on 06-14-201903:06 AM - edited on 10-15-202111:45 AM by Closed Account
Aaron S. Gajadhar1, Lauren E. Stopfer2, Cameron T. Flower2, Forest M. White2, Bhavin Patel3, Sebastien Gallien4, Romain Huguet1, Graeme McAlister1, Derek Bailey1, Shannon Eliuk1, Markus Kellmann5, Tabiwang N. Arrey5, Alexander Harder5, Daniel Lopez-Ferrer1, Andreas Huhmer1.
Purpose: We sought to develop a universal internal standard (IS)-guided LC-MS acquisition workflow for reproducible, sensitive and high-density absolute quantification of phosphotyrosine (pTyr) signaling.
Methods: We leveraged two-stage selective phosphotyrosine peptide enrichment, isotopically-labeled trigger peptides and the Thermo Scientific™ SureQuant™ IS targeted protein quantitation method to detect and quantify 356 pTyr targets per analysis using Thermo Scientific™ Orbitrap Exploris™ 480 and Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ Mass Spectrometers.
Results: Compared to traditional discovery-based proteomics approaches for pTyr analysis, the SureQuant pTyr acquisition workflow delivered more reproducible quantitation profiles across samples along with the ability to determine absolute concentrations of measured signaling components with high sensitivity.
Thermo Fisher Scientific, San Jose1, CA, USA; Rockford3, IL, USA; PMSC4, Cambridge, MA, USA; Bremen, Germany5; Department of Biological Engineering2, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA