Mass Spectrometric Characterization of Human Serum Albumin Adducts Formed with N-Oxidized Metabolites of 2-Amino-1-methylphenylimidazo[4,5- b ]pyridine in Human Plasma and Hepatocytes

Article abstract of DOI:10.1021/acs.chemrestox.5b00075

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic heterocyclic aromatic amine formed in cooked meats, is metabolically activated to electrophilic intermediates that form covalent adducts with DNA and protein. We previously identified

Full text of DOI:10.1021/acs.chemrestox.5b00075

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Mass Spectrometric Characterization of Human Serum Albumin
Adducts Formed with N‑Oxidized Metabolites of 2‑Amino-1-
methylphenylimidazo[4,5‑b]pyridine in Human Plasma and
Hepatocytes
Yi Wang,† Lijuan Peng,‡ Medjda Bellamri,§,∥ Sophie Langouet,§ and Robert J. Turesky*,†
̈
†Masonic Cancer Center and Department of Medicinal Chemistry, Cancer and Cardiology Research Building, University of
Minnesota, 2231 6th Street, Minneapolis, Minnesota 55455, United States
‡School of Chemical and Environmental Engineering, Wuhan Polytechnic University, ChangQing Garden, Hankou, Wuhan 430023,
P. R. China
§Institut National de la Sante
(IRSET), Universite de Rennes 1, UMS 3480 Biosit, F-35043 Rennes, France
∥ANSES Laboratoire de Fouger
̀
es, La Haute Marche-Javene, BP 90203, 350302 Fougeres, France
́ ́ ́
et de la Recherche Medicale (Inserm), U.1085, Institut de Recherche Sante Environnement et Travail
́
̀
́
S
* Supporting Information
ABSTRACT: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]-
pyridine (PhIP), a carcinogenic heterocyclic aromatic amine
formed in cooked meats, is metabolically activated to electro-
philic intermediates that form covalent adducts with DNA and
protein. We previously identified an adduct of PhIP formed at
the Cys34 residue of human serum albumin following reaction
of albumin with the genotoxic metabolite 2-hydroxyamino-1-
methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP). The
major adducted peptide recovered from a tryptic/chymotryptic
digest was identified as the missed-cleavage peptide
LQQC*[SO PhIP]PFEDHVK, a [cysteine-S-yl-PhIP]-S-dioxide
2
linked adduct. In this investigation, we have characterized the
albumin adduction products of N-sulfooxy-2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (N-sulfooxy-PhIP), which is thought to be a major genotoxic metabolite of PhIP formed in vivo.
Targeted and data-dependent scanning methods showed that N-sulfooxy-PhIP adducted to the Cys34 of albumin in human plasma
to form LQQC*[SO PhIP]PFEDHVK at levels that were 8−10-fold greater than the adduct levels formed with N-(acetyloxy)-2-
2
amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP) or HONH-PhIP. We also discovered that N-sulfooxy-PhIP
forms an adduct at the sole tryptophan (Trp214) residue of albumin in the sequence AW*[PhIP]AVAR. However, stable adducts of
PhIP with albumin were not detected in human hepatocytes. Instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo-
[4,5-b]pyridine (5-HO-PhIP), a solvolysis product of the proposed nitrenium ion of PhIP, were recovered during the proteolysis,
suggesting a labile sulfenamide linkage had formed between an N-oxidized intermediate of PhIP and Cys34 of albumin. A stable
adduct was formed at the Tyr411 residue of albumin in hepatocytes and identified as a deaminated product of PhIP,
Y*[desaminoPhIP]TK, where the 4-HO-tyrosine group bound to the C-2 imidazole atom of PhIP.
characterized as N-(deoxyguanosin-8-yl)-PhIP (dG-C8-
PhIP).10,11 The dG-C8-PhIP adduct has been employed as a
biomarker in human studies.12,13
The measurement of DNA adducts in human biospecimens
remains an analytical challenge because many DNA lesions are
repaired and the trace levels of DNA adducts present in tissues
may be difficult to measure, even by sensitive LC/MS-based
methods.9,14 The same reactive metabolites of HAAs (or other
INTRODUCTION
■
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is
a carcinogenic heterocyclic aromatic amine (HAA) formed
in cooked meat.1−4 PhIP undergoes metabolic activation by
cytochrome P450s, to form 2-hydroxyamino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (HONH-PhIP).5,6 HONH-
PhIP undergoes esterification by sulfotransferase (SULTs) or
N-acetyltransferase (NATs) to form the corresponding esters,7
which undergo heterolytic cleavage to produce the presumed
nitrenium ion that binds to DNA.8,9 The major DNA adduc-
tion product of N-oxidized metabolites of PhIP has been
Received: February 17, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.chemrestox.5b00075
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Figure 1. Structures of PhIP and N-oxidized metabolites.
