Unleashing Single-Cell Proteomics: How scPICO Revolutionizes Absolute Quantification with F.SIGHT and I.DOT Technology

In proteomics, accurately quantifying proteins at the single-cell level is essential for advancements in fields like cancer research and drug discovery. Traditional methods like Western blotting and ELISA, though commonly used, fall short in sensitivity and throughput, which hinders their effectiveness in high-demand settings.

Our customers at Actome developed single-cell protein interaction coupling (scPICO) assays, integrated with CYTENA's F.SIGHT Single Cell Isolator and DISPENDIX's I.DOT Non-Contact Dispenser, which offer a groundbreaking solution that addresses these challenges by enabling precise, reference-free absolute quantification (AQ) of proteins.

The Need for Single-Cell Precision in Proteomics

Understanding the diverse cellular landscape within tissues requires analyzing protein expressions at an individual cell level. However, traditional methods, such as mass spectrometry and flow cytometry, often struggle with single-cell applications due to complex sample preparation and high costs. To overcome these limitations, Actome has developed the single-cell protein interaction coupling (scPICO) assay, which brings accuracy, efficiency, and sensitivity to single-cell proteomics.

Key Components: CYTENA F.SIGHT & DISPENDIX I.DOT Technologies

The scPICO workflow (Fig. 1) is powered by two advanced BICO Group technologies:

• CYTENA F.SIGHT Single-Cell Dispenser: This system precisely isolates single cells into 384-well plates, enhancing accuracy in sample preparation. F.SIGHT’s gentle handling and single-use cartridges reduce contamination risks and preserve cell integrity.

• DISPENDIX I.DOT Non-Contact Dispenser: I.DOT’s precision in dispensing tiny volumes (down to nanoliters) ensures accurate and efficient antibody preparation, critical for maintaining assay sensitivity and specificity.

Figure 1. The scPICO workflow. A. Triangular PICO assay, i.e., three assays are set up using pairwise readout of three concurrently binding 4EBP1 (EIF4EBP1 4F3-H2, Invitrogen; 4EBP1 60246-1-Ig, Proteintech) and p-4EBP1 (Phospho-4EBP1 4EB1T37T46-A5, Invitrogen) antibodies at 5.56e-11 M. The assays are indicated with arrows and corresponding colors, while the colors of antibodies match the dPCR fluorescent color channels. The 4EBP1 protein is read using red and yellow antibodies, while phosphorylation of 4EBP1 is measured by two pairs (self-confirmatory assay) red-green and green-yellow, respectively. B. a. Dispensing 12 nL volume of the antibody mix (ABX) in lysis buffer (ACTOME LBTW) under hydrophobic oil (Qiagen Vapor-Lock) in a 384-well plate (Eppendorf Microplate 384/V-PP), using the DISPENDIX I.DOT Liquid Handler, b. and isolating optically interrogated single U937 cells in 150 pL volume and into the 384-well using the CYTENA F.SIGHT Single-Cell Dispenser. The previous two steps were fluorescently traced by FITC, to judge successful liquid handling steps. c. After overnight incubation (d) 2% of the material of the single cell was dPCR amplified on the QIAcuity system, and (e) PICO data analysis was carried out to gain the absolute number of detected copies of proteoforms in the dPCR volume.

Advantages of scPICO for Absolute Quantification

The scPICO assay enables proteomics researchers to detect and quantify specific proteins, post-translational modifications, and protein-protein interactions with high sensitivity and zero background noise. Its reference-free AQ method is particularly beneficial, as it eliminates the need for external standards, saving time and reducing error.

Real-World Application: Assessing Cellular Heterogeneity

In a recent study, scPICO was used to analyze protein responses in U937 cells treated with a PI3K/mTOR inhibitor. The assay detected variations in p-4EBP1 protein levels across individual cells, revealing significant heterogeneity in treatment responses—a capability unmatched by traditional bulk assays (Fig. 2).

Download the full application note here. 

Figure 2. Comparing Bulk and Single-cell PICO. A. scPICO - copies of detected couplexes. scPICO assesses a single cell in a 12 nL volume. U937 cells were treated with dactolisib (5.6 μM) for 4 h or left untreated (DMSO) and subjected to the scPICO workflow. Dactolisib treatment led to a significant reduction in p-4EBP1 signals but not in the 4EBP1 protein signal. Marked heterogeneity in the treatment response was observed among the cell population. B. scPICO - corresponding AQ signal of target proteoforms. C. Bulk PICO assays - 20,000 cells in a 4 uL volume, measuring absolute quantitative amounts (AQ) of 4EBP1 and p-4EBP1 in U937 bulk lysate at 5e-10 M of antibody concentration, treated with λ-phosphatase (PPase) [6] or left untreated (mock). Untreated samples showed comparable 4EBP1 and p-4EBP1 AQ levels, while λ-phosphatase treatment significantly reduced the p-4EBP1 signal, as expected. D. Evaluation of the true heterogeneity of scPICO. p-4EBP1 AQ levels in U937 single cells were compared to equivalent amounts of bulk lysate, both analyzed using the scPICO workflow depicting the raw couplex signal (single-cell lysate mock). E. ip-ABC - in-plate negative antibody binding control, using 12 nL volume ABX processed analog to single cells in scPICO but no cell added demonstrating zero signal with no background.

Conclusion

The scPICO assay, powered by F.SIGHT and I.DOT, is transforming single-cell proteomics with its innovative approach to AQ. Its high sensitivity, accuracy, and ability to capture cellular heterogeneity make it invaluable for researchers in precision medicine and other fields that rely on detailed protein analysis.

Download the full application note here. 

For those interested in bringing unparalleled precision to their proteomics research, scPICO technology enhanced by CYTENA's F.SIGHT Single-Cell Dispenser and DISPENDIX's I.DOT Non-Contact Dispenser is a game-changer. Download the I.DOT brochure today!