Abstract 223: Targeting critical signaling nodes using multiplexed antibody based phosphopeptide enrichment with iMRM validation
Authors: Farnsworth Charles L. , Zhu Yiying , Stokes Matthew 
Published: July 2017

Abstract
Introduction: A challenge for biomedical researchers is to develop assays to analyze complex systems that interrogate whole cellular signaling networks. Here we show the results of employing antibody based immunoaffinity enrichment of phosphopeptides derived from critical signaling nodes to quantitate changes in phosphorylation upon kinase inhibitor treatment. Human cancer cell lines, including gastric and breast adenocarcinomas were treated with kinase inhibitors and cellular extracts were digested with trypsin and peptides were purified. A pool of phosphorylation dependent site-specific antibodies was employed to enrich for known sites of phosphorylation. Following antibody based enrichment these peptides were identified and quantified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Phosphopeptides were identified using SEQUEST, and differences in abundance were determined by employing label-free MS1 based quantification using Progenesis from Non-Linear Dynamics. We also show validation data for a number of the antibodies employed using immuno-multiple reaction monitoring, or iMRM. Employing a QExactive mass Spectrometer (Thermo Scientific) we developed calibration curves for a number of the targeted tryptic phosphopeptides using combinations of heavy and light peptides with endogenous cancer cell line peptides as a background matrix.
Experimental Results: Cell lines were treated with DMSO or inhibitors to which the cell lines should be sensitive/insensitive. Following peptide purification, a pool of antibodies were multiplexed and used to enrich for phosphopeptides derived from signaling proteins across a number of signaling pathways. The results show significant changes in abundance for many proteins/sites, including activation loop tyrosine residues of tyrosine kinases, changes in sites on proteins in the AKT/PI3K pathway, sites in the MAPK pathway, and other critical regulators of cellular signaling such as sites on PKC family members. Targeted LC-MS/MS methods using iMRM were used to confirm these results and show the promise of using such targeted strategies for quantifying changes in phosphorylation consistently across many samples.
Conclusion:The strategy of multiplexing antibodies for immuno-enrichment allows for simultaneous detection and quantification of hundreds to thousands of known phosphorylation sites that have been shown to be important nodes of regulation. Targeted LC-MS/MS methods such as iMRM will allow researchers to confidently profile changes in both total levels as well as phosphorylation site occupancy for critical signaling proteins.
Citation Format: Charles L. Farnsworth, Yiying Zhu, Matthew Stokes. Targeting critical signaling nodes using multiplexed antibody based phosphopeptide enrichment with iMRM validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 223. doi:10.1158/1538-7445.AM2017-223