Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography

María Ibáñez-Vea, Honggang Huang, Xabier Martínez De Morentin, Estela Pérez, Maria Gato, Miren Zuazo, Hugo Arasanz, Joaquin Fernández-Irigoyen, Enrique Santamaría, Gonzalo Fernandez-Hinojal, Martin R. Larsen, David Escors, Grazyna Kochan*

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Resumé

Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO2) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

OriginalsprogEngelsk
TidsskriftJournal of Proteome Research
Vol/bind17
Udgave nummer3
Sider (fra-til)1172-1182
ISSN1535-3893
DOI
StatusUdgivet - 2018

Fingeraftryk

Organophosphonates
Macrophages
Chromatography
Cysteine
Protein S
Proteome
Labeling
Mass spectrometry
Nitric Oxide
Proteins
Cells
Throughput
Peptides
Cell Line
Oxidation-Reduction

Citer dette

Ibáñez-Vea, María ; Huang, Honggang ; Martínez De Morentin, Xabier ; Pérez, Estela ; Gato, Maria ; Zuazo, Miren ; Arasanz, Hugo ; Fernández-Irigoyen, Joaquin ; Santamaría, Enrique ; Fernandez-Hinojal, Gonzalo ; Larsen, Martin R. ; Escors, David ; Kochan, Grazyna. / Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography. I: Journal of Proteome Research. 2018 ; Bind 17, Nr. 3. s. 1172-1182.
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title = "Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography",
abstract = "Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO2) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.",
keywords = "immune system, macrophages, post-translational modifications (PTMs), proteomics, S-nitrosylation",
author = "Mar{\'i}a Ib{\'a}{\~n}ez-Vea and Honggang Huang and {Mart{\'i}nez De Morentin}, Xabier and Estela P{\'e}rez and Maria Gato and Miren Zuazo and Hugo Arasanz and Joaquin Fern{\'a}ndez-Irigoyen and Enrique Santamar{\'i}a and Gonzalo Fernandez-Hinojal and Larsen, {Martin R.} and David Escors and Grazyna Kochan",
year = "2018",
doi = "10.1021/acs.jproteome.7b00812",
language = "English",
volume = "17",
pages = "1172--1182",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "3",

}

Ibáñez-Vea, M, Huang, H, Martínez De Morentin, X, Pérez, E, Gato, M, Zuazo, M, Arasanz, H, Fernández-Irigoyen, J, Santamaría, E, Fernandez-Hinojal, G, Larsen, MR, Escors, D & Kochan, G 2018, 'Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography', Journal of Proteome Research, bind 17, nr. 3, s. 1172-1182. https://doi.org/10.1021/acs.jproteome.7b00812

Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography. / Ibáñez-Vea, María; Huang, Honggang; Martínez De Morentin, Xabier; Pérez, Estela; Gato, Maria; Zuazo, Miren; Arasanz, Hugo; Fernández-Irigoyen, Joaquin; Santamaría, Enrique; Fernandez-Hinojal, Gonzalo; Larsen, Martin R.; Escors, David; Kochan, Grazyna.

I: Journal of Proteome Research, Bind 17, Nr. 3, 2018, s. 1172-1182.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography

AU - Ibáñez-Vea, María

AU - Huang, Honggang

AU - Martínez De Morentin, Xabier

AU - Pérez, Estela

AU - Gato, Maria

AU - Zuazo, Miren

AU - Arasanz, Hugo

AU - Fernández-Irigoyen, Joaquin

AU - Santamaría, Enrique

AU - Fernandez-Hinojal, Gonzalo

AU - Larsen, Martin R.

AU - Escors, David

AU - Kochan, Grazyna

PY - 2018

Y1 - 2018

N2 - Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO2) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

AB - Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO2) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

KW - immune system

KW - macrophages

KW - post-translational modifications (PTMs)

KW - proteomics

KW - S-nitrosylation

U2 - 10.1021/acs.jproteome.7b00812

DO - 10.1021/acs.jproteome.7b00812

M3 - Journal article

C2 - 29338241

AN - SCOPUS:85042863606

VL - 17

SP - 1172

EP - 1182

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 3

ER -