 85 millimolar stocks of tandem mass tags out of the minus 80 degrees Celsius freezer.

Before opening them, warm the tubes to room temperature and dilute the labels to 22 millimole in anhydrous acetonitrile. Using this labeling strategy, add 0.5 microliters of diluted tandem mass tags to each well of the 384-well plate, using either a liquid dispensing robot or a manual pipette. Seal the plate, vortex for five seconds, and spin down in a benchtop plate spinner at 18 to 22 degrees Celsius.

Let the labeling reaction proceed at 18 to 22 degrees Celsius for one hour. For quenching of tandem mass tagging reaction, add 0.2 microliters of 0.5%hydroxylamine, diluted in HPLC-grade water, to each well of the 384-well plate using a liquid handler. Seal the plate, vortex for five seconds, and spin down in a benchtop plate spinner before keeping the plate at 18 to 22 degrees Celsius for 30 minutes.

LC-MS analysis preparation is started by removing the combined carrier or reference material from the minus 80 degrees Celsius freezer. For each set, pipette one microliter of carrier and reference material into the first well, will be part of a single set. Pipette the complete volume from the first well to the subsequent well.

Continue pipetting the complete volume from one well to the next until all wells included in a single set have been combined in the final well. For each set, add five microliters of 50%acetonitrile diluted in HPLC-grade water for the first single cell well to be included in each set. Pipette 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 included in a single set have been combined in the final well. Now transfer each set into their autosampler glass inserts. Dry the samples once all sets are combined and placed into glass inserts.

Prior to LC-MS-MS analysis, add 1.2 microliters of 0.1%formic acid to each set to resuspend the labeled peptides. Place the autosampler inserts into glass autosampler vials, and close each sample with a cap. Vortex each vial for five seconds to make sure resuspension is complete, and then spin down in a vial spinner at 18 to 22 degrees Celsius.

Ensure that each sample is at the bottom of the insert rather than splashed on the sides. Place the vials in the autosampler tray and for each set, inject a one microliter sample for LC-MS-MS analysis. Digestion and labeling efficiency of the single cells may not be directly assayed as with the carrier, but DO-MS software provides plots that estimate the digestion and labeling efficiency.

Poor digestion is indicated by a high ratio of the intensity of miscleaved peptides to their completely cleaved counterparts. Another factor to consider in the sets is the fraction of missing data in the median reporter ion intensity in each tandem mass tagging channel. When single cells are successfully prepared, missing data per cell and positive control are much lower than negative control samples.

Careful liquid handling during LC-MS-MS sample preparation is critical for maximum recovery of peptides. Small samples of approximately 100 picograms to 100 nanograms per well can be prepared for proteomics analysis using an isobaric carrier. SCoPE2 makes single-cell proteomics measurements accessible to researchers worldwide by using only common reagents and commercially-available equipment.

It is amenable to manual processing or high throughput automation by robotic liquid handling.