A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides.
A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides.
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DOI：
10.1016/j.ab.2011.07.024
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发表时间：
2011-11-15
影响因子：
2.9
通讯作者：
Schöneich C
中科院分区：
文献类型：
--
作者：
Dremina ES;Li X;Galeva NA;Sharov VS;Stobaugh JF;Schöneich C
We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO2)LER, was employed as a model for method validation. Further, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of non-derivatized peptides, and to enrich them for fluorescent detection and MS identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.
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