Capturing labile sulfenamide and sulfinamide serum albumin adducts of carcinogenic arylamines by chemical oxidation

Article abstract of DOI:10.1021/ac3028273

Aromatic amines and heterocyclic aromatic amines (HAAs) are a class of structurally related carcinogens that are formed during the combustion of tobacco or during the high temperature cooking of meats. These procarcinogens undergo metabolic activation by

Full text of DOI:10.1021/ac3028273

Article
pubs.acs.org/ac
Capturing Labile Sulfenamide and Sulfinamide Serum Albumin
Adducts of Carcinogenic Arylamines by Chemical Oxidation
Lijuan Peng and Robert J. Turesky*
Division of Environmental Health Sciences, Wadsworth Center, New York State Department of Health, Albany, New York 12201,
United States
S
* Supporting Information
ABSTRACT: Aromatic amines and heterocyclic aromatic
amines (HAAs) are a class of structurally related carcinogens
that are formed during the combustion of tobacco or during
the high temperature cooking of meats. These procarcinogens
undergo metabolic activation by N-oxidation of the exocyclic
amine group to produce N-hydroxylated metabolites, which
are critical intermediates implicated in toxicity and DNA
damage. The arylhydroxylamines and their oxidized arylnitroso
derivatives can also react with cysteine (Cys) residues of
glutathione or proteins to form, respectively, sulfenamide and
sulfinamide adducts. However, sulfur−nitrogen linked adducted proteins are often difficult to detect because they are unstable
and undergo hydrolysis during proteolytic digestion. Synthetic N-oxidized intermediates of 2-amino-1-methyl-6-phenylimidazo-
[4,5-b]pyridine (PhIP), a carcinogenic HAA produced in cooked meats, and 4-aminobiphenyl, a carcinogenic aromatic amine
present in tobacco smoke, were reacted with human serum albumin (SA) and formed labile sulfenamide or sulfinamide adducts
at the Cys34 residue. Oxidation of the carcinogen-modified SA with m-chloroperoxybenzoic acid (m-CPBA) produced the
arylsulfonamide adducts, which were stable to heat and the chemical reduction conditions employed to denature SA. The
sulfonamide adducts of PhIP and 4-ABP were identified, by liquid chromatography/mass spectrometry, in proteolytic digests of
denatured SA. Thus, selective oxidation of arylamine-modified SA produces stable arylsulfonamide-SA adducts, which may serve
as biomarkers of these tobacco and dietary carcinogens.
romatic amines and heterocyclic aromatic amines (HAAs)
are a class of structurally related carcinogens that are
and follow the kinetics of the lifetime of Hb or half-life of SA,
during chronic exposure, and augment the sensitivity of adduct
detection.8
A
formed during the combustion of tobacco or during the high
temperature cooking of meats.1,2 Certain aromatic amines, such
as 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA), are
classified as human carcinogens (Group 1), and several
prevalent HAAs, including 2-amino-1-methyl-6-phenylimidazo-
[4,5-b]pyridine (PhIP), which is present in tobacco smoke and
well-done cooked meats, have been listed as probable or
possible human carcinogens (Group 2A or 2B), on the basis of
toxicity data reviewed by the International Agency for Research
on Cancer.3,4 Thus, there is much concern about the health risk
associated with the exposure to these structurally related classes
of chemicals.
There is a critical need to establish long-term biomarkers that
can be implemented in molecular epidemiology studies to
evaluate the health risk of HAAs and aromatic amines. DNA
and protein adducts are a measure of the biologically effective
dose and have been used for human risk assessment of
environmental and dietary genotoxicants.5,6 However, DNA
adduct measurements are often precluded by the unavailability
of biopsy samples in large scale human studies. Protein
carcinogen adducts of hemoglobin (Hb) and serum albumin
(SA) have been used as an alternative to DNA adducts for
biomarkers of several different classes of genotoxicants.6,7
Stable carcinogen protein adducts are expected to accumulate
Arylnitroso compounds are reactive metabolites of aryl-
amines that bind to Cys residues of glutathione and proteins to
form arylsulfinamide adducts.6,9,10 The occurrence of arylsulfi-
namide adducts in intact proteins has been shown by mass
spectrometric analysis.11,12 However, arylsulfinamide adducts
undergo hydrolysis to form the arylamines and cysteine sulfinic
acid during the heat and chemical reduction conditions
employed to denature proteins13,14 or during proteolysis
(Scheme 1).6,12 The site of carcinogen adduction is inferred
by tandem mass spectrometry (MS/MS) sequencing and
identification of the sulfinic acid residue in peptides from
protease digests.11,13,14 In the case of 4-ABP, the location and
identity of the sulfinamide adduct at the human Hb-Cys93β
chain was proven by X-ray crystallography.15 Following that
hallmark study, the sulfinamide adducts of several arylamines
were measured indirectly as the parent amines following mild
acid treatment of Hb.16 However, covalent binding of N-
Received: October 10, 2012
Accepted: December 14, 2012
Published: December 14, 2012
© 2012 American Chemical Society
1065
dx.doi.org/10.1021/ac3028273 | Anal. Chem. 2013, 85, 1065−1072

Analytical Chemistry
Article
a
Scheme 1. Formation of SA-Cys34 Sulfenamide, Sulfinamide, and Sulfonamide Adducts with N-Oxidized Metabolites of PhIP
a
The genotoxic metabolite HONH-PhIP undergoes oxidation to form Nitroso-PhIP, which forms a sulfinamide adduct, following rearrangement of
the semimercaptal. HONH-PhIP forms reactive sulfate or acetate esters following conjugation reactions with phase II enzymes. These esters of
HONH-PhIP react with SA to form the sulfenamide adduct at Cys34. The superoxide generated during the oxidation of HONH-PhIP to Nitroso-
PhIP, or other reactive oxygen species (ROS), can convert the labile sulfenamide and sulfinamide adducts of SA-Cys34-PhIP to the stable
sulfonamide linkage.
