NDLI: Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration

Content Provider	Springer Nature Link
Author	Sharov, Victor S. Dremina, Elena S. Galeva, Nadezhda A. Gerstenecker, Gary S. Li, Xiaobao Dobrowsky, Rick T. Stobaugh, John F. Schöneich, Christian
Copyright Year	2009
Abstract	Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(aminomethyl)benzenesulfonic acid (ABS) in the presence of K3Fe(CN)6 to form a highly fluorescent 2-phenylbenzoxazole product. Synthetic 3-NT peptide (0.005-1 μM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 μM K3Fe(CN)6 in 0.1 M Na2HPO4 buffer (pH 9.0) was converted with yields >95% to a single fluorescent product incorporating two ABS molecules per 3-NT residue, with fluorescence excitation and emission maxima at 360 ± 2 and 490 ± 2 nm, respectively, and a quantum yield of 0.77 ± 0.08, based on reverse-phase LC with UV and fluorescence detection, fluorescence spectroscopy and LC–MS–MS analysis. This protocol was successfully tested for quantitative analysis of in vitro Tyr nitration in a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from C2C12 cultured cells exposed to peroxynitrite, with a detection limit of ca. 1 pmol 3-NT by fluorescence spectrometry, and an apparent LOD of 12 and 40 pmol for nitropeptides alone or in the presence of 100 μg digested cell proteins, respectively. LC–MS–MS analysis of ABS tagged peptides revealed that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration in proteomic studies. Fluorogenic tagging with ABS also can be instrumental for detection and visualization of protein 3-NT in LC and gel-based protein separations.
Starting Page	37
Ending Page	53
Page Count	17
File Format	PDF
ISSN	00095893
Journal	Chromatographia
Volume Number	71
Issue Number	1-2
e-ISSN	16121112
Language	English
Publisher	Vieweg Verlag
Publisher Date	2009-11-21
Publisher Place	Wiesbaden
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Column liquid chromatography–mass spectrometry Reversed phase UV and fluorescence detection Protein 3-nitrotyrosine Measurement Science and Instrumentation Plant Sciences Biochemistry Pharmacy Organic Chemistry Analytical Chemistry
Content Type	Text
Resource Type	Article
Subject	Organic Chemistry Analytical Chemistry Biochemistry Clinical Biochemistry

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