TY - JOUR

T1 - Analysis of protein chlorination by mass spectrometry

AU - Nybo, Tina

AU - Davies, Michael J.

AU - Rogowska-Wrzesinska, Adelina

PY - 2019

Y1 - 2019

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 mu M, 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 mu M, 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 - Protein chlorination

KW - Hypochlorous acid

KW - Myeloperoxidase

KW - Inflammation

KW - Extracellular matrix proteins

KW - Mass spectrometry

KW - Proteomics

U2 - 10.1016/j.redox.2019.101236

DO - 10.1016/j.redox.2019.101236

M3 - Journal article

C2 - 31181457

VL - 26

JO - Redox Biology

JF - Redox Biology

SN - 2213-2317

M1 - UNSP 101236

ER -