Lossless Single-Molecule Counting To Absolute Quantify Proteoforms

A novel immunoassay, termed Protein Interaction Coupling (PICO), is introduced to deliver unequivocal, reference-free quantification of proteoforms - absolute quantification. PICO employs a compartmentalized, homogeneous single-molecule assay with a lossless and highly sensitive signal generation, capable of detecting down to a few molecules per reaction. Additionally, it utilises a background-free, digital enumeration principle, known as the decouplexing. PICO is presented as exact mathematical theories, providing a theoretical comprehension of its chemistry. Consequently, PICO demonstrates absolute quantification, as exemplified with recombinant and non-recombinant ErbB2 and multi-tagged peptide rTRX targets, validating absolute quantification against internal and external references in both analytical and cellular matrices. Furthermore, PICO enables combinatorial multiplexing (cplex), a readout between any two antibodies, demonstrated by an 8-plex antibody, 12-cplex PICO, measuring functional changes of ErbB pathway upon mock and dactolisib treatment delivering absolute quantitative cellular stoichiometry. PICO possesses immense potential for versatile, standardized, and accurate protein measurements, offering insights into physiological and perturbed cellular processes.