A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides

Article abstract of DOI:10.1016/j.ab.2011.07.024

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. Furthermore, 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 nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (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. Copyright

Full text of DOI:10.1016/j.ab.2011.07.024

Analytical Biochemistry 418 (2011) 184–196
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
A methodology for simultaneous fluorogenic derivatization and boronate
affinity enrichment of 3-nitrotyrosine-containing peptides
Elena S. Dremina a,1, Xiaobao Li a,1,2, Nadezhda A. Galeva b, Victor S. Sharov a, John F. Stobaugh a,
Christian Schöneich a,
⇑
a Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA
b Mass Spectrometry Laboratory, University of Kansas, Lawrence, KS 66047, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfo-
nyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) res-
idues 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. Furthermore, this
derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to per-
oxynitrite 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 detec-
tion 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 sulfon-
amide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the
derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them
for fluorescent detection and mass spectrometry (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 nitra-
tion at low concentrations of 3-NT in complex protein mixtures.
Received 26 April 2011
Received in revised form 1 July 2011
Accepted 18 July 2011
Available online 28 July 2011
Keywords:
3-Nitrotyrosine
(3R,4S)-1-(4-
(Aminomethyl)phenylsulfonyl)pyrrolidine-
3,4-diol (APPD)
Fluorogenic derivatization
Fluorescence
Boronate affinity chromatography
Mass spectrometry
Proteomics
Ó 2011 Elsevier Inc. All rights reserved.
Protein tyrosine (Tyr)3 nitration to 3-nitrotyrosine (3-NT) is a ni-
tric oxide (NO)-dependent posttranslational modification (PTM)
associated with a number of diseases and biological aging [1–5].
To correlate protein Tyr nitration mechanistically with specific phys-
iological and pathological conditions, it is important to identify pro-
tein target sequences and to quantify protein–3-NT residues on
these proteins. Several proteomic studies have provided important
information about protein targets for nitration in vivo [6–11]. How-
ever, it appears that most of these approaches yielded data covering
only a small fraction of possible protein targets. Some strategies for
the enrichment of 3-NT-containing peptides and proteins have been
developed, based on the reduction of 3-NT to 3-aminotyrosine
(3-AT) as a critical step, followed by either differential detection of
3-AT (instead of 3-NT) [11] or the reaction of 3-AT with N-hydroxy-
succinimide esters coupled to biotin [12] or a functional group avail-
able for further derivatization [13]. However, these derivatization
approaches can suffer from side reactions of N-hydroxysuccinimide
esters with other primary amines, especially when the abundance
of 3-NT is low. Moreover, the published methods [11–13] do not al-
low quantification other than through integration of mass spectrom-
⇑
Corresponding author. Fax: +1 785 864 5735.
E-mail address: schoneic@ku.edu (C. Schöneich).
These authors contributed equally to this work.
Present address: Pharmaceutical Division, Integrated Analytical Laboratories,
1
2
Randolph, NJ 07869, USA.
etry (MS) signals. Therefore, we have developed
a selective
3
Abbreviations used: Tyr, tyrosine; 3-NT, 3-nitrotyrosine; NO, nitric oxide; PTM,
derivatization strategy of 3-AT with benzylamine that yields a highly
fluorescent benzoxazole product available for quantitative assess-
ment by fluorescence spectrometry or high-performance liquid chro-
matography (HPLC) with fluorescence detection [14]. This reagent
does not form a fluorescent product with either peptide N-terminal
or Lys amino groups and, furthermore, allows relative quantification
of original 3-NT-containing peptides through stable isotopic labeling
posttranslational modification; 3-AT, 3-aminotyrosine; MS, mass spectrometry; HPLC,
high-performance liquid chromatography; ABS, 4-(aminomethyl)benzenesulfonic
acid; APPD, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl) pyrrolidine-3,4-diol; Ph-b,
phosphorylase b; ESI, electrospray ionization; ACN, acetonitrile; ONOOÀ, peroxyni-
trite; SDS, sodium dodecyl sulfate; LC, liquid chromatography; MS/MS, tandem MS;
UV, ultraviolet; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-
buffered saline; TFA, trifluoroacetic acid; MALDI–TOF, matrix-assisted laser desorp-
tion/ionization time-of-flight; FTICR, Fourier transform ion cyclotron resonance;
capLC, capillary LC; LOD, limit of detection.
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2011.07.024

Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
185
using a couple of ‘‘light’’ and ‘‘heavy’’ reagents [14]. The chromato-
graphic properties of benzylamine-tagged peptides can be controlled
through substituents on the original benzylamine, as demonstrated
in a comparison between benzylamine and 4-(aminomethyl)ben-
zenesulfonic acid (ABS) [15]. We have now extended the derivatiza-
tion strategy to incorporate a functional group for the affinity
enrichment of 3-NT-containing peptides using a novel synthetic ben-
zylamine derivative with a substituent suitable for boronate affinity
chromatography. Boronate affinity separation is based on the pH-
dependent formation of a pentacyclic complex of boronic acid with
cis-diol compounds that has been employed in the separation of bio-
logical molecules such as catechols, nucleotides, nucleosides, carbo-
hydrates, glycoproteins, and some enzymes [16–20]. Recently, some
tagging methods for specific residues have been reported for the sep-
aration of peptides/proteins by boronate affinity chromatography.
For example, the guanidine group of arginine in peptides/proteins
was covalently modified with 2,3-butanedione to yield cis-diol-con-
taining triazolidine derivatives amenable for separation by boronate
affinity [21]. In the current article, we describe the synthesis and
application of (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrroli-
dine-3,4-diol (APPD) for fluorogenic derivatization and boronate
affinity enrichment of 3-NT in proteins and peptides. Our results
suggest that the method is suitable for quantitative proteomic anal-
ysis of protein nitration.
vale, CA, USA). Peptide purity was greater than 99%, as
characterized by electrospray ionization (ESI)–MS and analytical
reverse-phase HPLC analyses.
Synthesis of APPD tagging reagent
The synthetic steps for the synthesis of APPD are summarized in
Fig. 1.
(3R,4S)-1-Benzyl-3,4-dihydroxypyrrolidine-2,5-dione 1
N-Benzylmaleimide was prepared by reaction of benzylamine
with maleic anhydride followed by dehydration in acetic anhy-
dride containing sodium acetate. The crude product was purified
by dissolving in ethanol and subsequently precipitating with water
(45% yield). To a stirred solution of 6.5 g of N-methylmorpholine N-
oxide and 6 ml of osmium tetraoxide solution (2.5 wt% in tert-butyl
alcohol) in a mixture of 50 ml of acetone and 10 ml of deionized
water was added 10.0 g of N-benzylmaleimide (note that osmium
tetraoxide is toxic [22,23] and must be handled and disposed of
with appropriate precautions). The mixture was stirred at 45 °C
for 24 h. After the reaction mixture was cooled to room tempera-
ture, 3.0 g of Na2SO3 was added and stirred for 30 min and filtered
with celite through a sintered glass funnel. The filtrate was concen-
trated by evaporation under reduced pressure and extracted with
3 Â 50 ml of ethyl acetate. The combined organic extract was dried
over Na2SO4 and evaporated under reduced pressure, affording a
deep brown gum that was allowed to granulate for 4 h at room
temperature. The resulting solid was washed with 30 ml of chloro-
form and dried in vacuum to give compound 1 (5.5 g, 46% yield) as
white powder. Melting point: 125–127 °C. 1H NMR (DMSO-d6): d
7.32–7.24 (m, 5H), 6.02 (d, 2H), 4.55 (s, 2H), 4.42 (d, 2H).
Materials and methods
Reagents
All chemicals for the synthesis of APPD were purchased from
Sigma–Aldrich (St. Louis, MO, USA) and used as received unless
otherwise stated. Solvents were of HPLC purity.
(3aR,6aS)-5-Benzyl-2,2-dimethyl-3aH-[1,3]dioxolo[4,5-c]pyrrole-
4,6(5H,6aH)-dione 2
Synthesis and characterization of standard 3-NT-containing peptide
To a slurry of 8.5 g of compound 1 in 40 ml of 2,2-dimethyloxy-
propane was added 0.02 g of p-toluenesulfonic acid, and the mix-
ture was stirred at room temperature for 10 h. The reaction
mixture was then stirred with 0.5 g of sodium bicarbonate for
10 min, filtered, and concentrated by evaporation under reduced
pressure. The residue was dissolved in 80 ml of chloroform and
washed with 1 M Na2CO3 solution, dried over Na2SO4, and evapo-
rated under reduced pressure. The solid that formed was washed
A 3-NT-containing peptide representing the partial sequence of
mouse muscle phosphorylase b, 545FSAY(NO2)LER551, was synthe-
sized on an ACT 90 instrument (Advanced ChemTech, Louisville,
KY, USA) by means of a solid-phase technique using Fmoc-pro-
tected amino acids and 3-NT purchased from Bachem Bioscience
(King of Prussia, PA, USA). The peptide was purified by semiprepar-
ative HPLC performed on a Summit HPLC system (Dionex, Sunny-
Fig.1. Synthesis of APPD. Reagents and conditions: (a) OsO4, NMO, H2O/acetone (5:1, v/v), 50 °C; (b) 2,2-dimethyloxypropane/TyOH; (c) LiAlH4, THF, 80 °C; (d) H2, Pt/C,
MeOH; (e) 4-cyanobenzene-1-sulfonyl chloride, Et3N, MeCN; (f) H2, Pt/C, MeOH, 10% concentrated HCl.

