Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)-benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration.
Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)-benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration.
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DOI：
10.1365/s10337-009-1409-0
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发表时间：
2010-01-01
期刊：
影响因子：
1.7
通讯作者：
Schöneich C
中科院分区：
文献类型：
--
作者：
Sharov VS;Dremina ES;Galeva NA;Gerstenecker GS;Li X;Dobrowsky RT;Stobaugh JF;Schöneich C
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-(amino-methyl)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.
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影响因子：
--
作者：
MORRIS, JV;MAHANEY, MA;HUBER, JR
通讯作者：
HUBER, JR
影响因子：
5.6
作者：
Dairou, Julien;Pluvinage, Benjamin;Rodrigues-Lima, Fernando
通讯作者：
Rodrigues-Lima, Fernando
影响因子：
3.5
作者：
Girgis, AS;Kalmouch, A;Hosni, HM
通讯作者：
Hosni, HM
DOI：
10.1073/pnas.221269198
发表时间：
2001-10-09
影响因子：
11.1
作者：
Aulak, KS;Miyagi, M;Stuehr, DJ
通讯作者：
Stuehr, DJ
影响因子：
4.1
作者：
Ikehata, Keisuke;Duzhak, Tatyana G.;Hanzlik, Robert P.
通讯作者：
Hanzlik, Robert P.