Chapter 17
Rapid Shotgun Phosphoproteomics Analysis
Mo ´ nica Carrera, Benito Can ˜ as, and Daniel Lopez-Ferrer
Abstract
In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The
strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by highintensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO 2 ) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid
chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer
(LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat
T-cells. Using this accelerated workflow, HIFU-TiO 2 -SCX-LC-MS/MS, 15,367 phosphorylation sites
from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently
identified in less than 15 h.
Key words Phosphoproteomics, High-intensity focused ultrasound (HIFU), Titanium dioxide
(TiO 2 ), Strong cation exchange chromatography (SCX), Proteomics, Mass spectrometry (MS), Jurkat
T-cells
1 Introduction
The rapid analysis of the global phosphoproteome profiling is
extremely important to obtain early answers to many biological
questions, such as the control of the activation/inhibition status
of specific protein activities, the understanding of different cellular
signaling networks, and the diagnosis of several diseases [1–3]. To
date, substantial efforts have been directed toward the identification and characterization of the maximum number of phosphorylation sites per experiment in the minimum possible time [4–6].
In this chapter, a new strategy for the rapid global phosphoproteome analysis of Jurkat T-cells is presented (Fig. 1). The proposed methodology is based on the use of (a) cell lysis and protein
extraction (time: 45 min), (b) accelerated in-solution tryptic digestion using HIFU (time: 10 min), (c) a single TiO 2 phosphopeptide
enrichment step (time: 90 min), (d) off-line fractionation of phosphopeptides using SCX (time: 60 min), (e) LC-MS/MS analysis on
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
259
Rapid Shotgun Phosphoproteomics Analysis
Mo ´ nica Carrera, Benito Can ˜ as, and Daniel Lopez-Ferrer
Abstract
In this chapter, we describe a rapid workflow for the shotgun global phosphoproteomics analysis. The
strategy is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field by highintensity focused ultrasound (HIFU) coupled to titanium dioxide (TiO 2 ) selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX), and analysis by liquid
chromatography-tandem mass spectrometry (LC-MS/MS) in a high-resolution mass spectrometer
(LTQ-Orbitrap XL). The strategy was optimized for the global phosphoproteome analysis of Jurkat
T-cells. Using this accelerated workflow, HIFU-TiO 2 -SCX-LC-MS/MS, 15,367 phosphorylation sites
from 13,029 different phosphopeptides belonging to 3,163 different phosphoproteins can be efficiently
identified in less than 15 h.
Key words Phosphoproteomics, High-intensity focused ultrasound (HIFU), Titanium dioxide
(TiO 2 ), Strong cation exchange chromatography (SCX), Proteomics, Mass spectrometry (MS), Jurkat
T-cells
1 Introduction
The rapid analysis of the global phosphoproteome profiling is
extremely important to obtain early answers to many biological
questions, such as the control of the activation/inhibition status
of specific protein activities, the understanding of different cellular
signaling networks, and the diagnosis of several diseases [1–3]. To
date, substantial efforts have been directed toward the identification and characterization of the maximum number of phosphorylation sites per experiment in the minimum possible time [4–6].
In this chapter, a new strategy for the rapid global phosphoproteome analysis of Jurkat T-cells is presented (Fig. 1). The proposed methodology is based on the use of (a) cell lysis and protein
extraction (time: 45 min), (b) accelerated in-solution tryptic digestion using HIFU (time: 10 min), (c) a single TiO 2 phosphopeptide
enrichment step (time: 90 min), (d) off-line fractionation of phosphopeptides using SCX (time: 60 min), (e) LC-MS/MS analysis on
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
259