186
Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
with petroleum ether and dried under vacuum overnight, affording
product 2 (9.6 g, 95% yield) as white powder. NMR (CDCl3): d 7.41–
7.28 (m, 5H), 4.86 (s, 2H), 4.69 (s, 2H), 1.43 (s, 3H), 1.30 (s, 3H).
(3R,4S)-1-((4-(Aminomethyl)phenyl)sulfonyl)pyrrolidine-3,4-diol 6
To a solution of 5.0 g of compound 5 in 50 ml of methanol con-
taining 1 ml of acetic acid was carefully added 0.5 g of 10% Pd/C
catalyst. The mixture was hydrogenated at 40 psi for 18 h at room
temperature and subsequently filtered to remove the catalyst (note
that the hydrogenation must be carried out with appropriate care).
The filtration cake was washed with 20 ml of methanol. The com-
bined organic solutions were evaporated under reduced pressure.
To the residue was added 40 ml of 2 M HCl in aqueous solution,
and the mixture was heated to 70 °C and stirred for 2 h. The solvent
was removed by evaporation under reduced pressure to give prod-
uct product 6, which was washed with chloroform and ether and
then dried under vacuum (3.4 g, 68%). 1H NMR (D2O): d 7.94 (d,
2H), 7.71 (d, 2H), 4.31 (s, 2H), 4.14 (t, 2H), 3.53 (dd, 2H), 3.23
(dd, 2H). 13C NMR (50% CD3OD/50% H2O): 139.58 (s, 1C), 137.22
(s, 1C), 130.83 (s, 2C), 129.16 (s, 2C), 71.47 (s, 2C), 52.68 (s, 2C),
43.62 (s, 1C). MS (ES+, 20 eV): m/z 273.06 ([M+H]+), calculated
for C11H16N2O4S: 272.08.
(3aR,6aS)-5-Benzyl-2,2-dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-
c]pyrrole 3
To a solution of 9.5 g of compound 2 in 20 ml of diethyl ether
was added in portions a total of 5.0 g of LiAlH4 (note that LiAlH4
must be handled with appropriate precautions). The slurry was
heated to 80 °C, refluxed for 24 h, and then cooled in an ice bath.
To the reaction mixture were slowly added 30 ml of ethyl acetate
and then 20 ml of 30% NaOH solution under stirring. After
30 min, the mixture was filtered and the filtration cake was
washed twice with 30 ml of ethyl acetate. The combined organic
layer was dried over Na2SO4 and evaporated under reduced pres-
sure to give product 3 as pale yellow oil. Yield: 8.2 g, 92%. 1H
NMR (CDCl3): d 7.36–7.25 (m, 5H), 4.66 (d, 2H), 3.63 (s, 2H), 3.0
(dd, 2H), 2.1 (dd, 2H), 1.59 (s, 3H), 1.34 (s, 3H).
Preparation of peroxynitrite for protein nitration in vitro
(3aR,6aS)-2,2-Dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyrrole 4
To a stirred solution of 8.0 g of compound 3 in 50 ml of metha-
nol was carefully added 1.5 g of 20% Pd(OH)2/C catalyst under ar-
gon. The slurry mixture was hydrogenated at 20 psi for 18 h at
room temperature and then filtered to remove the catalyst (note
that the hydrogenation must be carried out with appropriate care),
and the filtration cake was washed with 20 ml of methanol. The
combined organic solutions were evaporated under reduced pres-
sure. The residue was dissolved in 50 ml of diethyl ether, and 10 ml
of 1.0 M HCl in ether was added. The crystalline precipitate that
formed was collected by filtration, washed with ether, and dried
under vacuum to give compound 4 (5.42 g, 89%) as a white powder.
1H NMR (DMSO-d6): d 4.65 (d, 2H), 3.08 (dd, 2H), 2.50 (dd, 2H),
1.44 (s, 3H), 1.31 (s, 3H).
Peroxynitrite (ONOOÀ) was prepared by the ozonolysis of so-
dium azide solutions as described previously [24]; the ONOOÀ con-
tent in stock solutions containing 0.1% NaOH was quantified using
e
302 = 1670 MÀ1 cmÀ1 [4].
Protein nitration in vitro
Prior to protein tyrosine nitration, 1 mg/ml (ꢀ10
lM) rabbit
muscle Ph-b (Prozyme, San Leandro, CA, USA) in a buffer contain-
ing 1% (w/v) sodium dodecyl sulfate (SDS) and 50 mM NH4HCO3
(pH 7.8) was reductively alkylated by incubation with 2 mM dithi-
othreitol for 30 min at 50 °C, followed by the addition of iodoacetic
acid at a final concentration of 5 mM and incubation for an addi-
tional 30 min at room temperature. The alkylated protein was sep-
arated from unreacted compounds and side products by
precipitation in 10 volumes of cold ethanol for 4 h at À20 °C, fol-
lowed by centrifugation at 14,000g for 2 min. This step was neces-
sary to remove IÀ, which otherwise could react with ONOOÀ to
form I2, leading to Tyr iodination as assessed through the forma-
tion of iodo-Tyr-modified peptides by liquid chromatography
(LC)–tandem MS (MS/MS) analysis (data not shown). Reconstituted
4-(((3aR,6aS)-2,2-Dimethyldihydro-3aH-[1,3]dioxolo[4,5-c]pyrrol-
5(4H)-yl)sulfinyl)benzonitrile 5
To a solution of 3.0 g of compound 4 in 50 ml of acetonitrile
(ACN) were added 5.0 ml of triethylamine and 3.4 g of 4-cyanoben-
zene-1-sulfonyl chloride, and the mixture was stirred for 1 h and
subsequently filtered. The filtrate was washed with 10 ml of a sat-
urated aqueous solution of NaCl and evaporated under reduced
pressure to give product 5 (5.6 g, 89% yield) as a white solid. 1H
NMR (DMSO-d6): d 7.92 (d, 2H), 7.82 (d, 2H), 4.64 (s, 2H), 3.59
(dd, 2H), 2.97 (dd, 2H), 1.29 (s, 3H), 1.22 (s, 3H).
100-
ing 1% SDS were then nitrated by the addition of 10
stock solution of ONOOÀ (final concentration of 3 mM) while vor-
l
l (100-lg) Ph-b aliquots in 0.1 M phosphate buffer contain-
l
l alkaline
Fig.2. Derivatization pathway of peptide 3-AT with APPD.

Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
187
texing to yield approximately 100
3-NT on Ph-b was determined by ultraviolet (UV) spectroscopy
430 = 4400 M–1 cm–1 at pH > 9.0) using non-nitrated protein sam-
ples for background control [4]. Ph-b samples containing desired
concentrations of 3-NT were prepared by mixing of nitrated and
control proteins at specific ratios.
l
M protein 3-NT. The content of
phase A). After washing for at least 5 min under control of the
baseline with UV detection, peptides were eluted by a steep linear
gradient increasing ACN in 0.1% TFA from 0% to 80% (v/v) within
5 min, followed by an additional 5-min elution with 80% (v/v)
ACN in 0.1% aqueous TFA. Fractions containing peptides (6–
10 min) were collected and dried in a SpeedVac before reconstitu-
tion in the media compatible with the next analysis or derivatiza-
tion step.
(e
Protein nitration in cell culture
C2C12 murine myoblasts were grown in Petri dishes as mono-
layers to approximately 50% confluence in Dulbecco’s modified Ea-
gle’s medium (DMEM) supplemented with 10% fetal bovine serum
and penicillin/streptomycin at 37 °C and 5% CO2. The resulting
monolayers were washed with phosphate-buffered saline (PBS),
trypsinized, washed with DMEM and PBS, and collected by centri-
fugation at 500 g for 2 min. A cell suspension in PBS containing
1.5 mg/ml protein was exposed to peroxynitrite (final concentra-
tion 3 mM) under vortexing for 5 s in 1.5-ml Eppendorf tubes. Cells
were lysed by the addition of 1% SDS to PBS buffer and sonication
for 30 s at 10% power output of the probe sonicator Sonic Dismem-
brator 500 (Fisher Scientific, Pittsburgh, PA, USA). The supernatant
was used for the analysis of protein nitration.
300
A
200
100
0
Preparation of protein tryptic digests
0
50
K3Fe(CN)6 (
100
M)
150
Following the nitration, C2C12 cell proteins were subjected to
reductive alkylation of Cys residues (in the presence of 1% SDS)
and cold ethanol precipitation as described above for Ph-b sample
preparation. After centrifugation, protein pellets were collected
and reconstituted in digestion buffer containing 50 mM NH4HCO3
(pH 7.8), 10% (v/v) ACN, and trypsin (sequence grade, Promega,
Madison, WI, USA) at a 1:20 (w/w) ratio to proteins. Digestion
was carried out overnight at 37 °C.
µ
1000
500
0
B
Derivatization of 3-NT peptides with APPD
Prior to APPD tagging, 3-NT peptides were reduced to 3-AT pep-
tides by incubation with 10 mM sodium dithionite (Na2S2O4) for
2 min at room temperature, and the resultant 3-AT-containing
peptides were immediately separated from the reagent by re-
verse-phase HPLC (see below). Tagging with APPD was carried
out in 0.1 M sodium phosphate buffer (pH 9.0) in the presence of
K3Fe(CN)6. Concentrations of the reagents were experimentally
evaluated (see below). A typical procedure for the derivatization
would be as follows: to 0.25 ml of peptide solution in 0.1 M PBS
0
5
10
FSAY(3-NO2)LER (
15
20
µM)
800
600
400
200
0
20
C
(pH 9.0), 10 mM APPD and 100 lM K3Fe(CN)6 are added and the
reaction mixture is incubated at room temperature for 1 h. The
reaction was monitored by fluorescence spectrometry and re-
verse-phase HPLC with UV and fluorescence detection.
10
Fluorescence measurements
5
2
0.5
0
After derivatization, fluorescence spectra were measured in 0.5-
ml, 1-cm light-pass fluorescence cuvettes (Hellma, Plainview, NY,
USA) using a Shimadzu RF-5000U fluorescence spectrophotometer
with excitation and emission bandwidths set at 5 nm. Spectra were
then digitized, and fluorescence intensities were calculated from
the spectra.
400
450
500
550
600
Wavelength (nm)
Fig.3. Dependence of fluorescence intensity after tagging of FSAY(3-NO2)LER with
APPD on the concentration of K3Fe(CN)6 (A) or 3-NT peptide (B) and fluorescence
Reverse-phase HPLC
spectra at different concentrations of 3-NT (C). (A) FSAY(3-NO2)LER (5 lM) was
reduced to FSAY(3-NH2)LER and incubated with 10 mM APPD and different
concentrations of K3Fe(CN)6 in 0.1 M sodium phosphate buffer (pH 9.0) for 1 h. (B
and C) Fluorescence intensities (B) and spectra (C) at different concentrations of the
Synthetic and tryptic peptides, as well as their derivatives, were
separated by reverse-phase HPLC using Vydac C18 columns and
ACN gradients. Desalting of peptides after 3-NT reduction to 3-AT
was achieved on guard columns (4.6 mm i.d. Â 10 mm length)
equilibrated with 0.1% aqueous trifluoroacetic acid (TFA) (mobile
3-NT peptide tagged with 10 mM APPD and 100
Fluorescence was measured in 0.5-ml (1-cm) cuvette using excitation and
emission wavelengths of 360 and 510 nm, respectively.
lM K3Fe(CN)6 as described above.
a

188
Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
679.69
A
100
90
80
70
60
50
40
30
20
10
0
6.4E+4
1421.97
701.68
1166.83
1149.80
500
800
1100
1400
1700
2000
Mass (m/z)
2.0
B
2
1
1.5
1.0
0.5
0.0
0
10
20
30
40
Elution time (min)
Fig.4. Analysis by MALDI–TOF MS (A) and reverse-phase HPLC detected by fluorescence (B) of APPD-tagged FSAY(3-NO2)LER. After reduction of 3-NT to 3-AT, the peptide
(10 M) was incubated for 1 h with 10 mM APPD and 20 M K3Fe(CN)6 (curve 1) or 100 M K3Fe(CN)6 (curve 2) in 0.1 M sodium phosphate buffer (pH 9.0) at room
temperature. Chromatograms were detected using excitation and fluorescence wavelengths of 360 and 510 nm, respectively.
l
l
l
For analytical HPLC of APPD-tagged FSAY⁄LER (where Y⁄
denominates various derivatives of the tyrosine residue such as
3-NT, 3-AT, and derivatization products), samples were injected
onto a Vydac C18 column (4.6 mm i.d. Â 250 mm length) equili-
brated with 10% ACN/90% H2O/0.1% TFA (v/v/v) and peptides were
separated by a linear increase of ACN in the mobile phase (1%/min)
followed by a washing step for 10 min at 80% ACN. UV and fluores-
cence detection were achieved using a photodiode array detector
SPD-M10AVP and a fluorescence detector RF-10AXL (both from
Shimadzu USA Manufacturing, Canby, OR, USA), respectively.
of the APPD-tagged peptides and water (ꢀ pH 5.8) for elution in the
presence or absence of 30% (v/v) ACN. The flow rate was 1.0 ml/
min, and the effluent was monitored with UV and fluorescence
detection.
A TSK-Gel Boronate-5PW column (1000 Å, 10 lm,
7.5 mm i.d. Â 75 mm length) was purchased from Tosoh Biosci-
ences (Montgomeryville, PA, USA).
MALDI–TOF mass spectrometry
For matrix-assisted laser desorption/ionization time-of-flight
(MALDI–TOF) MS analysis, either desalted samples (using C18
ZipTips, Millipore, Bedford, MA, USA) or nontreated samples were
Boronate affinity chromatography
directly applied onto the stainless-steel sample plate with 2
ll of
Sulfonamide-phenylboronic acid-functionalized (0.45 mmol/g)
50% (v/v) acetonitrile/0.1% TFA saturated with -cyano-4-
a
silica gel (Nucleosil, 5
l
m, 500 Å, Macherey–Nagel, Düren, Ger-
hydroxycinnamic acid. Mass spectra were acquired in the mass
range between 500 and 3000 amu on a Voyager-DE STR mass
spectrometer (PerSeptive Biosystems, Framingham, MA, USA)
equipped with a 337-nm nitrogen laser. The instrument was
operated in the positive reflector mode with the following param-
eters: accelerating voltage, 20,000 V; grid voltage, 70–75%; mirror
voltage ratio, 1.12; guide wire, 0.02%; extraction delay time,
150 ns.
many) was prepared as described previously [25] and used as bor-
onate affinity solid phase in this work. The solid phase was slurry
packed (0.5 g silica phase in 50 ml methanol) into an empty stain-
less-steel chromatography column (4.6 mm i.d. Â 50 mm) under a
pressure of 8000 psi with an air-driven fluid pump (Haskel, Bur-
bank, CA, USA). Boronate affinity chromatography was conducted
using 50 mM NH4HCO3 buffer (pH 7.8) as mobile phase for binding

Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
189
capLC–MS analysis for protein mapping
and a parent ion tolerance of 10 ppm. Trypsin specificity and up
to two missed cleavage sites were used in the search parameters.
To find all possible products (delta masses) of derivatization reac-
tion between APPD and nitropeptides, we used P-Mod software
[27] to map experimental data. Based on those data, variable pro-
tein modifications for Sequest and Mascot searches were set to be
+16 on M (oxidation), +15 on Y (aminotyrosine), +45 on Y (nitra-
tion), +264 on Y, +281 on Y, and +536 on Y (APPD derivatives).
An additional shift of +80 on Y for all of the above Y modifications
was considered to take into account a potential sulfation of tyro-
sine residues during the reduction of 3-NT to 3-AT with sodium
dithionite. Carboxymethylation of cysteine residues was consid-
ered as a fixed modification.
Sequest and Mascot results were imported into Scaffold 3.0
software (Proteome Software, Portland, OR, USA; a free viewer is
available at http://www.proteomesoftware.com) for analyzing
with the X!Tandem search algorithm and statistical validation of
peptide/protein identities. Peptide identifications were accepted
if they could be established at greater than 95% probability as spec-
ified by the Peptide Prophet algorithm as described previously
[28]. Protein identifications were accepted if they could be estab-
lished at greater than 95% probability and contained at least two
identified peptides. Protein probabilities were assigned by the Pro-
tein Prophet algorithm [29]. Proteins that contained similar pep-
tides and could not be differentiated based on MS/MS analysis
alone were grouped to satisfy the principles of parsimony.
Samples were desalted using Oasis HLB columns (Waters,
Milford, MA, USA) as suggested by the manufacturer and were
introduced into an LTQ-FT hybrid linear quadrupole ion trap
Fourier transform ion cyclotron resonance (FTICR) mass spec-
trometer (ThermoFinnigan, Bremen, Germany) via capillary LC
(capLC), analogous to a published procedure [26]. In brief, pep-
tides were separated on a reverse-phase LC Packings PepMap
C18 column (0.300 Â 150 mm) at a flow rate of 10
ll/min with
a linear gradient rising from 0% to 65% ACN in 0.06% aqueous
formic acid over a period of 55 min using an LC Packings Ulti-
mate Chromatograph (Dionex). LC–MS experiments were per-
formed in a data-dependent acquisition manner using Xcalibur
2.0 software (Thermo Scientific). The five most intensive precur-
sor ions in a survey MS1 spectrum acquired in the FTICR over a
mass range of m/z 300–2000 were selected and fragmented in
the linear ion trap by collision-induced dissociation. The ion
selection threshold was 500 counts.
Raw experimental files were processed by TurboSequest search
using BioWorks 3.2 (ThermoFinnigan). The resulting MS/MS peak
lists in .dta format were combined within each experiment using
an in-house written Perl script and submitted for peptide/protein
identification to the Mascot 2.2 database-searching program (Ma-
trix Science, London, UK) using the most recent Swiss–Prot or IPI
mouse database with a fragment ion mass tolerance of 0.20 amu
y5+2H
594.5?
100%
75%
50%
25%
F
R
S
A
Y+536
L
E
1,420.54 AMU, +2 H (Parent Error: -0.72 ppmR)
E
L
Y+536
A
S
F
y5
OH
OH
O
S
N
O
parent+2H-H2O
y6+2H
HO
HO
O
S
N
y5+1
N
N
H
O
O
y3
y4
b2
y6
b6
b4
1000
y2
b3
b5
0%
0
500
y5
100%
F
R
S
A
Y+281
parent+2H-H2O
L
E
1,165.48 AMU, +2 H (Parent Error: -2.4 ppmR)
E
L
Y+281
A
S
F
75%
50%
25%
y7-NH3-H2O+2H
OH
O
S
N
O
y3
y5+1
y4
N
567.2?
565.3?
H2N
O
OH
y4+1
y6
y2
b2
b4
b5
y1
b6
1000
b3
0%
0
250
500
750
b4
HN
100%
F
R
S
Y+216,4148.46 AMU, +2 HL(Parent Error: -1.3 ppm)
E
R
EA
L
Y+264
A
S
F
y3
y7-H2O-H2O+2H
75%
50%
25%
0%
y2
parent-H2O+2H
557.4?
N
O
566.5?
482.4?
549.2?
b5
b4+1
517.9?
b6
y1
O
S
N
O
y4 y5
0
250
500
750
1000
m/z
HO
OH
Fig.5. ESI–MS/MS identification of different benzoxazole products after nitro-to-amino reduction of FSAY(3-NO2)LER (10
lM) and tagging with 10 mM APPD and 100 lM
K3Fe(CN)6. The structures shown in insets contain different substitutions in position 6 of the original 3-NT ring (the fluorescent 2-phenylbenzoxasole core is circled).

190
Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
Statistical analysis
is suggested based on previous structural analysis of benzylamine
reaction with 4-methylcatechol [14]. Importantly, no significant
remaining amounts of FSAY(3-NH2)LER (m/z 900.5) or FSAY(3-
NO2)LER (m/z 930.4) were observed after the reaction. A small
yield (ꢀ7%) of a product with m/z 1149.8 (product III) was ob-
served in the MALDI–TOF MS spectrum (Fig. 4A). This mass is con-
sistent with the intramolecular addition of the N-terminal amino
group to position 6 in the final benzoxazole, a hypothetical struc-
ture of which is shown in the inset of Fig. 5C and based on the ob-
served MS/MS fragmentation pattern. Analogous products have
been observed on derivatization of 3-AT-containing peptides with
ABS [32]. Importantly, product III will contribute to the total fluo-
rescence at 510 nm and nearly coelutes with products I and II in
the reverse-phase chromatogram (HPLC fractions collected be-
tween 26 and 29 min in Fig. 4B), as confirmed by capLC–FTICR–
MS analysis (data not shown).
For quantitative analysis, all measurements were performed in
triplicate and the data are presented as means standard devia-
tions. The limits of detection were calculated from concentration
dependencies that included at least seven data points over a lim-
ited concentration range as LOD = 3sB/b, where sB (standard error
of blank) was obtained from the linear regression analysis
(y = a + bx) as a standard deviation of y and b is the slope of the best
linear fit line from the linear regression [30].
Results
Synthesis of tagging reagent (APPD)
The synthetic scheme for the preparation of APPD is shown in
Fig. 1. The key intermediate, cis-diol pyrrolidine, was derived from
(3R,4S)-1-benzyl-3,4-dihydroxypyrrolidine-2,5-dione 1, which was
prepared by the OsO4-catalyzed enantioselective dihydroxylation
of N-benzyl maleimide [31]. Compound 1 was protected with
2,2-dimethyloxypropane, which was sequentially reduced by
LiAlH4 at 80 °C and by hydrogenation over 20% Pd hydroxide/C cat-
alyst to remove the benzyl group, affording the protected pyrrole 4.
The crude product 4 was purified by precipitating its hydrochloride
salt in ether, and the overall yield of the first four steps was 33%.
Product 4 was treated with 4-cyanobenzene-1-sulfonyl chloride
to give compound 5 in 87% yield. The conversion of compound 5
to the target molecule 6 was achieved by the hydrogenation over
10% Pd/C catalyst in methanol containing 2% acetic acid. The
deprotection of cis-diol with 2.0 M HCl solution afforded the target
compound 6.
A
0.4
3
pH 7.8
2
pH 5.8
0.2
0.0
3
1
1
2
5
Fluorogenic derivatization of 3-NT-containing peptides with APPD
0
10
Elution time (min)
15
20
The reaction sequence for the fluorogenic derivatization of a 3-
AT-containing peptide with APPD is shown in Fig. 2, in accord with
the published reaction scheme for benzylamine [15]. Prior to fluo-
rescent tagging, 3-NT-containing peptides (mass shift of +45 amu
relative to Tyr) are completely reduced by 10 mM sodium dithio-
nite to respective 3-AT-containing sequences (mass shift of +15
relative to Tyr), as confirmed by MS analysis (data not shown).
Concentrations of reagents for fluorogenic derivatization were
optimized based on yields of fluorescent products assessed by fluo-
B
0.020
0.015
0.010
0.005
0.000
4
pH 7.8
3
2
pH 5.8
rescence spectrometry. For a 3-NT concentration of 5 lM, an in-
crease of APPD concentration of more than 10 mM (i.e., at more
than 1000-fold molar excess of the reagent) did not significantly
affect the yield of a fluorescent product with emission maximum
at 510 nm and excitation maximum at 360 nm (data not pre-
sented). A concentration dependence of the fluorescence intensity
at 510 nm on the concentration of oxidant, K3Fe(CN)6, showed sat-
1
uration behavior at approximately 100
FSAY(3-NH2)LER with 10 mM APPD and 100
sulted in a nearly linear dependence of the fluorescence intensity
at 510 nm on the peptide concentration between 0 and 20
l
M (Fig. 3A). Tagging
0
5
10
Retention time (min)
15
20
l
M K3Fe(CN)6 re-
Fig.6. Boronic affinity chromatography of APPD-tagged synthetic peptide (A) and of
tryptic digest of nitrated rabbit Ph-b mixed with proteins from lysate of C2C12
cultured cells (B) detected by fluorescence (excitation and emission wavelengths of
360 and 510 nm, respectively). In panel A, 200
reduction of 3-NT to 3-AT and incubation with 10 mM APPD and 100
for 1 h was injected onto the phenylboronic column equilibrated with buffer A
containing 50 mM NH4HCO3 (pH 7.8) in the absence (trace 1) or presence (traces 2
and 3) of 30% (v/v) acetonitrile. The arrow indicates switching from buffer A to
water in the mobile phase without changing the percentage of organic solvent.
Trace 2 represents a blank injection (washing run) after the separation performed in
the absence of organic solvent (trace 1). Panel B shows boronic affinity separation of
lM
(Fig. 3B and C). MALDI–TOF MS analysis (Fig. 4A) shows that APPD
tagging produced two major benzoxazole products of the peptide
with m/z 1421.9 (product I) and 1166.8 (product II), which corre-
spond to peptide derivatives of +536 and +281 amu, respectively.
The respective structures for these products are displayed in the
insets of Fig. 5A and B. LC–MS/MS analysis confirmed that in the
sequence of tagged peptides these characteristic mass shifts were
located on the Tyr residue, Tyr+536 and Tyr+281, respectively,
based on the mass difference between the y4 and y3 fragments
in the spectra (Figs. 5A and B). Here the addition of APPD and
ammonia, respectively, to the 6-position of the benzoxazole ring
ll of 10
lM FSAY(3-NO2)LER after
l
M K3Fe(CN)6
tryptic peptides from nitrated Ph-b (1 lg protein containing 100 pmol 3-NT) only
(trace 1) and in the presence of C2C12 cell proteins (trace 2: 0.2 mg total protein;
trace 3: 0.5 mg total protein; trace 4: 1 mg total protein) reduced and tagged in the
same conditions.

Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
191
A
100
50
3.5E+4
701.7
1166.6
1421.7
741.6
905.8
1252.7
0
700
960
1220
1480
1740
2000
y5+2
594.3?
y5
100%
50%
B
C
A
Y+536
1,420.54 LAMU, +2 H (Parent Error: -1.8 ppm)
E
E
L
Y+536
A
S
RF
594.9?
y5+1
parent+2H-H2O
y6+2
638.0?
703.0?
y3
y4
b5
y6
b2
y2
E
b4
0%
250
500
750
1000
1250
y5
100%
S
A
Y+281
L
1,165.48 AMU, +2 HE(Parent Error: -2.9 ppm)
R
parent+2H-H2O
L
Y+281
A
S
F
y7-NH3-H2O+2H
y3
y5+1
y4
50%
0%
567.2?
565.3?
y6
y4+1
b5
y2
b3
b2
b4
y1
b6
200
400
600
800
1000
m/z
Fig.7. Analysis of fluorescent boronate affinity HPLC fractions (13–18 min in chromatogram 3, Fig. 6A) by MALDI–TOF (A) and capLC–FTICR–MS (B and C). Panels B and C
represent MS/MS spectra for APPD-tagged FSAY⁄LER ions with mass shifts of Y+536 (m/z 1421.5) and Y+281 (m/z 1166.6), respectively, present in panel A.
In addition to the 510-nm fluorescence (benzoxazole), fluores-
cence spectrometry detected the formation of another fluorescent
species with an emission maximum at approximately 430 nm that
is also present in the control samples (reagents without peptide;
see spectra in Fig. 3C). We did not focus on the identification of
fluorescent side products in this study; however, reverse-phase
HPLC shows the presence of several fluorescent peaks besides the
major peak with a retention time of approximately 27 min that ap-
pear at increasing concentrations of the oxidant, K3Fe(CN)6 (cf.
chromatograms 1 and 2 in Fig. 4B). This is the reason for using rel-
ure, the relative contribution of this background fluorescence is
higher at lower concentrations of nitropeptides, defining a detec-
tion limit of approximately 0.1 lM 3-NT by fluorescence spectrom-
etry [32]. However, specifically for APPD tagging, we have the
unique opportunity to separate the APPD-tagged peptides from
fluorescence side products by boronic acid affinity chromatogra-
phy and to monitor them by their specific fluorescence at 510 nm.
Boronate affinity separation and enrichment of a fluorescent APPD-
tagged synthetic model peptide
atively low concentrations of the oxidant (not more than 100
lM;
see Fig. 3A), particularly at 3-NT concentrations below 1 M. These
l
The synthetic APPD reagent contains two separate functional
groups: the benzylamine group for formation of fluorescent benz-
oxazole and the cis-diol group that associates in a pH-dependent
manner with boronate anion. The latter properties can be used
for enrichment of the APPD-derivatized 3-NT peptides by boronate
affinity chromatography. We first tested the boronate affinity
enrichment of an APPD-tagged model peptide on a modified silica
solid phase containing a sulfonamide-substituted phenylboronic
acid with a boronate pKa of 7.5 [25]. This solid phase displays an
enhanced boronate binding of alkyl- and aryl-cis-diol compounds
results are in accord with our previous study on 3-AT tagging with
another benzylamine derivative, ABS, where we determined that
maximum yields of benzoxazole product can be achieved at a ratio
of K3Fe(CN)6 to 3-AT of approximately 5:1 and that a further in-
crease in the oxidant concentration results mostly in fluorescent
side product formation [32]. The background fluorescence after
tagging with ABS was attributed to the reagent background and,
potentially, to the formation of protein oxidation products during
the course of the derivatization procedure. Irrespective of its nat-

