When Protein Interaction Coupling (PICO) meets QIAcuity digital PCR
The current number of published protein-coding genes is 19,969 that are transcribed into 86,245 different transcripts encoding proteins1. These proteins, however, are not static bystanders in their environment but are part of a complex post-translationally regulated protein interaction network, also termed the interactome. But how do we study proteins and protein interactions nowadays? The main approaches for analytical protein detection, even today, are the workhorse methods, such as western blot and ELISA, co-immunoprecipitation (co-IP) and pull-down assays, as well as proximity assays such as proximity ligation assay (PLA) for protein interaction detection. All of these techniques have different advantages and disadvantages. Most of them, however, have limited sensitivity, high background noise, or lack options for parallel measurements. Finding the optimal technique is not easy.
Therefore, Actome developed the Protein Interaction Coupling (PICO) technology that translates complex protein status into DNA barcodes that can be amplified and detected using digital PCR on the QIAcuity system. PICO is an ultra-sensitive immunoassay for detecting and quantifying proteins, protein interactions, and post-translational modifications. The workflow is simple and enables parallel measurements.
In a recent webinar, Actome’s co-founder and the inventor of the PICO technology Dr. Csaba Jeney explained how they combine the advantages of immunoassays with digital PCR technology, by enabling a digital immunoassay that counts molecules. PICO enables previously unimaginable assays in the field of protein and interactomics studies.
To give you a glimpse, here are some of the frequently asked questions about this technology. If you couldn’t attend but would like to view the recording, you can sign up to watch it here.
What can PICO measure?
With PICO you can measure: 1) proteins, 2) protein interactions, and 3) post-translational modifications from any soluble biological sample. For a consistent nomenclature, we call them 'targets'. For the PICO assay, a pair of antibodies of your choice is required for each target.
What are the advantages of PICO compared to other protein detection assays?
PICO is designed to outperform existing protein detection and quantification solutions (e.g. western blot, co-immunoprecipitation, ELISA, or PLA) and to introduce powerful experimental possibilities:
- Ultra-high sensitivity: the limit of detection (LOD) of PICO is in the femtomolar range, meaning with mediocre antibodies you can detect 100 molecules per cell. To provide a practical example for such a low amount of target, 2 μl of lysed material containing 10,000 cells (less than a single 96-well amount of cells) can provide the necessary protein concentration that can be detected by PICO with high confidence. PICO can detect what has been undetectable to date.
- Zero background noise: Other immunoassays have background reactions (signal without antibody binding), which interfere with clear, sensitive measurements. PICO is free from it: during the dPCR step, the sample is compartmentalized to exclude the possibility of target-independent background reactions. Therefore, there is zero signal in case no target is present in the dPCR reaction.
- Parallelism: The QIAcuity dPCR System can detect five fluorescent colors meaning five different antibodies with different PICO DNA Labels. Thus, PICO offers extensive flexibility in designing parallel assays. For example, measurement of a phosphorylation-dependent protein interaction is possible in one PICO experiment, for which you need three PICO DNA-labeled antibodies.
What is a couplex?
A couplex is a target bound by two PICO DNA-labeled antibodies and is the molecular detection unit of the PICO assay. The couplexes are detected and counted using the QIAcuity Digital PCR System and the number of target molecules is calculated using Actome's web-based AMULATOR software.
How much more sensitive is PICO compared to a western blot?
The webinar highlights a study where we detected the oncoprotein HER2 in two different breast cancer cell lines, BT474 and MCF7. Using the therapeutic antibodies trastuzumab (TTZ) and pertuzumab (PTZ) for detection showed that PICO, in this example, is approximately 200-fold more sensitive than a western blot.
How much lab work is required for the PICO assay?
The PICO assay itself is a two-day process. On the first day, the sample is lysed and combined with the PICO DNA-labeled antibody mix for overnight incubation. The next day the sample is highly diluted and mixed with the dPCR Master Mix, followed by the dPCR run on the QIAcuity instrument.
Labeling of the antibodies with the PICO DNA Labels is an additional two days effort. However, it is necessary to label each pair of antibodies only once since antibody labeling generates sufficient material for hundreds of PICO assays. Once labeled, the antibodies are stable for at least 6 months at 4°C. Thus, the entire PICO workflow takes 4 days including antibody labeling.
What are the requirements/recommendations concerning antibodies?
For the PICO assay, a pair of antibodies is required for each target and the user is free to choose any antibody pair for their PICO assays. The PICO assay works with both monoclonal and polyclonal antibodies. However, we recommend using monoclonal antibodies, since they are often better characterized and defined by the antibody producer and the batch-to-batch variability is minimal. As the antibodies need to bind concurrently, choosing antibodies raised against different parts of the protein is recommended.
How stable are PICO DNA-labeled antibodies?
The PICO DNA-labeled antibodies are stable for at least six months at 4°C.
What kinds of samples can be used in a PICO assay?
In general, every liquid, molecularly dispersed sample can be analyzed with the PICO assay, e.g. cell lysates, supernatant, blood serum, etc. The PICO Amplification Core Kit contains a lysis buffer that is recommended for cell culture samples.
How does data quantification work?
Data analysis is performed using Actome’s web-based AMULATOR software. First, the raw dPCR data is exported from the QIAcuity Software Suite. Together with a Sample Definition file provided by Actome, it is uploaded to AMULATOR for data analysis and basic statistical evaluation.
Where can I purchase PICO products?
The PICO kits are available for purchase in Actome’s webshop.*
Reference:
1. Nurk S et al., (2022) The complete sequence of a human genome. Science 376, 44-53
* Through our webshop, PICO products are currently only available in Europe. Please get in touch with Actome to find out about product availability in the rest of the world.