A new strategy for the Rapid Phosphoproteomics Analysis is presented in this work (Figure 1). The performance of the method was established for the global phosphoproteome analysis of un-stimulated human Jurkat leukemia T cells (E6.1). The proposed method is based on six main steps: (a) cell lysis and protein extraction (time: 45 min), (b) in-solution trypsin digestion accelerated under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) (time: 10 min), (c) a single step of phosphopeptide enrichment using TiO2 (time: 90 min), (d) fractionation by strong cation exchange chromatography (SCX) (time: 60 min), (e) analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a high-resolution LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) (time: 60 min/run), and (f) data analysis using BYONIC and SEQUEST-HT (Proteome Discoverer 2.1, Thermo Fisher Scientific) (time: 60 min). Using this accelerated workflow, 15,367 phosphorylation sites from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins were efficiently identified with high-throughput and reproducibility in less than 15h [1]. Results demonstrates that the present strategy, HIFU-TiO2-SCX-LC-MS/MS, is the fastest analytical method reported to date for generating reproducible large-scale phosphoproteomics datasets in a limited time (<15h)

This work was supported by the EU Marie Curie actions (FP7-PEOPLE-2012- IEF, ref 332274) and by the Ramón Areces Foundation (XVII National Grant)

6th International Congress on Analytical Proteomics, Caparica, 8th-11th July 2019

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