procarcinogens) also may bind to proteins to form stable
covalent adducts.15,16 The employment of protein carcinogen
adducts as biomarkers is a promising approach to assess exposure
to hazardous chemicals because stable covalent protein adducts
are not repaired and expected to accumulate during chronic
exposure and follow the kinetics of the lifetime of the protein
in vivo.17,18
The formation of covalent adducts of carcinogens with
proteins such as albumin is thought to be driven not only by
the abundance and relative nucleophilicities of the different
amino acid residues available for reaction but also by noncovalent
interactions of the reactive metabolite with different receptor
regions of proteins, which can direct the sites of carcinogen
adduct formation.41 For example, the major adduct formed
between the activated hydroxamic acid of 4-ABP and rat albumin
occurred at the sole tryptophan residue, which is situated in
a fatty acid binding site in a relatively hydrophobic position
deep within the native protein.42 Studies employing genetically
engineered mammalian cell lines expressing human NATs
showed that HONH-PhIP is poorly bioactivated by NAT1 and
NAT2.43−45 In comparison, cell lines expressing SULT1A1 were
highly effective in the bioactivation of HONH-PhIP, to form the
presumed reactive metabolite N-sulfooxy-2-amino-1-methyl-
6-phenylimidazo[4,5-b]pyridine (N-sulfooxy-PhIP), which
adducted to DNA.46,47 Thus, the contribution of N-sulfooxy-
PhIP to the chemical modification protein (or DNA) may be
much greater than that of N-acetoxy-PhIP. N-Sulfooxy-PhIP is
appreciably more polar than HONH-PhIP and N-acetoxy-PhIP,
and it exists as a negatively charged species at physiological pH.
Therefore, the sites of reactivity of N-sulfooxy-PhIP with
albumin may be different from other N-oxidized PhIP
metabolites.37−39 To the best of our knowledge, studies of the
covalent binding of N-sulfooxy-PhIP with protein or DNA have
not been reported.
Hemoglobin (Hb) and serum albumin are the two most
abundant proteins in blood and have long life times. The
chemistry of reaction of Hb and albumin with many electrophilic
genotoxicants and toxicants has been investigated.16,19−21 Like
structurally related arylamines, N-oxidation is a major pathway
of metabolism of PhIP in humans.22−24 Hb adducts of
aromatic amines are formed by a co-oxidation reaction of the
N-hydroxylated arylamine metabolites with oxy-Hb to form the
arylnitroso intermediates, which selectively bind at the Hb-Cys93β
residue and form arylsulfinamide adducts.18,25,26 Arylsulfinamide
adducts are labile and undergo hydrolysis at acidic or alkaline pH
to produce the parent arylamine. The labile nature of the Hb
arylsulfinamide bond has been exploited to monitor occupational
and environmental exposures to a number of primary aryl-
amines.27 In the case of 4-aminobiphenyl (4-ABP), the structure
of the Hb sulfinamide linkage was proven by X-ray crystallo-
graphy.28 However, a study with radiolabeled [14C]PhIP showed
that the level of binding of PhIP to Hb was very low in humans
and Hb-PhIP adducts do not appear to be promising candidate
biomarkers.29 In contrast, the binding of PhIP to albumin
occurred at levels up to several percent of the ingested dose29,30
and may be sufficient to biomonitor albumin-PhIP adducts in
humans by LC/MS-based methods. However, the structure(s) of
the albumin-PhIP adduct(s) formed in humans is unknown.
Human albumin contains only one reduced Cys residue.31
Cys34 of albumin is a strong nucleophile and also serves as a
powerful antioxidant and scavenger of reactive oxygen species.21,32
The Cys34 residues of rodent and human albumin trap reactive
electrophiles of HAAs, e.g., 2-amino-3-methylimidazo[4,5-f ]-
quinoline,33 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline,34
and PhIP,35−39 and other toxic electrophiles.21 We have shown
that human albumin forms adducts with HONH-PhIP and
2-nitroso-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO-PhIP)
at Cys34 via a sulfinamide linkage.37,39 N-(Acetyloxy)-2-amino-1-
methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-PhIP), a
metabolite of PhIP that forms covalent DNA adducts,10,40 reacts
with albumin to form an unstable sulfenamide linkage at Cys34.38
We also identified several adducts formed between human
albumin and 2-nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine
(NO2-PhIP).38
Our goal is to comprehensively characterize the adduction
products of N-oxidized metabolites of PhIP with human albumin
and develop albumin-PhIP adducts as biomarkers for molecular
epidemiology studies that seek to understand the role of
PhIP in human cancer. In this study, we report our findings
on the relative reactivity of human albumin with HONH-PhIP,
N-acetoxy-PhIP, and N-sulfooxy-PhIP, all of which produce the
proposed PhIP nitrenium ion (Figure 1).8
MATERIALS AND METHODS
■
Caution. PhIP is a carcinogen and should be handled in a well-
ventilated fume hood with the appropriate protective clothing.
