Large numbers of cells are generally required for quantitative global proteome profiling due to the significant surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for ‘all-in-one’ one-pot sample preparation. This ‘all-in-one’ method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus significantly improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.

Fluorescence-assisted cell sorting (FACS) of single cells. Prior to cell collection, PCR tubes or 96-well PCR plates were pretreated with 0.1% DDM for coating the surface and then the DDM solution was removed. The pretreated PCR tubes or 96-well PCR plates were air-dried in the fume hood. To avoid cell clumping, after detaching they were dispersed into a single-cell suspension by passing three times through a 25-gauge needle. The cells were suspended in PBS, and then pelleted by centrifuging 5 min at 500g. This process was repeated five times to remove the remaining PBS and trypsin. After that the cells were resuspended in PBS and passed through a 35 μm mesh cap (BD Biosciences, Canaan, CT) to remove large aggregates. A BD Influx flow cytometer (BD Biosciences, San Jose, CA) was used to deposit cells into the precoated PCR tubes. Alignment into a Hard-Shell 96-well PCR plate (Bio-Rad, Hercules, CA) was done using fluorescent beads (Spherotech, Lake Forest, IL), after which the coated PCR tubes were placed into the plates for cell collection. For unstained MCF10A cells, forward and side scatter detectors were used for cell identification. Once sorting gates were established, cells were sorted into the PCR tubes using the 1-drop single sort mode. After isolation of the desired number of cells into the PCR tube, the isolated cells were immediately centrifuged at 1000g for 10 min at 4 °C to keep the cells at the bottom of the tube to avoid potential cell loss. The PCR tubes with the isolated cells were stored in a -80 °C freezer until further analysis.

Single cell sorting of patient CTCs from PCDXs and early metastases to the lungs. Cells from dissociated tumor and lung tissues were washed in PBS and then centrifuged at 300 g for 5 min at 4 C. Samples were resuspended in 2% FBS in PBS. MDA-MB-231 cells were collected and suspended in 2% FBS in PBS to serve as a tdTomato (L2T)-negative control for flow analysis. Cancer cells from the tumor and lung samples were sorted based on L2T expression. L2T+ tumor cells of the lung metastases were initially sorted into 10% FBS in PBS prior to single cell sorting, and each of the L2T+ single cells from the primary tumor and lung metastases was sorted into 5 µL H2O in a single tube of a 96-tube PCR plate. Plates were sealed, briefly spun on a microplate centrifuge, and stored at -80 C until later SOP-MS analysis.