oxidized metabolites of HAAs to Hb is low and alternative
protein carcinogen adduct biomarkers have been sought.2
Many genotoxicants also bind covalently to human SA.6−8,17
PhIP was reported to bind to human SA in vivo, but structures
of the PhIP-SA adduct(s) are unknown.18 We and others have
shown that N-oxidation products of PhIP covalently bind to
human SA in vitro.14,19−21 The genotoxic metabolites 2-
hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(HONH-PhIP) and 2-nitroso-1-methyl-6-phenylimidazo[4,5-
b]pyridine (NO-PhIP) were reacted with SA in vitro to
produce the N2-[cystein-S-yl-PhIP]-S-oxide at Cys34.14 This
sulfinamide adduct is also labile toward Pronase or even a weak
acid, and some portion of the PhIP bound to SA in vivo may
exist as the sulfinamide.18 N-(Acetyloxy)-2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP), a penultimate
metabolite of PhIP that forms covalent adducts with DNA, did
not form stable adducts with human SA. Instead, PhIP and 2-
amino-1-methyl-6-(5-hydroxy)-phenylimidazo[4,5-b]pyridine
(5-HO-PhIP), an aqueous reaction product of the proposed
nitrenium ion of PhIP, were identified in the proteolytic digest
of modified SA.21 We surmise that N-acetoxy-PhIP reacted with
SA to from an unstable sulfenamide adduct at Cys34, because
the pretreatment of SA with thiol specific-reagents prior to
reaction with N-acetoxy-PhIP greatly decreased the recovery of
PhIP and 5-HO-PhIP (Scheme 1).19−21
During the course of characterizing SA adduction products
with PhIP, we discovered that a portion of the sulfinamide
adduct at Cys34 underwent oxidation to the sulfonamide, N2-
[cystein-S-yl-PhIP]-S-dioxide.14,21 In contrast to the instability
of many arylsulfenamide and arylsulfinamide linkages, the
sulfur−nitrogen bond of the SA-PhIP sulfonamide adduct is
stable toward acid, heat, and reducing agents (Peng, L.,
unpublished observations). We discovered that 4-hydroxami-
nobiphenyl (HONH-4-ABP), the carcinogenic metabolite of 4-
ABP,22 also reacts with SA to form a sulfinamide linkage and
undergoes oxidation to produce the sulfonamide. Thus,
selective chemical oxidation of SA may allow us to biomonitor
labile Cys34 adducts of HAAs and arylamines through these
newly discovered sulfonamide adducts.
EXPERIMENTAL SECTION
■
Caution: PhIP and 4-ABP are carcinogens and should be handled
in a well-ventilated fume hood with the appropriate protective
clothing.
Chemicals and Materials. PhIP and 2-amino-1-[2H3]-
methyl-6-phenylimidazo[4,5-b]pyridine ([2H3]-PhIP, 99% iso-
1066
dx.doi.org/10.1021/ac3028273 | Anal. Chem. 2013, 85, 1065−1072

Analytical Chemistry
Article
topic purity) were purchased from Toronto Research
Chemicals (Toronto, ON, Canada). 2-Amino-1-methyl-6-
[2H5]-phenylimidazo[4,5-b]pyridine ([2H5]-PhIP, 99% isotopic
purity) was a gift from Dr. Mark Knize and Dr. Kristen Kulp,
(Lawrence Livermore National Laboratory, Livermore, CA).
Human SA, β-mercaptoethanol, iodoacetamide (IAA), dithio-
threitol (DTT), m-chloroperoxybenzoic acid (m-CPBA), and 4-
nitrobiphenyl were obtained from Sigma-Aldrich (St. Louis,
MO). LQQCPF was obtained from Biosynthesis (Lewisville,
TX). Sequencing grade trypsin and chymotrypsin were
purchased from Promega (Madison, WI). Pronase E, leucine
aminopeptidase, and prolidase were purchased from Sigma (St.
Louis, MO). All solvents were high-purity B & J Brand from
Honeywell Burdick and Jackson (Muskegon, MI). Isolute C18
SPE columns (25 mg) were from Biotage (Charlotte, NC).
Spire PEP tips were a gift from Thermo Scientific (Bellefonte,
PA).
