Sebastien Gallien1,2, Aaron Gajadhar3, Bhavin Patel4, Tabiwang Arrey5, Dave Sarracino2, Sarah Trusiak2, Yue Xuan2,5, Emily Chen2
Purpose: High-throughput mass spectrometry-based protein assays were developed, based on multiplex-immunoprecipitation, fast LC separations, and advanced PRM acquisition schemes, to support signaling pathway analysis. They were applied to the monitoring of the main protein components of AKT/mTOR signaling pathway, under“total-” or “phosphorylated-” forms.
Methods: The analyses were performed on a Thermo Scientific™ Q Exactive™ HF-X hybrid quadrupole-Orbitrap™ mass spectrometer and a Thermo Scientific™ Q Exactive™ HF hybrid quadrupole-Orbitrap mass spectrometer operated with several PRM-based acquisition schemes (using instrument programming interface in some cases). Chromatographic separations were carried out using an Evosep One system and a Thermo Scientific™ UltiMate™ 3000 RSLC system equipped for capillary flow. Various gradient lengths and MS acquisition parameter settings were employed to analyze samples of high complexity, e.g., digests of human cell lines, and samples of low complexity obtained through multiplexed immunoprecipitation targeting proteins of AKT/mTOR pathway.
Results: The developed set-ups exhibited the ability to quantify with high sensitivity several dozens of endogenous peptides in one hundred samples within one day under high efficiency acquisition modes. Advanced PRM methods allowed further increases in analytical throughput without compromising the quality of quantification data when combined with multiplexed immunoprecipitation, and minor sensitivity decrease without enrichment.
1 Thermo Fisher Scientific, Paris, France
2 Thermo Fisher Scientific, PMSC, Cambridge, MA
3 Thermo Fisher Scientific, San Jose, CA
4 Thermo Fisher Scientific, Rockford, IL
5 Thermo Fisher Scientific, Bremen, Germany High
United States
