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Fast Global Phosphoproteome Profiling of Jurkat T cells by HIFU-TiO2-SCX-LC-MS/MS

AuthorsCarrera, Mónica ; Cañas, Benito; López-Ferrer, Daniel
High-intensity focused ultrasound (HIFU)
Strong cation exchange chromatography (SCX)
Mass spectrometry (MS)
Human Jurkat leukemia T cells
Issue Date2017
PublisherAmerican Chemical Society
CitationAnalytical Chemistry - Columbus 89(17): 8853-8862 (2017)
AbstractWe propose a new workflow for fast phosphoproteome profiling. The workflow is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) combined with an inverse strategy based on TiO2 selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX) and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a high-resolution mass spectrometer. The performance of the method was established for the global phosphoproteome analysis of un-stimulated human Jurkat leukemia T cells (E6.1). 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 15 h. The functional analysis revealed significant phosphorylation-based networks that are implicated in immune function and tumor development pathways. The present strategy, HIFU-TiO2-SCX-LC-MS/MS, is the fastest analytical method reported to date for generating large-scale phosphoproteomics datasets (<15 h)
Description10 pages, 6 figures, 1 table
Publisher version (URL)https://doi.org/10.1021/acs.analchem.7b01321
Appears in Collections:(IIM) Artículos
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