192
Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
under neutral conditions. Fig. 6A shows the boronate affinity HPLC
with fluorescence detection at 510 nm of APPD-tagged FSAY(3-
NO2)LER using a column (4.6 i.d. Â 50 mm length) packed with
the synthetic sulfonamide-substituted phenylboronic acid. The
APPD-tagged peptide was bound to the column in 50 mM
NH4HCO3 buffer (pH 7.8) and washed for 10 min (at 1 ml/min)
with this buffer. Subsequently, the tagged peptide was eluted with
water (ꢀpH 5.8) during the next 10 min (trace 1 in Fig. 6A). Further
acidification of the elution buffer is not necessary because it does
not yield greater amounts of APPD-tagged peptides but requires
additional efforts to clean up samples for subsequent MS analysis.
To account for nonspecific binding to the column material due to
hydrophobic interactions, increasing concentrations of ACN were
added to both binding and elution buffers. We found that the addi-
tion of 30% (v/v) ACN effectively blocks nonspecific affinity during
both washing and elution steps (Fig. 6A, trace 3). Without organic
solvent, a significant accumulation of the fluorescent peptide oc-
curs on the column and can be washed out by 30% ACN in the mo-
bile phases during a subsequent blank run (trace 2 in Fig. 6A). The
fluorescent fractions eluting after acidification (between 13 and
18 min) were collected and analyzed by MALDI–TOF MS (Fig. 7A)
and capLC–FTICR–MS (Fig. 7B and C), which confirmed that the
eluate contained predominantly APPD-tagged FSAY⁄LER in the
form of product I (m/z 1421.97) and product II (m/z 1166.83)
(the respective structures are shown in the insets of Fig. 5A and
B). It appears that both APPD derivatives of FSAY⁄LER, containing
either one or two cis-diol groups, have a comparable affinity to
the column based on a comparable abundance of the respective
peptide ions in the MALDI mass spectrum (Fig. 7A).
specific 510-nm fluorescence detection (traces 1 and 2 in Fig. 8A).
capLC–FTICR–MS analysis followed by Sequest search against
mouse and rabbit protein databases identified native peptides from
more than 200 mouse proteins (not shown) and multiple native, but
no APPD-tagged, rabbit Ph-b peptides in the flow-through (from 1 to
10 min) fractions (Fig. 9A). In contrast, the fluorescent boronate
affinity chromatographic fractions contained only APPD-tagged
peptides from the rabbit Ph-b sequence (Fig. 9B).
The specific enrichment of APPD-tagged peptides via boronate
affinity chromatography can be demonstrated by comparative
analysis of the chromatograms obtained for nitropeptides tagged
with either APPD or ABS, the latter of which differs from APPD
by the lack of the cis-diol pyrrolidine ring. Tagging with ABS results
in the formation of a fluorescent derivative with emission maxi-
mum at 480 nm and approximately 2-fold higher chemical and/
or fluorescence quantum yields as compared with APPD
(Fig. 10A), in accord with our previous results [32]. The ABS deriv-
atives did not bind to the column and eluted during the washing
step immediately after injection, unlike the APPD derivatives
(Fig. 10B). These results clearly demonstrate the significance of
A
pH 7.8
1,2
0.3
0.2
0.1
0.0
pH 5.8
Qualitative enrichment of nitrated peptides from a biological matrix
It is well recognized that the presence of biological matrix may
interfere with, and even completely block, both the specific chem-
ical derivatization of low-abundant analytes and subsequent LC
and MS analysis [33]. We tested our APPD derivatization approach
using small aliquots of in vitro nitrated protein, Ph-b, mixed with
100- to 1000-fold excess of proteins derived from C2C12 cultured
cells to obtain levels of 100–2000 pmol protein-bound 3-NT/mg to-
tal protein in the mixture. In terms of molar ratios, the amount of
100 pmol 3-NT/mg protein would represent average Tyr nitration
levels of 0.005 mol 3-NT/mol protein with an average molecular
weight of 50 kDa. That is, statistically in these mixtures, only 1 of
200 proteins would contain a nitrated Tyr residue, and each ni-
trated protein would yield only 1 nitropeptide but, on average,
50–100 non-nitrated peptides on tryptic digestion. Fig. 6B shows
2
1
3
3
0
5
10
15
20
1.2
1.0
0.8
0.6
0.4
0.2
0.0
B
2
pH 7.8
pH 5.8
that a protein digest containing 1
lg (ꢀ10 pmol) of nitrated Ph-b
and 100 pmol of 3-NT can be derivatized with APPD to yield a fluo-
rescent product with a 510-nm emission maximum that is success-
fully enriched by boronic affinity chromatography and detected by
its specific fluorescence. Importantly, the presence of an excess
amount of non-nitrated peptides (trypsin digests from 0.2 to
1 mg C2C12 proteins) did not reduce the yield of fluorescent benz-
oxazole products based on the 510-nm fluorescence.
1
2
1
Additional proof for the specific binding of 510-nm fluorescent
products (benzoxazole) to the boronate column was obtained
through a proteomic analysis of nonretained peptides (HPLC
fractions from 1 to 10 min) and more tightly retained peptides
(13–18 min), as shown in Fig. 8. In this experiment, slightly higher
0
5
10
15
20
Retention time (min)
Fig.8. Boronate affinity chromatography of model nitrated protein, Ph-b, mixed
with high amounts of C2C12 cell lysate protein, through APPD tagging of digested
proteins and detected by specific fluorescence (A) or 280-nm absorbance (B).
amounts of nitrated Ph-b (5 lg) were used to obtain better coverage
Nitrated Ph-b (5 lg) was analyzed either alone (trace 1) or in the presence of 0.2 mg
of Ph-b peptides. The boronate affinity chromatograms after APPD
derivatization of nitrated Ph-b digest alone and in the presence of
0.2 mg of protein digest from C2C12 cells show significant differ-
ences in matrix peptide content in both fractions based on 280-
nm absorbance (traces 1 and 2 in Fig. 8B) but no changes in the yield
of fluorescent products in the boronate affinity fractions based on
of C2C12 proteins (trace 2); trace 3 represents a reagent control (APPD tagging in
the absence of 3-NT). 3-NT-containing peptides were reduced to corresponding 3-
AT peptides and tagged with 10 mM APPD and 50 lM K3Fe(CN)6 in 0.2 M NH4HCO3
(ABC) buffer at pH 9.0, bound to boronate affinity column in 0.1 M ABC buffer at pH
7.8 (0–10 min), and eluted with H2O at pH 5.8 (10–20 min) at a flow rate of 1 ml/
min. Boronate affinity (13–18 min) and flow-through fractions (1–10 min) were
collected for LC–MS analysis.

Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
193
A
B
Fig.9. Sequence coverage for Ph-b in flow-through (A) and boronate affinity (B) fractions collected during chromatographic run shown in Fig. 8 (trace 2). In panel B, APPD
derivatization products detected in the sample (Y+536, Y+264, and Y+281) are shown in bold, italic, and underlined font, respectively.
the cis-diol group in the APPD reagent for boronate affinity enrich-
ment. For comparison of our synthetic sulfonamide-substituted
phenylboronic acid, boronate affinity chromatography of the
APPD-tagged peptides was conducted on a commercially available
cence of APPD-tagged peptides (excitation and emission wave-
lengths of 360 and 510 nm, respectively). The dependence of the
peak areas on the original concentration of 3-NT is shown in
Fig. 11A. Importantly, the results are presented for relatively low,
column (TSK-Gel Boronate-5PW, 1000 Å, 10
l
m, 7.5 mm
biologically relevant 3-NT concentrations (<1 lM) in the presence
i.d. Â 75 mm length) containing immobilized m-aminophenylbo-
ronic acid under the same HPLC conditions. This column displayed
a similar affinity for the APPD-tagged peptides from C2C12 protein
digests as our synthetic column (data not presented), providing
additional evidence for the role of pH-dependent complex forma-
tion between the APPD cis-diol function and the boronate anion
of the stationary phase.
of highly abundant non-nitrated peptides (digest of 1 mg total pro-
tein from C2C12 cultured cell lysate). A linear regression analysis
gave the LOD of approximately 68 nM 3-NT, corresponding to
13 pmol of 3-NT per injection. This value is lower than the LOD
for fluorescence spectrometry analysis alone, namely 95 nM 3-NT
(19 pmol/sample) (Fig. 11B). Although the calculated values for
APPD tagging are comparable to those reported for ABS tagging
(0.09 lM and 40 pmol 3-NT [32]), quantitation through APPD tag-
ging/boronate affinity chromatography can be performed in the
Quantitative analysis of APPD-tagged 3-NT in complex peptide
mixtures from proteins nitrated in vitro by boronate affinity
chromatography with fluorescence detection
presence of at least 10-fold larger amounts of biological matrix
(1 mg for APPD vs. 100 lg for ABS). This feature offers an opportu-
nity of enrichment and accumulation of APPD-tagged peptides
from large amounts of biological material that is very important,
particularly for sequence-specific analysis of low-abundant protein
nitration in tissues.
In the previous section, we tested the qualitative enrichment of
APPD-tagged peptides from a complex biological matrix. To test
whether boronate affinity chromatography of APPD-tagged nitro-
peptides may represent a quantitative method to analyze low lev-
els of protein tyrosine nitration in complex peptide mixtures, we
nitrated Ph-b by peroxynitrite in vitro and mixed with an excess
of proteins from lysate of C2C12 cultured cells. 3-NT concentra-
tions were quantified through specific 430-nm absorbance as de-
scribed in Materials and methods. After trypsin digestion,
reduction of 3-NT with sodium dithionite, and derivatization with
APPD as described above, samples were separated by boronate
affinity chromatography and detected through the specific fluores-
Sequence-specific characterization of protein Tyr nitration by
fluorogenic tagging with APPD and boronate affinity chromatography
enrichment of tagged peptides
To test whether low-level protein tyrosine nitration in complex
protein mixtures can be assessed in a sequence-specific matter, we
analyzed by capLC–FTICR–MS the fluorescent boronate affinity
fractions of APPD-tagged peptides from peroxynitrite-exposed

