on 06-14-201901:54 AM - edited on 10-15-202111:45 AM by AnalyteGuru
Sebastien Gallien1,2, Jing Wang2, Aaron S. Gajadhar3, Bhavin Patel4, Markus Kellmann5, Tabiwang N. Arrey5, Alexander Harder5, Romain Huguet3, Graeme McAlister3, Derek Bailey3, Shannon Eliuk3, Yue Xuan5, Andreas Huhmer3, Emily L. Chen2
ASMS 2019 Poster Purpose: A novel global plasma proteome quantification workflow has been developed. It relies on a new high density targeted acquisition method, called ‘SureQuant’ method, implemented on next generation Orbitrap mass spectrometers: Thermo ScientificTM Orbitrap ExplorisTM 480 mass spectrometer and Thermo ScientificTM Orbitrap EclipseTM TribridTM mass spectrometer. In addition, the workflow leverages a large set of stable isotopically labeled (SIL) peptides, used as internal standards, to drive the systematic screening of more than 500 plasma proteins (PQ500 kit from Biognosys).
Methods: The SureQuant PQ500 analyses of undepleted plasma samples were performed using Orbitrap Exploris 480 and Orbitrap Eclipse Tribrid mass spectrometers, coupled to Thermo ScientificTM UltiMateTM 3000 RSCLC system or Thermo ScientificTM EASY-nLCTM 1200 system. An acquisition variant of the internal standard triggered targeted acquisition, dedicated to higher throughput analyses, was executed through an application programming interface (API) controlling a Thermo ScientificTM Q ExactiveTM HF-X hybrid quadrupole-OrbitrapTM mass spectrometer coupled to a Evosep One LC system. PQ500 kit from Biognosys was spiked into the plasma samples to drive the targeted acquisition.
Results: The SureQuant method has been adapted from the internal standard triggered PRM (IS-PRM) method in order to improve its usability and robustness, while retaining its analytical performance, and has been implemented in the native instrument control software of next generation Orbitrap mass spectrometers. The application of the method to the analysis of plasma samples, leveraging spiked-in SIL peptides from PQ500 kit, allowed robust and precise quantification of around 560 endogenous peptides used as surrogate of 400 plasma proteins using a 70-min LC gradient. Therefore, the method combined data quality of targeted analyses with proteome coverage of state-of-the-art profiling experiments. In addition, the availability of pre-set PQ500 SureQuant methods embedded into the instrument control software enables a ‘load and play’ execution of such plasma proteomics experiments.
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