Bekker-Jensen, DBMartínez-Val, ASteigerwald, SRüther, PFort, KLArrey, TNHarder, AMakarov, AOlsen, J2026-02-252026-02-252020-04-30Mol Cell Proteomics. 2020 Apr;19(4):716-729.https://hdl.handle.net/20.500.12105/27273State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.Part of this work has been funded as part of the MSmed project that has received funding from the European Union's Horizon 2020 Research and Innovation program under grant agreement no. 686547 and as part of EPIC-XS project under the grant agreement no. 823839. Work at The Novo Nordisk Foundation Center for Protein Research (CPR) is funded in part by a generous donation from the Novo Nordisk Foundation (NNF14CC0001). J.V.O. and P.L.R. are supported by the Marie Sklodowska Curie European Training Network "TEMPERA" (grant number 722606). The authors declare the following competing financial interest(s): K.L.F., T.N.A., A.H., and A.A.M. are employees of Thermo Fisher Scientific, the manufacturer of the Orbitrap Exploris 480 MS instrument used in this research. The authors have declared a conflict of interest.engVoRhttp://creativecommons.org/licenses/by-nc-nd/4.0/Attribution-NonCommercial-NoDerivatives 4.0 InternationalMOLECULAR & CELLULAR PROTEOMICSopen access