Synthesis of HONH-PhIP, 2-Nitroso-1-methyl-6-
phenylimidazo[4,5-b]pyridine (NO-PhIP), N-Acetoxy-PhIP, 5-
HO-PhIP, and HONH-4-ABP. HONH-PhIP and HONH-[2H5]-
PhIP, HONH-4-ABP were prepared by reduction of their
respective nitro derivatives with hydrazine, using Pd/C as a
catalyst.23 NO-PhIP was obtained by oxidation of HNOH-PhIP
with K3Fe(CN)6.14 N-Acetoxy-PhIP was prepared by reaction
of HONH-PhIP with acetic anhydride,21 and 5-HO-PhIP was
obtained by solvolysis of N-acetoxy-PhIP.21,24
Synthesis of PhIP Sulfinamide and Sulfonamide Adducts
with LQQCPF. LQQCPF was reacted with NO-PhIP in H2O to
form the PhIP sulfinamide LQQC*PF (C-[SO]-PhIP).14
The sulfonic acid of LQQCPF and the sulfonamide adduct of
PhIP were obtained by oxidation of LQQCPF or LQQC*PF
(C-[SO]-PhIP) (1 nmol) with m-CPBA (1.5 nmol) in 0.1 M
phosphate buffer (pH 7.4) at room temperature for 30 min.
Another 1.5 nmol of m-CPBA was added and the reaction
continued for 1 h. Thereafter, the peptides were enriched by
SPE and LQQC*PF(C-[SO2H]) and PhIP sulfonamide
LQQC*PF (C-[SO2]-PhIP) were purified by HPLC.14 The
identities of the adducts were confirmed by MS/MS (vide
infra).
also examined as a function of time with m-CPBA (protein/m-
CPBA molar ratio = 1:50) at room temperature for 1, 3, 5, or
17 h, followed by sonication for 1 h. Protein samples were
processed by gel filtration to remove the oxidant.
Proteolytic Digestion. Enzymes were prepared as described
previously.14 Two different proteolytic conditions were
employed to digest SA adducts modified by HONH-PhIP
and HONH-4-ABP.
(1) Trypsin/chymotrypsin digestion: The PhIP- or 4-ABP-
modified SA (10 μg, 150 pmol, in 50 μL 50 mM ammonium
bicarbonate buffer (pH 8.5) containing CaCl2 (1 mM) was
digested with trypsin at a protease:protein ratio of 1:50 (w/w)
and chymotrypsin at a protease:protein ratio of 1:12.5 (w/w) at
37 °C for 20 h, followed by adduct enrichment with SPE.14,21
In some instances, the PhIP-modified SA (0.5 mg) was
denatured prior to enzyme digestion by heating the protein in 6
M guanidine−HCl (pH 7.4, 0.5 mL) with DTT (20 mM) at 55
°C for 1 h, followed by alkylation with excess IAA.21 The SA
was then subjected to gel filtration chromatography with the
PD-10 column in 50 mM ammonium bicarbonate buffer (pH
8.5).
(2) Pronase E/leucine aminopeptidase/prolidase digestion:
PhIP- or 4-ABP-modified SA (10 μg, 150 pmol) in 50 mM
ammonium bicarbonate buffer (pH 8.5) containing 1 mM
MnCl2 was treated with Pronase E at a protease/protein ratio
of 1:2 (w/w), leucine aminopeptidase at a protease/protein
ratio of 1:30 (w/w), and prolidase at a protease/protein ratio of
1:8 (w/w). The digestion was conducted at 37 °C for 20 h,
followed by adduct enrichment with SPE.14,21
Estimates PhIP-Cys34 SA Adducts. LQQC*PF (C-[SO2]-
[2H5]-PhIP) sulfonamide (7.9 pmol), LQQC*PF (C-[SO]-
[2H5]-PhIP) sulfinamide (12.3 pmol), and [2H3]-PhIP (3.95
pmol) were added as internal standards to PhIP-modified SA
(10 μg protein) prior to proteolysis. The major adducts were
identified as the missed cleavage adducts LQQC*PFEDHVK
(vide infra). The ionization efficiencies of the mixed cleavage
peptide adducts were assumed to be comparable to the
ionization efficiencies of the internal standards of [2H5]-PhIP-
modified LQQC*PF.
Modification of Human SA with N-Oxidized Derivatives of
PhIP and 4-ABP. Human SA was pretreated with β-
mercaptoethanol to reduce mixed disulfides formed at
Cys34.13 Following PD-10 gel filtration in 0.1 M potassium
phosphate buffer (pH 7.4), the sulfhydryl content of SA was
determined using Ellman’s reagent.25 The SA reduced with ß-
mercaptoethanol contained a sulfhydryl content of 0.95 mol of
SH/mol SA. SA (1 mg, 15 nmol) was reacted with HONH-
PhIP, N-acetoxy-PhIP or HONH-4-ABP (15 nmol, 40 μL
C2H5OH) in 10 mM potassium phosphate buffer (1 mL, pH
7.4). The reactions of HONH-PhIP and HONH-ABP with SA
were performed at 37 °C for 18 h, whereas the reaction of N-
acetoxy-PhIP with SA was performed at 37 °C for 2 h.
Thereafter, unbound carcinogen was removed with ethyl
acetate, and the modified SA was run through a PD-10 gel
filtration column in 10 mM phosphate buffer (pH 7.4).
Oxidation of PhIP-Modified Human SA with m-CPBA.
