One of the major additions in MS technology has been the irruption of the Orbitrap mass analyzer, which has boosted the proteomics analyses of biological complex samples since its introduction. Here, we took advantage of the capabilities of the new Orbitrap Fusion Lumos Tribrid mass spectrometer to assess the performance of different data-dependent acquisition methods for the identification and quantitation of peptides and phosphopeptides in single-shot analysis of human whole cell lysates. Our study explored the capabilities of tri-hibrid mass spectrometers for (phospho-) peptide identification and quantitation using different gradient lengths, sample amounts, and combinations of different peptide fragmentation types and mass analyzers. Moreover, the acquisition of the same complex sample with different acquisition methods resulted in the generation of a dataset to be used as a reference for further analyses, and a starting point for future optimizations in particular applications.

The CRG/UPF Proteomics Unit is part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)” supported by grant PT13/0001 of ISCIII and Spanish Ministry of Economy and Competitiveness. We acknowledge support of the Spanish Ministry of Economy and Competitiveness, “Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016” (CTQ2016‐80364‐P) “Centro de Excelencia Severo Ochoa 2013-2017” (SEV‐2012‐0208), and from “Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya” (2014SGR678).