Use of Quantitative, Multiplex Immunoassays for Improved Exploration of EGFR Signalling

The ErbB or epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases consists of four members: EGFR/ErbB1/HER1, ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4. The ErbB receptors play crucial roles in propagating signals regulating cell proliferation, differentiation, motility, and apoptosis, contributing to pathological processes such as cancer. The ability to analyse the phosphorylation status of ErbB family members, as well as the phosphorylation status of receptor-related intracellular signal transduction proteins, is necessary for a thorough understanding of this signalling pathway.

To examine the phosphorylation status of the ErbB family and receptor–related intracellular proteins, several assay platforms are available. The Western blot has been the gold standard used to study pathway signalling. Other platforms include ELISA, reverse phase arrays, high content analysis and mass spectroscopy. Although some of these platforms yield absolute quantitative data, the assay is either limited to measuring only one analyte at a time, or there is a high level of difficulty and expense.

This article will describe a quantitative approach to multiplex immunoassays that simultaneously measures both total protein abundance and

site-specific phosphorylation at the peptide level with picomolar sensitivity. Data showing use of this technology for exploration of signalling pathways affected by epidermal growth factor receptor inhibition are presented.