Poster

  • P-III-0751

Single-molecule proteoform analysis of EGFR in cancer cells in response to therapeutic drug treatment

Presented in

New Technology: Non MS-based Proteomics

Poster topics

Authors

Zhengjian Zhang (San Carlos, CA / US), James Joly (San Carlos, CA / US), Vivek Budamagunta (San Carlos, CA / US), Ishwar Kohale (South San Francisco, CA / US), Daryl Bulloch (South San Francisco, CA / US), Mathew Rardin (South San Francisco, CA / US), Andreas Huhmer (San Carlos, CA / US), Greg Kapp (San Carlos, CA / US), Parag Mallick (San Carlos, CA / US)

Abstract

Alterations in both abundance and activation of the epidermal growth factor receptor (EGFR) have been correlatesd with poor prognosis in numerous cancers. Extensive studies of EGFR signaling have revealed dozens of phosphorylation sites that are believed to govern its role as a potent oncogene. Though numerous phosphorylation sites have been identified, the coordinated action of these sites within heterogeneous mixtures of proteoforms has yet to be elaborated. For example, it is not yet known how triple phosphorylation of EGFR at sites 1045,1086 and 1173 impacts cell behavior differently than triple phosphorylation of EGFR at sites 974, 992 and 1068. Here we describe the development and application of a high throughput single-molecule analysis platform to study EGFR proteoforms in cancer cells in response to ligand stimuli and drug treatments.

The goal of the assay is to quantify the abundances, with single-molecule resolution, of a discrete set of proteoforms of EGFR, as defined by a pre-determined set of site-specific post-translational modifications. This measurement is achieved through first immobilizing billions of individual protein molecules onto a patterned flow cell. In parallel, each molecule is iteratively probed with fluorescently labeled affinity reagents that bind to site-specific post-translational modifications. A computational framework, then takes these measurements, accounts for cross-reactivity, false-positive and false-negative binding and infers the abundance of each proteoform.

Titration studies have demonstrated that the assay is able to detect proteoforms that are present at less than 0.1% of the total population. To further enhance sensitivity, affinity purification strategies are used to enrich EGFR proteins from TCP 1016 lung cancer cells that have been treated with a monoclonal antibody therapeutic. The assay was first validated using in vitro phosphorylated recombinant EGFR (GST fusion of the intracellular domain, residuals 668-1210) as compared to un-phosphorylated control. Next, the assay was used to examine the impact of drug treatment on EGFR proteoform molecular heterogeneity.

Ultimately, this novel assay may enable a deeper understanding of the regulation and dysregulation of EGFR during oncogenesis and in response to therapeutic intervention.

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