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Fast phosphoproteomics by HIFU-TiO2-SCX-LC-MS/MS

AuthorsCarrera, Mónica ; Cañas, Benito; López-Ferrer, Daniel
Issue Date2019
Citation6th International Congress on Analytical Proteomics (2019)
AbstractA 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)
Description6th International Congress on Analytical Proteomics, Caparica, 8th-11th July 2019
Appears in Collections:(IIM) Comunicaciones congresos
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