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

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-(amino-methyl)benzenesulfonic acid (ABS) in the presence of K(3)Fe(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 K(3)Fe(CN)(6) in 0.1 M Na(2)HPO(4) 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.

Fig. 1

Formation of fluorescent 6-substituted 2-phenylbenzoxazole (products of types I, II, and III) from FSAY(3-NO₂)LER upon reaction with ABS and K₃Fe(CN)₆ (modified from [21])

Fig. 2

Reduction of 50 μM FSAY(3-NO₂)LER (a) to FSAY(3-NH₂)LER (b) by 10 mM Na₂S₂O₄ analyzed by RP–LC with UV detection (the chromatograms are recorded at 214 nm). The UV spectra of major peaks analyzed by a PDA detector are shown as inserts in a and b

Fig. 3

Fluorescence tagging of FSAY(3-NH₂)LER analyzed by RP–LC with UV detection at 214 nm (a) and by fluorescence detection with excitation and emission wavelengths at 360 and 490 nm, respectively (b). Chromatograms 1 and 2 are recorded before and after tagging of 50 μM FSAY(3-NH₂)LER with 10 mM ABS and 0.5 mM K₃Fe(CN)₆ for 1 h at room temperature. The UV spectra of major peaks analyzed by a PDA detector are shown in the insert to a; the fluorescence spectrum of a total sample is shown in the insert to panel b

Fig. 4

MS analysis of tagged FSAY(3-NH₂)LER (50 μM) after reaction with 10 mM ABS and 0.5 mM K₃Fe(CN)₆ by MALDI–TOF–MS (a) and CapLC–LTQ–FT–MS–MS (b and c). Panels b and c display sequence-indicating fragmentation of m/z 624.6 and 541.4 representing doubly charged ions of FSAY(A₂)LER (Y + 366) and FSAY(AN)LER (Y + 196), respectively

Fig. 5

The dependence of fluorescent product formation from FSAY(3-NH₂)LER on the ABS concentration, analyzed by fluorescence spectrometry (solid lines) and RP–LC (dashed line) with fluorescence detection (excitation and emission wavelengths were 360 and 480 nm, respectively). The reaction was performed in the presence of 0.5 mM K₃Fe(CN)₆ for 1 h at room temperature with different concentrations of ABS and 3-AT peptide as indicated in respective panels

Fig. 6

Effect of [K₃Fe(CN)₆] on fluorescent product formation from 1 (a) and 0.1 μM (b) FSAY(3-NH₂)LER assessed by RP–LC with fluorescence detection (excitation and emission wavelengths set at 360 and 480 nm, respectively) and by fluorescence spectrometry (c). The ABS concentration was 2 mM, the concentrations of FSAY(3-NH₂)LER are shown at the curves

Fig. 7

Determination of the fluorescence quantum yield for RP–LC-purified FSAY(A₂)LER (peak at ca. 16 min in the panel a) using a reference fluorescence peptide, the GSH-ThioGlo1 adduct. Panel b shows absorbance (Abs.), fluorescence excitation (Ex.) and emission (Em.) spectra for 1 μM FSAY(A₂)LER (solid lines) and 2 μM ThioGlo1-GSH (dashed lines) in 0.1 M NaH₂PO₄, pH 7.4

Fig. 8

Fluorescence spectra recorded after ABS tagging of low FSAY(3-NO₂)LER concentrations in the absence (a, b) or in the presence of tryptic peptides prepared from lysate of 100 μg C2C12 cell protein (c, d). Fluorescence tagging after nitropeptide reduction was performed with 2 mM ABS and 10 μM K₃Fe(CN)₆ in 0.1 M Na₂HPO₄ at 25°C. Concentrations of the nitropeptide in the respective samples (nM) are indicated for respective spectra. The dashed line traces represent spectra obtained through subtraction of respective background fluorescence spectra (in the absence of nitropeptide)