Unmodified or PhIP-modified SA (0.5 mg, 7.5 nmol) was
oxidized with m-CPBA for 4 h in 0.1 M phosphate buffer (pH
7.4, 1 mL) with a protein/m-CPBA molar ratio of 1:10, 1:50,
1:100, or 1:200. The samples were then incubated for 1 h with
a Branson 2510 model sonicator. Thereafter, the SA was run
through a PD-10 gel filtration column in 50 mM ammonium
bicarbonate (pH 8.5). The oxidation of PhIP-modified SA was
Ultraperformance Liquid Chromatography−Electrospray
Ionization/Multistage Mass Spectrometry (UPLC−ESI/MSn).
A NanoAcquity UPLC system (Waters Corp., Milford, MA)
equipped with a Magic C18AQ column (0.3 mm × 150 mm,
Michrom Bioresource Inc., Auburn, CA) was employed for
chromatography. The peptides were resolved with a linear
gradient starting from 90% A solvent (0.01% HCO2H) to 100%
B solvent (95% CH3CN containing 4.99% H2O and 0.01%
HCO2H) over 20 min, at a flow rate of 5 μL/min. MS spectra
were acquired with a linear quadrupole ion trap mass
spectrometer (LTQ, Thermo Fisher, San Jose, CA). Analyses
were conducted in the positive ionization mode and employed
an Advance CaptiveSpray source from Michrom Bioresource
Inc. The temperature of capillary tube was set at 200 °C; the
spray voltage was 1.5 kV; and the in-source fragmentation was
10 V. There was no sheath or auxiliary gas. The automatic gain
control (AGC) settings were full MS target 30 000 and MSn
target 10 000; the maximum injection time was 10 ms, and one
microscan was used for data acquisition. Xcalibur version 2.07
software was used for data manipulations. The tandem mass
spectra of peptides were interpreted manually and facilitated by
online software (Protein Prospector, Univ. of California, San
Francisco, http://us.expasy.org/proteomics).
1067
dx.doi.org/10.1021/ac3028273 | Anal. Chem. 2013, 85, 1065−1072

Analytical Chemistry
Article
Figure 1. (A) UV spectra of modified LQQC*PF peptides and ESI/MS product ion spectra of (B) LQQC*PF sulfonic acid ([M + H]+ at m/z
783.3), (C) LQQC*PF (C-[S=O]-PhIP) sulfinamide ([M + H]+ at m/z 973.3), and (D) LQQC*PF (C-[SO2]-PhIP) sulfonamide ([M + H]+ at
m/z 989.5).
to form a semimercaptal, which undergoes rearrangement to
form the more stable sulfinamide structure (Scheme 1). During
the course of analysis, the sulfonamide adduct of PhIP was also
identified.21 The mechanism of formation of this adduct is not
known. The superoxide anion slowly generated over the time,
by HONH-PhIP,26 may have oxidized a portion of the existing
N2-[cystein-S-yl-PhIP]-S-oxide linkage to the N2-[cystein-S-yl-
PhIP]-S-dioxide adduct (Scheme 1). Alternatively, a portion of
the N2-[cystein-S-yl-PhIP]-S-oxide adduct may have undergone
oxidation during enzymatic digestion of SA.
RESULTS AND DISCUSSION
■
The Cys34 residue of human SA is an important adduction site
for electrophilic N-oxidized metabolites of PhIP.14,21 Treatment
of SA or plasma with HNOH-PhIP produced the N2-
cysteinesulfinyl-PhIP adduct: greater than 90% of the
HONH-PhIP bound to the Cys34 of SA.14 The absence of
other PhIP-SA adducts signifies that the reactivity of HONH-
PhIP with SA is poor and adduct formation only occurs
following the oxidation of HONH-PhIP to NO-PhIP. The
sulfhydryl SH of Cys34 reacts with the NO bond of NO-PhIP
1068
dx.doi.org/10.1021/ac3028273 | Anal. Chem. 2013, 85, 1065−1072

Analytical Chemistry
Article
Figure 4. Reconstructed ion chromatograms of N-acetoxy-PhIP-
modified SA monitoring: PhIP ([M + H]+, m/z 225.1 → 210.1); 5-
HO-PhIP ([M + H]+, m/z 241.1 → 210.1); and LQQC*PFEDHVK
(C-[SO2]-PhIP) ([M + 3H]3+, m/z 533.2 → 225.2, 521.8, 670.5) in
(A) nonoxidized SA and (B) m-CPBA-oxidized SA, digested with
trypsin/chymotrypsin. The scales of the signals were fixed to the
maximum response of the respective analytes between the nonoxidized
and oxidized samples.
