Analysis of protein chlorination by mass spectrometry

Tina Nybo, Michael J. Davies, Adelina Rogowska-Wrzesinska*

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Resumé

Chlorination of tyrosine is a commonly known effect/consequence of myeloperoxidase activity at sites of inflammation, and detection of 3-chlorotyrosine has been used as biomarker for inflammatory diseases. However, few studies have addressed site specific chlorination in proteins, and no methods for large scale chloroproteomics studies have yet been published. In this study, we present an optimized mass spectrometry based protocol to identify and quantify chlorinated peptides from single proteins modified by HOCl (100 and 500 μM, within estimated pathophysiological levels), at a high level of sensitivity and accuracy. Particular emphasis was placed on 1) sensitive and precise detection of modification sites, 2) the avoidance of loss or artefactual creation of modifications, 3) accurate quantification of peptide abundance and reduction of missing values problem, 4) monitoring the dynamics of modification in samples exposed to different oxidant concentrations and 5) development of guidelines for verification of chlorination sites assignment. A combination of an optimised sample preparation protocol, and improved data analysis approaches have allowed identification of 33 and 15 chlorination sites in laminin and fibronectin, respectively, reported in previous manuscripts [1,2]. The method was subsequently tested on murine basement membrane extract, which contains high levels of laminin in a complex mixture. Here, 10 of the major chlorination sites in laminin were recapitulated, highlighting the utility of the method in detecting damage in complex samples.

OriginalsprogEngelsk
Artikelnummer101236
TidsskriftRedox Biology
Vol/bind26
Antal sider10
ISSN2213-2317
DOI
StatusUdgivet - 1. sep. 2019

Fingeraftryk

Chlorination
Mass spectrometry
Laminin
Proteins
Network protocols
Peptides
Manuscripts
Biomarkers
Complex Mixtures
Fibronectins
Oxidants
Peroxidase
Tyrosine
Guidelines
Monitoring

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title = "Analysis of protein chlorination by mass spectrometry",
abstract = "Chlorination of tyrosine is a commonly known effect/consequence of myeloperoxidase activity at sites of inflammation, and detection of 3-chlorotyrosine has been used as biomarker for inflammatory diseases. However, few studies have addressed site specific chlorination in proteins, and no methods for large scale chloroproteomics studies have yet been published. In this study, we present an optimized mass spectrometry based protocol to identify and quantify chlorinated peptides from single proteins modified by HOCl (100 and 500 μM, within estimated pathophysiological levels), at a high level of sensitivity and accuracy. Particular emphasis was placed on 1) sensitive and precise detection of modification sites, 2) the avoidance of loss or artefactual creation of modifications, 3) accurate quantification of peptide abundance and reduction of missing values problem, 4) monitoring the dynamics of modification in samples exposed to different oxidant concentrations and 5) development of guidelines for verification of chlorination sites assignment. A combination of an optimised sample preparation protocol, and improved data analysis approaches have allowed identification of 33 and 15 chlorination sites in laminin and fibronectin, respectively, reported in previous manuscripts [1,2]. The method was subsequently tested on murine basement membrane extract, which contains high levels of laminin in a complex mixture. Here, 10 of the major chlorination sites in laminin were recapitulated, highlighting the utility of the method in detecting damage in complex samples.",
keywords = "Extracellular matrix proteins, Hypochlorous acid, Inflammation, Mass spectrometry, Myeloperoxidase, Protein chlorination, Proteomics",
author = "Tina Nybo and Davies, {Michael J.} and Adelina Rogowska-Wrzesinska",
note = "Copyright {\circledC} 2019 The Authors. Published by Elsevier B.V. All rights reserved.",
year = "2019",
month = "9",
day = "1",
doi = "10.1016/j.redox.2019.101236",
language = "English",
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journal = "Redox Biology",
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}

Analysis of protein chlorination by mass spectrometry. / Nybo, Tina; Davies, Michael J.; Rogowska-Wrzesinska, Adelina.

I: Redox Biology, Bind 26, 101236, 01.09.2019.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Analysis of protein chlorination by mass spectrometry

AU - Nybo, Tina

AU - Davies, Michael J.

AU - Rogowska-Wrzesinska, Adelina

N1 - Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

PY - 2019/9/1

Y1 - 2019/9/1

N2 - Chlorination of tyrosine is a commonly known effect/consequence of myeloperoxidase activity at sites of inflammation, and detection of 3-chlorotyrosine has been used as biomarker for inflammatory diseases. However, few studies have addressed site specific chlorination in proteins, and no methods for large scale chloroproteomics studies have yet been published. In this study, we present an optimized mass spectrometry based protocol to identify and quantify chlorinated peptides from single proteins modified by HOCl (100 and 500 μM, within estimated pathophysiological levels), at a high level of sensitivity and accuracy. Particular emphasis was placed on 1) sensitive and precise detection of modification sites, 2) the avoidance of loss or artefactual creation of modifications, 3) accurate quantification of peptide abundance and reduction of missing values problem, 4) monitoring the dynamics of modification in samples exposed to different oxidant concentrations and 5) development of guidelines for verification of chlorination sites assignment. A combination of an optimised sample preparation protocol, and improved data analysis approaches have allowed identification of 33 and 15 chlorination sites in laminin and fibronectin, respectively, reported in previous manuscripts [1,2]. The method was subsequently tested on murine basement membrane extract, which contains high levels of laminin in a complex mixture. Here, 10 of the major chlorination sites in laminin were recapitulated, highlighting the utility of the method in detecting damage in complex samples.

AB - Chlorination of tyrosine is a commonly known effect/consequence of myeloperoxidase activity at sites of inflammation, and detection of 3-chlorotyrosine has been used as biomarker for inflammatory diseases. However, few studies have addressed site specific chlorination in proteins, and no methods for large scale chloroproteomics studies have yet been published. In this study, we present an optimized mass spectrometry based protocol to identify and quantify chlorinated peptides from single proteins modified by HOCl (100 and 500 μM, within estimated pathophysiological levels), at a high level of sensitivity and accuracy. Particular emphasis was placed on 1) sensitive and precise detection of modification sites, 2) the avoidance of loss or artefactual creation of modifications, 3) accurate quantification of peptide abundance and reduction of missing values problem, 4) monitoring the dynamics of modification in samples exposed to different oxidant concentrations and 5) development of guidelines for verification of chlorination sites assignment. A combination of an optimised sample preparation protocol, and improved data analysis approaches have allowed identification of 33 and 15 chlorination sites in laminin and fibronectin, respectively, reported in previous manuscripts [1,2]. The method was subsequently tested on murine basement membrane extract, which contains high levels of laminin in a complex mixture. Here, 10 of the major chlorination sites in laminin were recapitulated, highlighting the utility of the method in detecting damage in complex samples.

KW - Extracellular matrix proteins

KW - Hypochlorous acid

KW - Inflammation

KW - Mass spectrometry

KW - Myeloperoxidase

KW - Protein chlorination

KW - Proteomics

U2 - 10.1016/j.redox.2019.101236

DO - 10.1016/j.redox.2019.101236

M3 - Journal article

VL - 26

JO - Redox Biology

JF - Redox Biology

SN - 2213-2317

M1 - 101236

ER -