194
Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
A
A
ABS
500
400
300
200
100
0
1000
500
y = a + bx
Parameter Value Error
------------------------------------------
a
b
51.083 9.124
921.08 21.15
-----------------------------------------
R
SD
N
P
0.9987
18.96
7
<0.0001
APPD
LOD=0.068 µM (13 pmol)
0
300
400
500
600
700
0.0
0.5
3-NT (
1.0
Wavelength (nm)
1.0
0.5
0.0
µM)
B
pH 7.8
60
B
y = a + bx
Parameter Value Error
ABS
pH 5.8
----------------------------------------------
a
b
1.006 0.652
46.771 1.512
----------------------------------------------
40
20
0
R
SD
N
7
P
0.9974
1.355
<0.0001
APPD
LOD=0.095
0.5
µM (19 pmol)
0.0
1.0
0
5
10
15
20
Elution time (min)
3-NT (µM)
Fig.10. Comparison of fluorescence spectra (A) and boronate affinity chromato-
graphic runs detected by fluorescence (B) of nitrated C2C12 cultured cell protein
Fig.11. Validation of the fluorogenic APPD derivatization as quantitative method
for peptide 3-NT determination by boronate affinity chromatography with fluores-
cence detection (A) and by fluorescence spectrometry (B). 3-NT concentration in
digests (250
sodium dithionite and tagged with 10 mM of either APPD (solid lines) or ABS
(dotted lines) and 100 M K3Fe(CN)6 in 100 mM PBS (pH 9.0). Chromatographic
lg total protein containing 500 nmol 3-NT), reduced with 20 mM
samples (0–1
nitrated Ph-b, containing approximately 10 mol 3-NT/mol protein (1
2.5 g Ph-b), to 0.5 mg of C2C12 cell lysate protein. Samples were digested with
trypsin, reduced by 10 mm sodium dithionite, and derivatized with 2 mM APPD and
20 M K3Fe(CN)6 in 50 mM ammonium bicarbonate buffer (pH 9.0). Boronate
l
M) was achieved through the addition of different amounts of
l
l
M 3-NT/
conditions were as in the legend to Fig. 8.
l
l
affinity chromatography conditions were as in the legend to Fig. 8. SD, standard
deviation.
C2C12 cells. Protein nitration was achieved at a level of approxi-
mately 1 nmol of 3-NT per 0.5 mg of total protein. The efficiency
of APPD tagging/boronate affinity enrichment was addressed
through comparison of capLC–FTICR–MS data with those for non-
tagged or ABS-tagged samples prepared from the same amount
of material in similar conditions except for affinity separation of
tagged peptides. Analysis of nontagged or ABS-tagged nitrated
C2C12 protein digests identified more than 100 proteins at the
highest probability settings; no ABS-tagged peptides were revealed
(data not shown). In contrast, APPD tagging/boronate affinity
enrichment resulted in the detection of two APPD-tagged peptides
(Tyr+536 amu), indicating two tyrosine nitration sites in the se-
quence of mouse actin 1 (Fig. 12A and B). Importantly, no nontag-
ged peptides from actin 1 (Fig. 12C) or other proteins (data not
shown) can be identified in the samples after boronate affinity
enrichment, in contrast to nonpurified sample (Fig. 12D). Because
the yields of fluorescent benzoxazole products are higher in the
ABS-tagged samples (Fig. 10A), our data suggest that for successful
sequence-specific characterization of tagged 3-NT, the absolute
amounts of an analyte, which is tagged 3-NT, are less important
than the relative abundance in the sample. Therefore, our approach
of APPD tagging/boronate affinity enrichment of nitropeptides, in
addition to accumulation of APPD-tagged peptides from larger
amounts of biological material, offers the option of removal of non-
tagged matrix peptides, which suppress the sequence-specific MS
analysis of low-abundant protein tyrosine nitration.
Discussion
In this study, a novel chemical tagging reagent, APPD, was syn-
thesized and applied to fluorogenic derivatization and boronate
affinity enrichment of 3-NT-containing peptides. Based on the
derivatization of a synthetic peptide, FSAY(3-NO2)LER, we estab-
lished a tagging protocol optimized for maximum yields of fluores-
cence benzoxazole derivatives from 3-NT. Although different
fluorescent products of APPD-tagged 3-NT peptides are detected
by MS analysis, with different substitutions in position 6 of the
benzoxazole, they all contain the 2-benzoxazole core and contrib-
ute to the measured fluorescence with emission maximum at
510 nm and excitation maximum at 360 nm. Fluorescent 2-benz-
oxazole derivatives are formed via condensation with the benzyl-
amine moiety of APPD specifically from protein/peptide 3-AT
and, potentially, from 3-hydroxytyrosine (DOPA) [14,15] and 5-
hydroxytryptophan [32], two another important biomarkers of oxi-
dative protein modification. The latter two species do not require a

Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
195
A
B
C
D
Fig.12. Representative capLC–FTICR–MS/MS spectra of APPD-tagged peptides in the protein tryptic digest from peroxynitrite-exposed C2C12 cells obtained after boronate
affinity enrichment of a sample containing 1 nmol of 3-NT in 500 l
g of protein. Panels A and B represent MS/MS spectra of two peptides, SY⁄ELPDGQVITIGNER and
QEY⁄DESGPSIVHR (mass shift on both of +536 relative to Tyr) from the sequence of mouse cytoplasmic actin 1 (gene name: actb). Panel C shows protein sequence coverage.
For comparison, panel D shows sequence coverage for this protein observed without APPD tagging and boronate affinity enrichment.
reduction step to form benzoxazole on tagging with benzylamines
[14] and can be fluorometrically assessed separately from 3-NT
provided that the respective nonreduced controls are included. In
our experiments with C2C12 cells exposed to peroxynitrite, we
did not observe significant amounts of fluorescence products emit-
ting at 510 nm in nonreduced samples. However, unidentified fluo-
rescent side products with an emission maximum at 430 nm may
also form from APPD irrespective of the presence of 3-NT that
causes detection limits of 95 and 68 nM, respectively, for fluores-
cence spectroscopy and boronate affinity HPLC–fluorescence anal-
ysis of APPD-tagged 3-NT. These numbers are better than those
reported previously for fluorogenic 3-NT tagging with ABS, which
lacks the affinity functionality [32]. The critical advantage of APPD
tagging/boronate affinity enrichment is that APPD-tagged peptides
will be separated from nontagged material and eluted virtually in a
single peak. Therefore, our approach offers a possibility for the
accumulation of APPD-tagged 3-NT peptides for both quantitative
fluorescence spectrometry and sequence-specific MS analysis pro-
vided that the amount of material is not limited (a common situa-
tion in studies of tissue biomarkers), suggesting its potential
application in the characterization of protein nitration in complex
biological systems.
Acknowledgments
This research was supported by the National Institutes of
Health (NIH, AG23551, AG25350) and a grant from Pfizer. The
authors thank D. Yakovlev (Biochemical Research Service Labora-
tory, University of Kansas) for the synthesis and purification of
the nitropeptide FSAY(3-NO2)LER.
References
[1] I.V. Turko, F. Murad, Protein nitration in cardiovascular diseases, Pharmacol.
Rev. 54 (2002) 619–634.
[2] M.R. Reynolds, R.W. Berry, L. Binder, Nitration in neurodegeneration:
deciphering the ‘‘Hows’’ ‘‘nYs’’, Biochemistry 46 (2007) 7325–7336.

