Turnkey, Multi-pathway Signaling Analysis Using a Synthetic Phosphopeptide Panel, Standardized Sample Preparation Kits and SureQuant Internal Standard Targeted Quantitation

[Orbitrap_SciLib]

Aaron S. Gajadhar1; Bhavin Patel2; Penny Jensen2; Sebastien Gallien3,4; Romain Huguet1; Kay Opperman2; John C Rogers2; Andreas Huhmer1; Daniel Lopez-Ferrer1
ASMS 2020
Purpose: We sought to develop a universal phosphopeptide enrichment and internal standard (IS)-guided LC-MS acquisition workflow for reproducible, sensitive and high-density absolute quantification of biologically relevant phosphorylation sites in multiple biological pathways. Methods: We leveraged Sequential Metal Oxide Affinity Chromatography (SMOAC) for selective phosphopeptide enrichment, isotopically-labeled trigger peptides and the Thermo Scientific™ SureQuant™ IS targeted protein quantitation method to detect and quantify 138 pSTY targets per analysis using Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometers. Results: Compared to traditional discovery-based proteomics approaches for pSTY analysis, the SureQuant multi-pathway phosphopeptide acquisition workflow outperformed DDA and PRM for detection of the multi-pathway phosphopeptides panel.


1Thermo Fisher Scientific, San Jose, CA; 2Thermo Fisher Scientific, Rockford, IL; 3Thermo Fisher Scientific, PMSC, Cambridge, MA; 4Thermo Fisher Scientific, Paris, France