Fig. 9

a Calibration of FSAY*LER in the absence (open circles) and in the presence of 100 μg digested C2C12 cell proteins (solid circles). Solid squares connected with dashed line represent the result of background fluorescence subtraction. b The dependence of background fluorescence (in the absence of 3-NT peptide) on the amount of protein digest. Fluorescence was measured at excitation and emission wavelengths of 360 and 490 nm, respectively, after 3-NT reduction and tagging with 2 mM ABS and 20 μM K₃Fe(CN)₆ for 1 h at room temperature

Fig. 10

ABS-independent background (matrix) fluorescence originating from oxidative protein modification. a and b Spectra of samples containing 100 μg of (a) digested or (b) non-digested C2C12 proteins (1) before and (2) after 1 h incubation in 0.1 M PBS (pH 9) with 10 μM K₃Fe(CN)₆. c Fluorescence spectra of 10 (curves 1,3,4) and 2 μM (curve 2) kynurenin in 0.1 M PBS, pH 9 (curve 1), 50% PBS/50% ethanol (curves 2 and 3) and 100% ethanol (curve 4). Excitation wavelength was set at 360 nm

Fig. 11

Fluorogenic derivatization of 5-OH-Trp with ABS (modified from [35])

Fig. 12

Fluorescence spectra of ABS-tagged 5-hydroxytryptophan (a), the dependence of the fluorescence intensity (excitation and emission wavelengths of 360 and 490 nm, respectively) on 5-OH-Trp concentration (b), and fluorescence resulted from the incubation for 1 h of 50 μM Trp with 2 mM ABS and 10 μM K₃Fe(CN)₆) in 0.1 M PBS (pH 9) at room temperature (c). The excitation spectrum for 1 μM 5-OH-Trp (the emission wavelength was set at 500 nm) is shown in panel a (dashed line)

Fig. 13

The dependence of fluorescence intensity (excitation and emission wavelengths of 360 and 480 nm, respectively) on the concentration of 3-NT after 3-NT reduction and tagging of whole phosphorylase b (open squares) or protein digest (solid squares) with 2 mM ABS and 20 μM K₃Fe(CN)₆. The total amount of protein in the samples was maintained at 100 μg; the 3-NT content was altered through mixing of native and nitrated protein at certain ratios

Fig. 14

Representative MS–MS spectra for ABS-tagged tryptic peptides from rabbit muscle Ph-b demonstrating the formation of fluorescent PBO products type I (Y + 366 AMU) in the sequences DFY*ELEPHK (a) and IGEEY*SDLDQLR (b)

Fig. 15

Fluorescence imaging of ABS-tagged proteins after SDS-PAGE separation Line 1: molecular weight standard (Precision Plus Dual Color, Bio-Rad); line 2: 5 μg rabbit phosphorylase b (containing 50 pmol 3-NT), non-derivatized; line 3: same protein, exposed to ABS without 3-NT reduction; line 4: same protein, 3-NT reduced and ABS-tagged; line 5: lysate of C2C12 cells exposed to peroxynitrite (50 μg protein containing 500 pmol 3-NT), after 3-NT reduction and ABS-tagging; line 6: same preparation, non-reduced, ABS tagged; line 7: same preparation, non-reduced and non-tagged control

Fig. 16

Representative tandem MS spectra of ABS tagged nitrated C2C12 proteins obtained by nanoLC–FTICR–MS–MS analysis of in-gel digests. Reduction of 3-NT (with 10 mM Na₂S₂O₄) and tagging with 2 mM ABS and 20 μM K₃Fe(CN)₆ in 100 mM Na₂HPO₄, pH 9.0, was performed on non-digested protein samples (containing 100 μg protein and 5 μM 3-NT in a 100-μL volume). MS–MS spectra show the type I product (Y + 366 AMU) of ABS tagged sequences for murine actin peptide GY*SFTTTAER (a), and murine lactate dehydrogenase peptide QVVDSAY*EVIK (b)