Biotechnology for detecting oxidative modifications to fibrinogen

Abstract

Systemic inflammation has been recognized as a risk factor for a number of diseases, including type 2 diabetes. There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. Patients with inflammatory disease have higher levels of plasma protein 3-nitrotyrosine than healthy controls. Fibrinogen is an abundant plasma protein, and has been shown to be highly susceptible to such oxidative modifications, and is therefore a potential marker. Oxidative damage and modifications to fibrinogen offer potential as biomarkers of disease, and for diagnosis and a greater understanding of the pathology. The aim of this study was to map the in-vitro locations of oxidatively-modified fibrinogen, and test the possibility of producing antibodies that show increased binding to protein-specific modification sites. Fibrinogen was nitrated using the peroxynitrite generator SIN-1, and analyzed by liquid chromatography tandem mass spectrometry. Several modified peptides that consistently occurred were identified with the Mascot© search engine and manually validated. Based on literature or mass spectrometry results, peptides with the core amino acid sequence STSYGTGC or DYEDDQQKQLC, either unmodified, or containing 3-chlorotyrosine or 3-nitrotyrosine were synthesized and subsequently antibodies were produced. Each subsequently produced sheep anti-serum was tested against various native and modified peptides containing a variety of different amino acid sequences. Anti-STSY-(NO2)-GTGC serum showed modification site specificity, whereas anti-DY-(Cl)-EDDQQKQLC serum showed sequence specificity but was unable to distinguish between 3-chlorotyrosine and 3-nitrotyrosine. This cross-reactivity between chlorinated and nitrated modification sites might allow broader detection of modified fibrinogen in inflammation. The anti-STSY-(NO2)-GTGC serum was also tested against plasma from healthy control volunteers and type 2 diabetic patients, but showed limited binding and ability to discriminate the disease state. In conclusion, it has been shown that antibodies can be produced to differentiate between native and modified proteins; however, further work is required to understand sequence-specificity better and monoclonal antibodies might be better suited to achieving sequence-specificity.