5. Using the present rapid shotgun phosphoproteomics workflow
(HIFU-TiO 2 -SCX-LC-MS/MS), 15,367 phosphorylation
sites from 13,029 different phosphopeptides belonging to
3,163 phosphoproteins from Jurkat T-cells can be identified
in less than 15 h. To the best of our knowledge, the present
dataset is the largest phosphoproteomics dataset compiled to
date for a particular cellular sample in a limited time frame.
6. Due to its easy and rapid nature, the method presented here
might be applied in parallel for sample preparation with mass
spectrometers of new generation to increase the number of
phosphopeptide identifications and reduce the time of analysis.
In consequence, the present strategy might be applied to analyze the global phosphoproteome profiles of any control or
pathological biological tissue or cell type in a limited time
frame.
Acknowledgments
The authors wish to express their gratitude to the proteomics
laboratory at the Institute of Molecular Systems Biology (IMSB),
Zu ¨rich, Switzerland, mainly to Prof. Dr. Ruedi Aebersold and
Dr. Matthias Gstaiger, for allowing them to perform the
LTQ-Orbitrap XL analysis in their facilities and for their excellent
scientific recommendations. This work was supported by the EU
Marie Curie actions (FP7-PEOPLE-2012-IEF, ref. 332274), by
the Ramo ´ n Areces Foundation (XVII National grant), by the
GAIN-Xunta de Galicia Project (IN607D 2017/01) and by the
Spanish AEI/EU-FEDER (PID2019-103845RB-C21) project.
References
1. de Graauw M, Hensbergen P, van de Water B
(2006) Phospho-proteomic analysis of cellular
signaling. Electrophoresis 27:2676–2686
2. Ptacek J, Snyder M (2006) Charging it up:
global analysis of protein phosphorylation.
Trends Genet 22:545–554
3. Chong PK, Lee HY, Kong JWF, Loh MCS,
Wong CH, Lim YP (2008) Phosphoproteomics, oncogenic signaling and cancer research.
Proteomics 8:4370–4382
4. Macek B, Mann M, Olsen JV (2009) Global
and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev
Pharmacol Toxicol 49:199–221
5. Humphrey SJ, Karayel O, James DE, Mann M
(2018) High-throughput and high-sensitivity
phosphoproteomics with the EasyPhos platform. Nat Protoc 13:1897–1916
6. Carrera M, Can ˜as B, Lopez-Ferrer D (2017)
Fast global phosphoproteome profiling of Jurkat T cells by HIFU-TiO 2 -SCX-LC-MS/MS.
Anal Chem 89:8853–8862
7. Carrera M, Can ˜as B, Lo ´ pez-Ferrer D,
Pin ˜eiro C, Va ´zquez J, Gallardo JM (2011)
Fast monitoring of species-specific peptide biomarkers using high-intensity-focused-ultrasound-assisted tryptic digestio ´ n and selected
MS/MS ion monitoring. Anal Chem
83:5688–5695
8. Bodenmiller B, Aebersold R (2010) Quantitative analysis of protein phosphorylation on a
system-wide scale by mass spectrometry-based
proteomics. Methods Enzymol 470:317–334
9. Beausoleil SA, Jedrychowski M, Schwartz D,
Elias JE, Villen J, Li J, Cohn MA, Cantley LC,
Gygi SP (2004) Large-scale characterization of
Rapid Shotgun Phosphoproteomics Analysis
267
(HIFU-TiO 2 -SCX-LC-MS/MS), 15,367 phosphorylation
sites from 13,029 different phosphopeptides belonging to
3,163 phosphoproteins from Jurkat T-cells can be identified
in less than 15 h. To the best of our knowledge, the present
dataset is the largest phosphoproteomics dataset compiled to
date for a particular cellular sample in a limited time frame.
6. Due to its easy and rapid nature, the method presented here
might be applied in parallel for sample preparation with mass
spectrometers of new generation to increase the number of
phosphopeptide identifications and reduce the time of analysis.
In consequence, the present strategy might be applied to analyze the global phosphoproteome profiles of any control or
pathological biological tissue or cell type in a limited time
frame.
Acknowledgments
The authors wish to express their gratitude to the proteomics
laboratory at the Institute of Molecular Systems Biology (IMSB),
Zu ¨rich, Switzerland, mainly to Prof. Dr. Ruedi Aebersold and
Dr. Matthias Gstaiger, for allowing them to perform the
LTQ-Orbitrap XL analysis in their facilities and for their excellent
scientific recommendations. This work was supported by the EU
Marie Curie actions (FP7-PEOPLE-2012-IEF, ref. 332274), by
the Ramo ´ n Areces Foundation (XVII National grant), by the
GAIN-Xunta de Galicia Project (IN607D 2017/01) and by the
Spanish AEI/EU-FEDER (PID2019-103845RB-C21) project.
References
1. de Graauw M, Hensbergen P, van de Water B
(2006) Phospho-proteomic analysis of cellular
signaling. Electrophoresis 27:2676–2686
2. Ptacek J, Snyder M (2006) Charging it up:
global analysis of protein phosphorylation.
Trends Genet 22:545–554
3. Chong PK, Lee HY, Kong JWF, Loh MCS,
Wong CH, Lim YP (2008) Phosphoproteomics, oncogenic signaling and cancer research.
Proteomics 8:4370–4382
4. Macek B, Mann M, Olsen JV (2009) Global
and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev
Pharmacol Toxicol 49:199–221
5. Humphrey SJ, Karayel O, James DE, Mann M
(2018) High-throughput and high-sensitivity
phosphoproteomics with the EasyPhos platform. Nat Protoc 13:1897–1916
6. Carrera M, Can ˜as B, Lopez-Ferrer D (2017)
Fast global phosphoproteome profiling of Jurkat T cells by HIFU-TiO 2 -SCX-LC-MS/MS.
Anal Chem 89:8853–8862
7. Carrera M, Can ˜as B, Lo ´ pez-Ferrer D,
Pin ˜eiro C, Va ´zquez J, Gallardo JM (2011)
Fast monitoring of species-specific peptide biomarkers using high-intensity-focused-ultrasound-assisted tryptic digestio ´ n and selected
MS/MS ion monitoring. Anal Chem
83:5688–5695
8. Bodenmiller B, Aebersold R (2010) Quantitative analysis of protein phosphorylation on a
system-wide scale by mass spectrometry-based
proteomics. Methods Enzymol 470:317–334
9. Beausoleil SA, Jedrychowski M, Schwartz D,
Elias JE, Villen J, Li J, Cohn MA, Cantley LC,
Gygi SP (2004) Large-scale characterization of
Rapid Shotgun Phosphoproteomics Analysis
267