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Article
Fast Global Phosphoproteome Profiling of Jurkat T cells by HIFU-TiO-SCX-LC-MS/MS 2
Mónica Carrera, Benito Canas, and Daniel Lopez-Ferrer Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01321 • Publication Date (Web): 08 Aug 2017 Downloaded from http://pubs.acs.org on August 15, 2017
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Analytical Chemistry
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Fast Global Phosphoproteome Profiling of
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Jurkat T cells by
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HIFU-TiO2-SCX-LC-MS/MS
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Mónica Carrera1*, Benito Cañas2, Daniel Lopez-Ferrer3
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Spanish National Research Council (CSIC), 36208, Vigo, Spain
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Complutense University of Madrid (UCM), 28040, Madrid, Spain
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3
Thermo Fisher Scientific, 95134, San Jose, California, USA
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*
CORRESPONDING AUTHOR: Dr. Mónica Carrera
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Spanish National Research Council (CSIC). Eduardo Cabello 6, 36208 Vigo, Pontevedra,
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Spain. Tel.: +34 986 231930. Fax: +34 986 292762.
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E-mail:
[email protected] 9 10
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Analytical Chemistry
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ABSTRACT
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We propose a new workflow for fast phosphoproteome profiling. The workflow is
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based on the use of accelerated in-solution trypsin digestion under an ultrasonic field provided
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by high-intensity focused ultrasound (HIFU) combined with an inverse strategy based on
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TiO2 selective phosphopeptide enrichment, fractionation by strong cation exchange
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chromatography (SCX) and analysis by liquid chromatography tandem mass spectrometry
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(LC-MS/MS) using a high-resolution mass spectrometer. The performance of the method was
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established for the global phosphoproteome analysis of un-stimulated human Jurkat leukemia
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T cells (E6.1). Using this accelerated workflow, 15,367 phosphorylation sites from 13,029
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different phosphopeptides belonging to 3,163 different phosphoproteins were efficiently
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identified with high-throughput and reproducibility in less than 15 h. The functional analysis
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revealed significant phosphorylation-based networks that are implicated in immune function
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and tumor development pathways. The present strategy, HIFU-TiO2-SCX-LC-MS/MS, is the
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fastest analytical method reported to date for generating large-scale phosphoproteomics
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datasets (48 hours
Peptide Digestion
Time
Stimulated with or without PGE Multiple proteases (AspN, Chym., GluC, LysC, and Trypsin) Ti4+-IMAC LTQ-Orbitrap ELITE (CID and ETD) MS-GF+ 37,771 (13,476 trypsin) 18,430 5,326 (3,519 trypsin) >48 hours
Nguyen, 2016 Human Jurkat T cells E6.1
Carrera et al. Human Jurkat T cells E6.1
Stimulated with anti-CD3/anti-CD28 at ≠ times Trypsin 18 hours
Un-stimulated
Other Humphrey, 2015 Different mouse liver cell lines (Hepa 1-6, FL83B and HeLa S3) Stimulated with insulin at ≠ times TFE-based tryptic digestion 18 hours TiO2 in DWP wells Q-Exactive (HCD) MaxQuant
SCX-IMAC-TiO2
HIFU-based tryptic digestion 10 min TiO2-SCX
LTQ-Orbitrap XL (HCD and CID) SEQUEST+OMSSA +EasyProt 4,346
LTQ-Orbitrap XL (CID) BYONICTM+SEQUEST HTTM 13,029
--1,690
15,367 3,163
>10,000 ---
>48 hours