196
Derivatization of 3-NT-containing peptides / E.S. Dremina et al. / Anal. Biochem. 418 (2011) 184–196
[3] H. Ischiropoulos, Biological selectivity and functional aspects of protein
[18] N.L. Wilson, N.G. Karlsson, N.H. Packer, Enrichment and analysis of
glycoproteins in the proteome, in: G.B. Smejkal, A. Lazarev (Eds.), Separation
Methods in Proteomics, CRC Press, Boca Raton, FL, 2006, pp. 345–362.
[19] D.S. Hage, Clinical applications of high-performance affinity chromatography,
Adv. Chromatogr. 42 (2003) 377–420.
[20] R.P. Singhal, S. DeSilva, M. Shyamali, Boronate affinity chromatography, Adv.
Chromatogr. 31 (1992) 293–335.
[21] A. Foettinger, A. Leitner, W. Lindner, D. Christian, Solid-phase capture and
release of arginine peptides by selective tagging and boronate affinity
chromatography, J. Chromatogr. A 1079 (2005) 187–196.
[22] I.C. Smith, B.L. Carson, T.L. Ferguson, Osmium: an appraisal of environmental
exposure, Environ. Health Perspect. 8 (1974) 201–213.
[23] T. Najrana, Y. Saito, F. Uraki, K. Kubo, K. Yamamoto, Spontaneous and osmium
tetroxide-induced mutagenesis in an Escherichia coli strain deficient in both
endonuclease III and endonuclease VIII, Mutagenesis 15 (2000) 121–125.
[24] W.A. Pryor, R. Cueto, X. Jin, W.H. Koppenol, M. Ngu-Schwemlein, G.L.
tyrosine nitration, Biochem. Biophys. Res. Commun. 305 (2002) 776–783.
[4] R. Radi, Nitric oxide, oxidants, and protein tyrosine nitration, Proc. Natl. Acad.
Sci. USA 101 (2004) 4003–4008.
[5] P. Pacher, J.S. Beckman, L. Liaudet, Nitric oxide and peroxynitrite in health and
disease, Physiol. Rev. 87 (2007) 315–424.
[6] K.S. Aulak, M. Miyagi, L. Yan, K.A. West, D. Massillon, J.W. Crabb, D.J. Stuehr,
Proteomic method identifies proteins nitrated in vivo during inflammatory
challenge, Proc. Natl. Acad. Sci. USA 98 (2001) 12056–12061.
[7] J. Kanski, S.-J. Hong, C. Schöneich, Proteomic analysis of protein nitration in
aging skeletal muscle and identification of nitrotyrosine-containing sequences
in vivo by nanoelectrospray ionization tandem mass spectrometry, J. Biol.
Chem. 280 (2005) 24261–24266.
[8] J. Kanski, A. Behring, J. Pelling, C. Schöneich, Proteomic identification of 3-
nitrotyrosine-containing rat cardiac proteins: effects of biological aging, Am. J.
Physiol. 288 (2005) H371–H381.
[9] J. Kanski, M.A. Alterman, C. Schöneich, Proteomic identification of age-
dependent protein nitration in rat skeletal muscle, Free Radic. Biol. Med. 35
(2003) 1229–1239.
Squadrito, P.L. Uppu, R.M. Uppu, A practical method for preparing
peroxynitrite solutions of low ionic strength and free of hydrogen peroxide,
Free Radic. Biol. Med. 18 (1995) 75–83.
[10] C.A. Sacksteder, W.-J. Qian, T.V. Knyushko, H. Wang, M.H. Chin, G. Lacan, W.P.
Melega, D.G. Camp, R.D. Smith, D.J. Smith, T.C. Squier, D.J. Bigelow,
Endogenously nitrated proteins in mouse brain: links to neurodegenerative
disease, Biochemistry 45 (2006) 8009–8022.
[11] B. Ghesquiere, N. Colaert, K. Hensens, L. Dejager, C. Vanhaute, K. Verleysen, K.
Kas, E. Timmerman, M. Goethals, C. Libert, J. Vandekerckhove, K. Gevaert, In
vitro and in vivo protein-bound tyrosine nitration characterized by diagonal
chromatography, Mol. Cell. Proteomics 8 (2009) 2642–2652.
[25] X.-B. Li, J. Pennington, J.F. Stobaugh, C. Schöneich, Synthesis of sulfonamide-
and sulfonyl-phenylboronic acid modified silica phases for boronate affinity
chromatography at physiological pH, Anal. Biochem. 372 (2008) 227–236.
[26] K. Ikehata, T.G. Duzhak, N.A. Galeva, T. Ji, Y.M. Koen, R.P. Hanzlik, Protein
targets of reactive metabolites of thiobenzamide in rat liver in vivo, Chem. Res.
Toxicol. 21 (2008) 1432–1442.
[27] B.T. Hansen, S.W. Davey, A.J. Ham, D.C. Liebler, P-Mod: an algorithm and
software to map modifications to peptide sequences using tandem MS data, J.
Proteome Res. 4 (2005) 358–368.
[28] A. Keller, A.I. Nesvizhskii, E. Kolker, R. Aebersold, Empirical statistical model to
estimate the accuracy of peptide identifications made by MS/MS and database
search, Anal. Chem. 74 (2002) 5383–5392.
[29] A.I. Nesvizhskii, A. Keller, E. Kolker, R. Aebersold, A statistical model for
identifying proteins by tandem mass spectrometry, Anal. Chem. 75 (2003)
4646–4658.
[30] J.C. Miller, J.N. Miller, Statistics for Analytical Chemistry, Ellis Horwood,
London, 1992. pp. 115–117.
[31] A. Mitsugu, S. Kyozo, Characterization of a model compound for the lysine
tyrosylquinone cofactor of lysyl oxidase, Biochem. Byophys. Res. Commun. 218
(2001) 193–199.
[32] V.S. Sharov, E.S. Dremina, N.A. Galeva, G.S. Gerstenecker, X.-B. Li, R.T.
Dobrowsky, J.F. Stobaugh, C. Schöneich, Fluorogenic tagging of protein 3-
nitrotyrosine with 4-(aminomethyl) benzenesulfonic acid for quantitative
analysis of protein tyrosine nitration, Chromatographia 71 (2010) 37–53.
[33] T. Nuriel, R.S. Deeb, D.P. Hajjar, S.S. Gross, Protein 3-nitrotyrosine in complex
biological samples: quantitation by high-pressure liquid chromatography/
electrochemical detection and emergence of proteomic approaches for
unbiased identification of modification sites, Methods Enzymol. 441 (2008)
1–17.
[12] G. Nikov, V. Bhat, J.S. Wishnok, S.R. Tannenbaum, Analysis of nitrated proteins
by nitrotyrosine-specific affinity probes and mass spectrometry, Anal.
Biochem. 320 (2003) 214–222.
[13] Q. Zhang, W. Qian, T.V. Knyuschko, T.R.W. Clauss, S.O. Purvine, R.J. Moore, C.A.
Sacksteder, M.H. Chin, D.J. Smith, D.G. Camp II, D.J. Bigelow, R.D. Smith, A
method for selective enrichment and analysis of nitrotyrosine-containing
peptides in complex proteome samples, J. Proteome Res. 6 (2007) 2257–2268.
[14] J. Pennington, C. Schöneich, J.F. Stobaugh, Selective fluorogenic derivatization
with isotope coding of catechols and 2-aminophenol with benzylamine: a
chemical basis for the relative determination of 3-hydroxytyrosine and 3-
nitrotyrosine peptides, Chromatograhia 66 (2007) 649–659.
[15] V.S. Sharov, E.S. Dremina, J. Pennington, J. Killmer, C. Asmus, M. Thorson, S.J.
Hong, X.-B. Li, J.F. Stobaugh, C. Schöneich, Selective fluorogenic derivatization
of 3-nitrotyrosine and 3,4-dihydroxyphenylalanine (DOPA) in peptides:
a
method designed for quantitative proteomic analysis, Methods Enzymol. 441
(2008) 19–32.
[16] A. Bergold, W.H. Scouten, Borate chromatography, Chem. Anal. 66 (1983) 149–
187.
[17] X.-C. Liu, W.H. Scouten, Miscellaneous methods in affinity chromatography: I.
Boronic acids as selective ligands for affinity chromatography,
Biochromatography (2002) 307–317.