Quantification of 3-nitrotyrosine in biological tissues and fluids: Generating valid results by eliminating artifactual formation

Abstract

Reactive nitrogen species such as peroxynitrite can nitrate specific amino acids, whether free or protein bound, and 3-nitrotyrosine is believed to be one marker of this reaction. To examine the significance of this pathway in biological systems we have developed an accurate, sensitive, and specific assay for 3-nitrotyrosine based on combined liquid chromatography tandem mass spectrometry. Our approach allowed simultaneous analysis of both tyrosine and 3-nitrotyrosine and employs isotopomer standards (i.e., [15N1, 13C9]-tyrosine and [13C6]-3nitrotyrosine). Calibration curves were linear (r2 = 0.999) across the range 0.5-100 pg/microL (i.e., 2.2-442 fmol/microL), and the detection limit for standard samples was 0.5 pg/microL (2.2 fmol/microL, or 10 fmol on column; S/N = 5) or 1 pg/microL (4.4 fmol/microL) for extracted (biological) samples. As a component of this study we have undertaken an extensive investigation of artifactual formation of 3-nitrotyrosine under conditions that exist during sample extraction and derivatization. Our studies show that under appropriate conditions (low pH, elevated temperatures, and in the presence of a vast excess of the two substrates, tyrosine and the nitrate anion), 3-nitrotyrosine can readily be formed as an artifact.

Protein Nitration in a Mouse Model of Familial Amyotrophic Lateral Sclerosis

“…Tg SOD1 WT and G93A mice express transgenic SOD1 at the same level, as measured by densitometric analysis (data not shown), allowing accurate proteome map comparisons. Technical problems exist in the detection of nitrated proteins because (i) the modification is rare in tissues and in biological fluids, even in pathological conditions (10); (ii) the modification is lost in strongly reducing conditions and possibly also under the action of not yet identified denitrase enzymes (17,18); and (iii) common chemical and immunological methods may detect false-positive nitrated proteins (35,36). Therefore, special care was needed in each experiment to avoid under-or overestimation, as described under "Materials and Methods."…”

Phagocytes Produce 5-Chlorouracil and 5-Bromouracil, Two Mutagenic Products of Myeloperoxidase, in Human Inflammatory Tissue

“…Previous studies have demonstrated that the artifactual generation of halogenated and nitrated oxidation products is a major problem during the analysis of biological material (42)(43)(44)(45)(46)(47). Indeed, some studies have found that virtually all of the oxidation products detected in tissue were generated artifactually (42,46,47).…”

“…Indeed, some studies have found that virtually all of the oxidation products detected in tissue were generated artifactually (42,46,47). This is particularly a problem when acidic conditions are used for sample hydrolysis or derivatization.…”

Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

“…Regardless of the mechanism of formation in vivo, protein 3-NT levels provide a useful measure of the severity of tissue exposure to reactive nitrogen species and a telling indicator of inflammatory disease severity and progression Brodsky et al, 2004;HalejcioDelophont et al, 2001;Skinner et al, 1997;Upmacis et al, 2007). Semiquantitative immunological methods (Ischiropoulos, 1998), as well as quantitative high-pressure liquid chromatography (HPLC)-based methods that utilize ultraviolet-visible (UV/VIS) absorption, electrochemistry, and mass spectrometry (MS) for detection (Frost et al, 2000;Maruyama et al, 1996;Schwedhelm et al, 1999;Shigenaga et al, 1997;Yi et al, 2000), have all been employed for the quantification of protein 3-NT residues; each method offers its own particular strengths and limitations. Importantly, these alternative 3-NT detection methods differ widely with regard to their relative sensitivity, specificity, throughput, and accessibility (because of differing requirements for expensive and/or specialized instrumentation).…”