Figure 2. UPLC−ESI/MS2 chromatograms of Cys34 peptide adducts
recovered from 100 ng of SA digest of (A) HONH-PhIP-modified SA,
following trypsin/chymotrypsin digestion; (B) HONH-PhIP-modified
SA, which was denatured by heating the SA in 6 M guanidine−HCl
with DTT, followed by alkylation with IAA, prior to trypsin/
chymotrypsin digestion; and (C) HONH-PhIP-modified SA was
oxidized with m-CPBA prior to denaturation and digestion with
trypsin/chymotrypsin. Targeted UPLC−ESI/MS2 was done on the
following peptides: LQQC*PFEDHVK (C-[SO]-PhIP) as triply
charged species ([M + 3H]3+ at m/z 527.9); LQQC*PFEDHVK (C-
[SO2]-PhIP) ([M + 3H]3+ at m/z 533.2); LQQC*PF (C-[SO]-
the N2-cysteinesulfinyl-PhIP adduct of SA to form the
chemically stable sulfonamide linkage in high yield. Initially,
LQQCPF, a target peptide recovered from human SA digested
with trypsin and chymotrypsin,14,27 was treated with several
different oxidants. The oxidation of LQQCPF [MH+ observed
at m/z 735.3] with m-CPBA produced the sulfonic acid
LQQC*PF (C-[SO2H]) [MH+ observed at m/z 783.3] in
higher yields (80%) than the oxidation reactions conducted
with Fenton reagents (Fe2+/H2O2) or NaOCl (Peng, L.,
unpublished observations).28 Thereafter, the oxidation of PhIP
sulfinamide LQQC*PF (C-[SO]-PhIP) peptide was exam-
ined: treatment with m-CPBA converted LQQC*PF (C-[S
O]-PhIP) to the sulfonamide LQQC*PF (C-[SO2]-PhIP)
adduct [MH+ observed at m/z 989.5]. The UV and product ion
spectra of the oxidized sulfonic acid peptide LQQC*PF (C-
[SO2H]) and the sulfinamide and sulfonamide adducts of PhIP
formed with LQQC*PF are shown in Figure 1.
+
PhIP) ([M + H] at m/z 973.3); and LQQC*PF (C-[SO2]-PhIP)
([M + H]+ at m/z 989.4). The scale of the signals for all three
treatments were normalized to the response of LQQC*PFEDHVK
(C-[SO2]-PhIP) in panel C.
Oxidation of N2-cysteinesulfinyl-PhIP at Cys34 of
Human SA, by m-CPBA, to Form the N2-Cysteinesulfo-
namide-PhIP Adduct. The condition of oxidation of the SA-
N2-cysteinesulfinyl-PhIP adduct with m-CPBA was optimized
to produce the arylsulfonamide linkage in high yield. A 50-fold
mole excess of m-CPBA over protein produced the highest
amount of the sulfonamide adduct within a period of 6 h
(Supporting Information, Figure S-1).
The stability of the sulfinamide and sulfonamide adducts of
HOHN-PhIP-modified SA under the conditions of denatura-
tion, which included heat and chemical reduction of SA
disulfide bonds with DTT, followed by alkylation of the
unfolded SA with IAA, were assessed. The mass chromato-
grams of the Cys34 peptide adducts recovered from the trypsin/
chymotrypsin digests of SA are shown in Figure 2. The
sulfinamide LQQC*PF (C-[SO]-PhIP) and sulfonamide
LQQC*PF (C-[SO2]-PhIP) adducts as well as the missed
Figure 3. Estimates of LQQC*PFEDHVK adducts recovered from
trypsin/chymotrypsin digestion of m-CPBA-oxidized or nonoxidized
HONH-PhIP-modified SA, without or with denaturation. Adduct
formation was estimated by UPLC−ESI/MS2, employing LQQC*PF
(C-[SO2]-[2H5]-PhIP) sulfonamide and LQQC*PF (C-[SO]-
[2H5]-PhIP) sulfinamide as internal standards. The ionization
efficiencies of the mixed cleavage peptide adducts LQQC*PFEDHVK
were assumed to be comparable to the ionization efficiencies of the
internal standards of [2H5]-PhIP-modified LQQC*PF. Total PhIP
bound to SA was determined by UV measurement at 320 nm.14
Oxidation of LQQCPF and N2-Cysteinesulfinyl-PhIP
Peptide LQQC*PF (C-[SO-PhIP]). The goal was to oxidize
1069
dx.doi.org/10.1021/ac3028273 | Anal. Chem. 2013, 85, 1065−1072

Analytical Chemistry
Article
Figure 5. UPLC−ESI/MS2 chromatograms of Cys34 peptide adducts recovered from HONH-4-ABP-modified SA following digestion with trypsin/
chymotrypsin (left panel) and corresponding product ion mass spectra of (A) LQQC*PFEDHVK (C-[SO]-4-ABP) sulfinamide ([M + 3H]3+ at
m/z 509.6), (B) LQQC*PFEDHVK (C-[SO2]-4-ABP) sulfonamide ([M + 3H]3+ at m/z 514.9), (C) LQQC*PF (C-[SO]-4-ABP) sulfinamide
([M + H]+ at m/z 918.4, and (D) LQQC*PF (C-[SO2]-4-ABP) sulfonamide ([M + H]+ at m/z 934.4).
cleavage adducts LQQC*PFEDHVK (C-[SO]-PhIP) and
LQQC*PFEDHVK (C-[SO2]-PhIP) are detected in the
nonoxidized, nondenatured SA sample (Figure 2A). However,
the sulfinamide adduct underwent hydrolysis during protein
denaturation, and only the sulfonamide peptides LQQC*PF
(C-[SO2]-PhIP) and LQQC*PFEDHVK (C-[SO2]-PhIP)
were identified in the digest of denatured SA, either without
(Figure 2B) or with m-CPBA treatment (Figure 2C). The
missed cleavage adducts LQQC*PFEDHVK were the predom-
inant peptides identified in both nondenatured and denatured
samples. IAA-modified LQQC*PFEDHVK was also recovered
in higher amount than IAA-modified LQQC*PF in denatured
SA, and the sulfonic acid peptide LQQC*PFEDHVK (C-
[SO2H]) was recovered in higher yield than LQQC*PF (C-
[SO2H]) in m-CPBA-oxidized SA, irrespective of denaturation
of the protein (Supporting Information, Figure S-2). Thus, the
proline residue and not the PhIP-Cys adduct in
LQQC*PFEDHVK impeded the efficacy of digestion by
chymotrypsin. The employment of larger quantities of
chymotrypsin overcame some of this missed cleavage peptide
(Peng, L, unpublished observations).
