Sebastien Gallien1,2, Aaron S. Gajadhar3, Bhavin Patel4, Markus Kellmann5, Tabiwang N. Arrey5, Christian Thoeing5, Alexander Harder5, Romain Huguet3, Graeme McAlister3, Derek Bailey3, Shannon Eliuk3, Emily L. Chen2, Yue Xuan5, Andreas Huhmer3
Purpose: A novel internal standard triggered targeted quantification workflow, called ‘SureQuant’ workflow (Figure 1), has been developed and implemented in the native instrument control software of novel Orbitrap mass spectrometers: Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer and Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer. It aims at enabling large-scale target profiling with superior quantitative performance, robustness, and usability.
Methods: The SureQuant internal standard triggered-PRM (IS-PRM) acquisition scheme was developed and tested on Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid mass spectrometers. Chromatographic separations were carried out using Thermo Scientific™ UltiMate™ 3000 RSCLC system and Thermo Scientific™ EASY-nLC™ 1200 system. Thermo Scientific™ Pierce™ SureQuant™ AKT Pathway Multiplex Panel kit and PQ500 kit from Biognosys were used to evaluate SureQuant workflows applied to the analyses of cancer cell line models and undepleted plasma samples.
Results: The novel SureQuant IS-PRM method significantly enhanced targeted data acquisition efficiency in comparison with conventional PRM method (i.e., 80-90% of the instrument time dedicated to the collection of pertinent data), and exhibited strong robustness against chromatographic variations. Applied to the analyses of a variety of samples supplemented with different sets of stable isotopically labeled (SIL) peptides used as internal standards (IS) in various formats (30-800 IS), the method demonstrated the capability to quantify endogenous peptides in the low amol range at all scales. In addition, Embedding pre-set methods, associated with predefined kits of IS peptides, directly into the instrument control software turned out to be a decisive step towards the provision of turnkey targeted proteomics solutions.
1Thermo Fisher Scientific, Paris, France; 2Thermo Fisher Scientific, Precision Medicine Science Center, Cambridge, MA; 3Thermo Fisher Scientific, San Jose, CA; 4Thermo Fisher Scientific, Rockford, IL; 5Thermo Fisher Scientific, Bremen, Germany