Digital proteomics by DNA sequencing

The simultaneous measurement of many different proteins presents a major challenge to the fields of surgical and clinical pathology, and there is a genuine need for the development of a more direct, quantitative approach to multiplex analysis of proteins in tissues and clinical assays. Here we present a novel, high-throughput immunoassay, driven by the hypothesis that highly sensitive, multiplex protein characterization can be obtained by coupling the unique specificity of antibodies with massively parallel sequencing to detect and quantitate antigens from formalin-fixed, paraffin-embedded tissue (FFPE) and in solution. The method involves conjugating antibodies with a universal anchor oligonucleotide that is hybridized with a second oligonucleotide specifying the sample, antibody, and a unique molecule identifier, akin to “make, model and serial” number. Recovery of the data-rich oligonucleotide from mixtures of antibodies from samples such as histological tissue sections, (herein breast carcinoma) or extracts in an ELISA-type format, enabling error free-amplification that increases the sensitivity and expands the range of analysis. Overall design: To profile the abundance of select targets in breast carcinoma, libraries of antibody-oligonucleotide heteroconjugates of defined stoichiometry were constructed for ER, PR, Her2, and EGFR. Each conjugate was tagged with a barcode of nucleotides providing metadata to the cognate antigen for the antibody as well as for each antibody molecule in the library. Breast carcinoma tissue arrays were incubated with mixtures of different antibody libraries. Additionally, target antigens were evaluated in an ELISA-like assay. Following antibody binding in either case, the barcoded oligonucleotides were purified, amplified by PCR, and sequenced. The number of unique sequence identifiers was used to determine the relative amounts of each protein. Routine immunohistochemistry (IHC) and ELISAs were performed in parallel to sequencing experiments. The oligonucleotides_barcodes that used for identifying target unique oligios for this study are provided in the oligonucleotides_barcodes.txt.