[SO]-PhIP) to the sulfonamide LQQC*PFEDHVK (C-
[SO2]-PhIP). Comparable amounts of LQQC*PFEDHVK (C-
[SO2]-PhIP) were recovered from m-CPBA-oxidized SA for the
denatured and nondenatured SA samples, signifying that the
PhIP sulfonamide adduct is stable under the conditions
employed for denaturation. The level of LQQC*PFEDHVK
(C-[SO2]-PhIP) recovered from HONH-PhIP-modified SA
accounted for ≥95% of the PhIP bound to SA on the basis of
UV measurement of PhIP-modified SA at 320 nm.14
A
comparable estimate of the sulfonamide adduct was obtained
by measurement of tripeptide C*PF (C-[SO2]-PhIP) in m-
CPBA oxidized PhIP-modified SA, following digestion with
Pronase E/leucine aminopeptidase/prolidase (Supporting
Information, Figure S-3).
Trapping Labile Putative N2-Cysteinesulfenamide
Adduct of PhIP at Cys34 of Human SA, by m-CPBA
Oxidation, to Form the N2-Cysteinesulfonamide Adduct.
The mass chromatograms of the tryptic/chymotryptic digest of
N-acetoxy-PhIP-modified SA either without or with oxidation
by m-CPBA are shown in Figure 4. There was no evidence for
sulfur−nitrogen PhIP adducts (sulfenamide, sulfinamide, or
sulfonamide adducts of LQQC*PFEDHVK) in the non-
oxidized SA sample, whereas the sulfonamide
LQQC*PFEDHVK (C-[SO2]-PhIP) was identified in the m-
CPBA-treated SA sample. Moreover, the amount of PhIP and
5-HO-PhIP recovered in the m-CPBA treated SA sample was
∼80% lower than those levels present in the nonoxidized SA
sample. These findings suggest that a labile sulfenamide adduct
The estimates of LQQC*PFEDHVK (C-[SO]-PhIP) and
LQQC*PFEDHVK (C-[SO2]-PhIP) adduct formation in
HONH-PhIP-modified SA, either without or with oxidation
with m-CPBA and without or with denaturation of SA prior to
trypsin/chymotryspin digestion, are depicted in Figure 3. The
treatment of HONH-PhIP-modified SA with m-CPBA
efficiently oxidized the sulfinamide LQQC*PFEDHVK (C-
1070
dx.doi.org/10.1021/ac3028273 | Anal. Chem. 2013, 85, 1065−1072

Analytical Chemistry
Article
adduct of the HAA, 2-amino-3-methylimidazo[4,5-f]quinoline
formed with rat SA,13 and Cys34 adducts of human SA derived
from the reactive N-acetyl-p-benzoquinoneimine metabolite of
acetaminophen,29 sulfur mustard,30 and acrylamide31 do not
undergo digestion beyond the tripeptide stage of rat (C*PY) or
human (C*PF) SA treated with Pronase. Consistent with those
findings, the digestion of HONH-4-ABP-modified human SA
(nonoxidized and nondenatured) with Pronase E/leucine
aminopeptidase/prolidase also produced a prominent tripep-
tide adduct at CP*F. The UPLC−ESI/MS2 chromatogram
(Figure 6) of the digest shows the presence of the sulfonamide
CP*F (C-[SO2]-4-ABP) along with 4-ABP, but the sulfinamide
(C-[SO]-4-ABP), which is labile, underwent complete
hydrolysis during enzyme digestion with Pronase E.
CONCLUSIONS
■
Many arylsulfinamide protein adducts undergo extensive
hydrolysis during protein denaturation or proteolytic digestion
to form the arylamines and cysteine sulfinic acid of the protein.
