Detection and quantification of citrullinated chemokines.

<h4>Background</h4>Posttranslational deimination or citrullination by peptidylarginine deiminases (PAD) regulates the biological function of proteins and may be involved in the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This posttranslational...

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Autores principales:	Eva A V Moelants, Jo Van Damme, Paul Proost
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/bc72f8567a1e4d66b9d34ba3c6f19782
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spelling	oai:doaj.org-article:bc72f8567a1e4d66b9d34ba3c6f197822021-11-18T07:31:56ZDetection and quantification of citrullinated chemokines.1932-620310.1371/journal.pone.0028976https://doaj.org/article/bc72f8567a1e4d66b9d34ba3c6f197822011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22194966/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Posttranslational deimination or citrullination by peptidylarginine deiminases (PAD) regulates the biological function of proteins and may be involved in the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This posttranslational modification of arginine was recently discovered on inflammatory chemokines including CXCL8 and CXCL10, and significantly reduced their biological activity. To evaluate the importance of these modified chemokines in patients, methods for the detection and quantification of citrullinated chemokines are needed. Since citrullination only results in an increase of the protein mass with one mass unit and the loss of one positive charge, selective biochemical detection is difficult. Therefore, we developed an antibody-based method to specifically detect and quantify citrullination on a protein of interest.<h4>Methodology/principal findings</h4>First, the citrullinated proteins were chemically modified with antipyrine and 2,3-butanedione at low pH. Such selectively modified citrullines were subsequently detected and quantified by specific antibodies raised against a modified citrulline-containing peptide. The specificity of this two-step procedure was validated for citrullinated CXCL8 ([Cit(5)]CXCL8). Specific detection of [Cit(5)]CXCL8 concentrations between 1 and 50 ng/ml was possible, also in complex samples containing an excess of contaminating proteins. This novel detection method was used to evaluate the effect of lipopolysaccharide (LPS) on the citrullination of inflammatory chemokines induced in peripheral blood mononuclear cells (PBMCs) and granulocytes. LPS had no significant effect on the induction of CXCL8 citrullination in human PBMCs and granulocytes. However, granulocytes, known to contain PAD, were essential for the production of significant amounts of [Cit(5)]CXCL8.<h4>Conclusion/significance</h4>The newly developed antibody-based method to specifically detect and quantify chemically modified citrullinated proteins is proven to be effective. This study furthermore demonstrates that granulocytes were essential to obtain significant levels of [Cit(5)]CXCL8. For human PBMCs and granulocytes stimulation with LPS did not affect the citrullination of CXCL8.Eva A V MoelantsJo Van DammePaul ProostPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 12, p e28976 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Eva A V Moelants Jo Van Damme Paul Proost Detection and quantification of citrullinated chemokines.
description	<h4>Background</h4>Posttranslational deimination or citrullination by peptidylarginine deiminases (PAD) regulates the biological function of proteins and may be involved in the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This posttranslational modification of arginine was recently discovered on inflammatory chemokines including CXCL8 and CXCL10, and significantly reduced their biological activity. To evaluate the importance of these modified chemokines in patients, methods for the detection and quantification of citrullinated chemokines are needed. Since citrullination only results in an increase of the protein mass with one mass unit and the loss of one positive charge, selective biochemical detection is difficult. Therefore, we developed an antibody-based method to specifically detect and quantify citrullination on a protein of interest.<h4>Methodology/principal findings</h4>First, the citrullinated proteins were chemically modified with antipyrine and 2,3-butanedione at low pH. Such selectively modified citrullines were subsequently detected and quantified by specific antibodies raised against a modified citrulline-containing peptide. The specificity of this two-step procedure was validated for citrullinated CXCL8 ([Cit(5)]CXCL8). Specific detection of [Cit(5)]CXCL8 concentrations between 1 and 50 ng/ml was possible, also in complex samples containing an excess of contaminating proteins. This novel detection method was used to evaluate the effect of lipopolysaccharide (LPS) on the citrullination of inflammatory chemokines induced in peripheral blood mononuclear cells (PBMCs) and granulocytes. LPS had no significant effect on the induction of CXCL8 citrullination in human PBMCs and granulocytes. However, granulocytes, known to contain PAD, were essential for the production of significant amounts of [Cit(5)]CXCL8.<h4>Conclusion/significance</h4>The newly developed antibody-based method to specifically detect and quantify chemically modified citrullinated proteins is proven to be effective. This study furthermore demonstrates that granulocytes were essential to obtain significant levels of [Cit(5)]CXCL8. For human PBMCs and granulocytes stimulation with LPS did not affect the citrullination of CXCL8.
format	article
author	Eva A V Moelants Jo Van Damme Paul Proost
author_facet	Eva A V Moelants Jo Van Damme Paul Proost
author_sort	Eva A V Moelants
title	Detection and quantification of citrullinated chemokines.
title_short	Detection and quantification of citrullinated chemokines.
title_full	Detection and quantification of citrullinated chemokines.
title_fullStr	Detection and quantification of citrullinated chemokines.
title_full_unstemmed	Detection and quantification of citrullinated chemokines.
title_sort	detection and quantification of citrullinated chemokines.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/bc72f8567a1e4d66b9d34ba3c6f19782
work_keys_str_mv	AT evaavmoelants detectionandquantificationofcitrullinatedchemokines AT jovandamme detectionandquantificationofcitrullinatedchemokines AT paulproost detectionandquantificationofcitrullinatedchemokines
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Detection and quantification of citrullinated chemokines.

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