As a consequence, the analysis of arylsulfinamide peptide
adducts is a challenging analytical task. The sulfinamide adducts
of several one- and two-ring aromatic amines formed at the Hb-
Cys93β chain have been measured indirectly as their liberated
parent amines, following mild acid treatment of Hb (Scheme
1).16 However, aromatic amines and HAAs having a larger ring
system do not form Hb sulfinamide adducts in vivo, perhaps
because alternative metabolic pathways predominate or because
N-oxidized metabolites do not efficiently penetrate the
erythrocyte or do not react with the heme moiety to produce
the reactive nitrosoarene intermediates.6
The nucleophilic Cys34 residue of human SA reacts with
many genotoxicants and toxic electrophiles to form covalent
adducts,17,32 and several putative labile arylsulfinamide adducts
have been detected.6,13,14,33 The formation of arylamine
adducts with SA does not require the transport of the
arylhydroxylamine or the arylnitroso metabolite to the blood
since SA is synthesized in the hepatocyte,34 the cell where the
arylhydroxylamine metabolites are formed by cytochrome P450
enzymes.2 The Cys34 of SA is also a major scavenger of reactive
oxygen species (ROS) in plasma.35,36 The Cys34 sulfenic acid of
SA is a proposed transient intermediate that undergoes further
oxidation to form stable Cys34 sulfinic (Cys-SO2H) and sulfonic
(Cys-SO3H) acids.36,37 Moreover, the Cys34 sulfenic acid is
thought to be the intermediate of SA that is involved in the
formation of mixed disulfides with low molecular weight thiols,
such as Cys or glutathione: these mixed disulfides account for
about 30% of the circulating SA in plasma.34,35 The high level
of oxidation of Cys34 of SA by ROS suggests that
arylsulfinamide adducts of SA also may undergo oxidation in
vivo. The ability to trap labile sulfur−nitrogen arylamine
adducts at Cys34 of SA by chemical oxidation to form stable
arylsulfonamide adducts may allow us to simultaneously
biomonitor exposures to many carcinogenic aromatic amines
and HAAs that were not previously possible.
Figure 6. UPLC−ESI/MS2 chromatograms of (A) a putative C*PF
(C-[SO]-4-ABP) sulfinamide adducts ([M + H]+ at m/z 549.2),
(B) C*PF (C-[SO2]-4-ABP) sulfonamide ([M + H]+ at m/z 565.2),
and (C) 4-ABP ([M + H]+, m/z 170.1 → 153.1) recovered from
HONH-4-ABP-modified SA digested with Pronase E, leucine
aminopeptidase, and prolidase. The scale of the signal of the putative
C-[SO]-4-ABP adduct, which was not detected, was normalized to
the scale of response of (C-[SO2]-4-ABP). (D) Product ion mass
spectrum of C*PF (C-[SO2]-4-ABP) sulfonamide adduct.
had formed at Cys34 in N-acetoxy-PhIP modified SA (Scheme
1): the treatment of the modified SA with m-CPBA oxidized
the sulfur−nitrogen linkage to the stable PhIP sulfonamide
adduct.
Sulfinamide and Sulfonamide Adduct Formation of
HONH-4-ABP at Cys34 of Human SA. The reactivity of SA
with HONH-4-ABP, the carcinogenic metabolite of 4-ABP,22
was examined. Similar to the results obtained with HONH-
PhIP-modified SA, the missed cleavage sulfinamide (C-[SO]-
4-ABP) and sulfonamide (C-[SO2]-4-ABP) adducts of
LQQC*PFEDHVK were recovered in higher yields than the
LQQC*PF adducts in the trypsin/chymotrypsin digest of SA
(nonoxidized and nondenatured), as depicted in Figure 5.
Following oxidation with the m-CPBA, the total ion counts of
sulfonamide adduct (C-[SO2]-4-ABP) was 10-fold greater than
the ion counts of the sulfinamide linked adduct (C-[SO]-4-
ABP), demonstrating that the oxidation of the sulfur−nitrogen
linkage, by m-CPBA, was efficient (data not shown).
ASSOCIATED CONTENT
■
S
* Supporting Information
Pronase alone or in combination with leucine amino-
peptidase and prolidase has been employed to digest a number
of carcinogens adducted at the Cys34 of rat or human SA. For
many carcinogen adducts, including the N2-cysteinsulfinyl
Additional information as noted in text (Figure S-1−S-3). This
material is available free of charge via the Internet at http://
pubs.acs.org.
1071
dx.doi.org/10.1021/ac3028273 | Anal. Chem. 2013, 85, 1065−1072

Analytical Chemistry
Article
(25) Riener, C. K.; Kada, G.; Gruber, H. J. Anal. Bioanal. Chem. 2002,
373, 266−276.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 518-474-4151. Fax: 518-473-2095. E-mail: Rturesky@
wadsworth.org.
(26) Hiramoto, K.; Negishi, K.; Namba, T.; Katsu, T.; Hayatsu, H.
Carcinogenesis 1988, 9, 2003−2008.
(27) Aldini, G.; Regazzoni, L.; Orioli, M.; Rimoldi, I.; Facino, R. M.;
Carini, M. J. Mass Spectrom. 2008, 43, 1470−1481.
(28) Wang, Y.; Vivekananda, S.; Men, L.; Zhang, Q. J. Am. Soc. Mass
Spectrom. 2004, 15, 697−702.
Notes
The authors declare no competing financial interest.
(29) Damsten, M. C.; Commandeur, J. N.; Fidder, A.; Hulst, A. G.;
Touw, D.; Noort, D.; Vermeulen, N. P. Drug Metab. Dispos. 2007, 35,
1408−1417.
ACKNOWLEDGMENTS
■
This research was supported by Grant R01 CA122320 (L.P.
and R.J.T.)
(30) Noort, D.; Hulst, A. G.; de Jong, L. P.; Benschop, H. P. Chem.
Res. Toxicol. 1999, 12, 715−721.
(31) Noort, D.; Fidder, A.; Hulst, A. G. Arch. Toxicol. 2003, 77, 543−
545.
(32) Rappaport, S. M.; Li, H.; Grigoryan, H.; Funk, W. E.; Williams,
REFERENCES
■
(1) Sugimura, T.; Wakabayashi, K.; Nakagama, H.; Nagao, M. Cancer
Sci. 2004, 95, 290−299.
E. R. Toxicol. Lett. 2012, 213, 83−90.
(33) Lynch, A. M.; Murray, S.; Zhao, K.; Gooderham, N. J.; Boobis,
A. R.; Davies, D. S. Carcinogenesis 1993, 14, 191−194.
(34) Peters, T., Jr. Adv. Clin. Chem. 1970, 13, 37−111.
(35) Turell, L.; Botti, H.; Carballal, S.; Ferrer-Sueta, G.; Souza, J. M.;
Duran, R.; Freeman, B. A.; Radi, R.; Alvarez, B. Biochemistry 2008, 47,
358−367.
(2) Turesky, R. J.; Le Marchand, L. Chem. Res. Toxicol. 2011, 24,
1169−1214.
(3) IARC Monographs on the Evaluation of Carcinogenic Risks to
Humans: Tobacco Smoking, International Agency for Research on
Cancer: Lyon, France, 1986.
(4) IARC Monographs on the Evaluation of Carcinogenic Risks to
Humans. Some Naturally Occurring Substances: Food Items and
Constituents, Heterocyclic Aromatic Amines and Mycotoxins; Interna-
tional Agency for Research on Cancer: Lyon, France, 1993.
(5) Jarabek, A. M.; Pottenger, L. H.; Andrews, L. S.; Casciano, D.;
Embry, M. R.; Kim, J. H.; Preston, R. J.; Reddy, M. V.; Schoeny, R.;
Shuker, D.; Skare, J.; Swenberg, J.; Williams, G. M.; Zeiger, E. Crit. Rev.
Toxicol. 2009, 39, 659−678.
(36) Turell, L.; Botti, H.; Carballal, S.; Radi, R.; Alvarez, B. J.
Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2009, 877, 3384−3392.
(37) Grigoryan, H.; Li, H.; Iavarone, A. T.; Williams, E. R.;
Rappaport, S. M. Chem. Res. Toxicol. 2012, 25, 1633−1642.
(6) Skipper, P. L.; Tannenbaum, S. R. Carcinogenesis 1990, 11, 507−
518.
(7) Liebler, D. C. Environ. Health Perspect 2002, 110 (Suppl1), 3−9.
(8) Tornqvist, M.; Fred, C.; Haglund, J.; Helleberg, H.; Paulsson, B.;
Rydberg, P. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2002,
778, 279−308.
(9) Dolle, B.; Topner, W.; Neumann, H. G. Xenobiotica 1980, 10,
527−536.
(10) Kiese, M.; Taeger, K. Naunyn Schmiedebergs Arch. Pharmacol.
1976, 292, 59−66.
(11) Guo, Z.; Wagner, C. R.; Hanna, P. E. Chem. Res. Toxicol. 2004,
17, 275−286.
(12) Liu, L.; Wagner, C. R.; Hanna, P. E. Chem. Res. Toxicol. 2008,
21, 2005−2016.
(13) Turesky, R. J.; Skipper, P. L.; Tannenbaum, S. R. Carcinogenesis
1987, 8, 1537−1542.
(14) Peng, L.; Turesky, R. J. Chem. Res. Toxicol. 2011, 24, 2004−
2017.
(15) Ringe, D.; Turesky, R. J.; Skipper, P. L.; Tannenbaum, S. R.
Chem. Res. Toxicol. 1988, 1, 22−24.
(16) Bryant, M. S.; Vineis, P.; Skipper, P. L.; Tannenbaum, S. R. Proc.
Natl. Acad. Sci. U.S.A. 1988, 85, 9788−9791.
(17) Rubino, F. M.; Pitton, M.; Di, F. D.; Colombi, A. Mass Spectrom.
Rev. 2009, 28, 725−784.
(18) Dingley, K. H.; Curtis, K. D.; Nowell, S.; Felton, J. S.; Lang, N.
P.; Turteltaub, K. W. Cancer Epidemiol. Biomarkers Prev. 1999, 8, 507−
512.
(19) Reistad, R.; Frandsen, H.; Grivas, S.; Alexander, J. Carcinogenesis
1994, 15, 2547−2552.
(20) Chepanoske, C. L.; Brown, K.; Turteltaub, K. W.; Dingley, K. H.
Food Chem. Toxicol. 2004, 42, 1367−1372.
(21) Peng, L.; Dasari, S.; Tabb, D. L.; Turesky, R. J. Chem. Res.
Toxicol. 2012, 25, 2179−2193.
(22) Beland, F. A.; Beranek, D. T.; Dooley, K. L.; Heflich, R. H.;
Kadlubar, F. F. Environ. Health Perspect. 1983, 49, 125−134.
(23) Turesky, R. J.; Lang, N. P.; Butler, M. A.; Teitel, C. H.;
Kadlubar, F. F. Carcinogenesis 1991, 12, 1839−1845.
(24) Langouet, S.; Paehler, A.; Welti, D. H.; Kerriguy, N.; Guillouzo,
̈
A.; Turesky, R. J. Carcinogenesis 2002, 23, 115−122.
1072
dx.doi.org/10.1021/ac3028273 | Anal. Chem. 2013, 85, 1065−1072