Chemicals and Materials. PhIP was purchased from Toronto
Research Chemicals (Toronto, ON). 2-Amino-1-methyl-6-[2H5]-
phenylimidazo[4,5-b]pyridine ([2H5]PhIP, 99% isotopic purity) was
a gift from M. Knize and K. Kulp (Lawrence Livermore National
Laboratory, Livermore, CA). Human serum albumin, trypsin,
chymotrypsin, Pronase E, leucine aminopeptidase, prolidase,
m-chloroperoxybenzoic acid, and LC/MS grade formic acid were
purchased from Sigma-Aldrich (St. Louis, MO). LC/MS grade solvents
were purchased from Fisher Scientific (Pittsburgh, PA). All other
B
DOI: 10.1021/acs.chemrestox.5b00075
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chemicals were ACS grade and purchased from Sigma-Aldrich unless
stated. Isolute C18 solid-phase extraction (SPE) columns (25 mg) were
from Biotage (Charlotte, NC). The Pierce Albumin Depletion Kit was
from Thermo Fisher (Rockford, IL). Amicon Ultra centrifugal filter units
(10000 molecular weight cutoff) were from Millipore (Billerica, MA).
Human plasma was purchased from Bioreclamation LLC (Hicksville, NY).
Synthesis of HONH-PhIP, N-Acetoxy-PhIP, and 2-Amino-1-
methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine (5-HO-
PhIP), N-Sulfooxy-PhIP, and N-(Deoxyguanosin-8-yl)-PhIP
(dG-C8-PhIP). The syntheses of HONH-PhIP and N-acetoxy-PhIP
were previously reported.10,38,48 5-HO-PhIP was obtained by the
decomposition of N-acetoxy-PhIP in phosphate buffer (pH 7.4).35,49
Two methods were employed for the synthesis of N-sulfooxy-PhIP. The
first method employed sulfur trioxide/pyridine.50 HONH-PhIP (5 μg,
21 nmol) was reacted with a 1.3-fold molar excess of sulfur trioxide in
50 μL of anhydrous pyridine at room temperature for 18 h. The
second method was described by Beland and Miller for the synthesis
of 2-acetylaminfluorene N-sulfate.51 A 5-fold molar excess of N,N′-
dicyclohexylcarbodiimide (DCCI) dissolved in DMF previously dried
over 3 Å molecular sieves was added to HONH-PhIP (5 μg, 21 nmol),
followed by a H2SO4/HONH-PhIP molar ratio of 1.2. The mixture was
purged with argon and then mixed for 6 h at room temperature.
Thereafter, the mixtures were vacuum centrifuged to dryness using a
high-vacuum CentriVap Cold Trap (Labconco). The N-sulfooxy-PhIP
residues were dissolved in a 1/1 H2O/CH3CN mixture, sonicated for
1 min, and then immediately subjected to infusion into the MS source,
or reacted with DNA or albumin. The UV spectra of HONH-PhIP,
N-acetoxy-PhIP, and PhIP were acquired in CH3OH, employing an
Agilent 8453 spectrophotometer (Agilent Technologies, Santa Clara,
CA) (Figure S-1 of the Supporting Information). The spectral data are
consistent with previous results.37 The ESI-MSn product ion spectra
support the proposed structure of N-sulfooxy-PhIP ([M + H]+
m/z 321.1 → [M + H − SO3]+ m/z 241.1; [M − H]− m/z 319.1 →
[M − H − SO3]− m/z 239.1). Further mass spectral data are provided
(vide infra). dG-C8-PhIP and its internal standard, [13C10]dG-C8-PhIP,
were synthesized as described previously.10,11
Trapping N-Sulfooxy-PhIP, N-Acetoxy-PhIP, or HONH-PhIP
with 2′-Deoxyguanosine (dG). The reaction condition of N-acetoxy-
PhIP with dG to form dG-C8-PhIP was previously described10,11
and employed to trap other electrophiles of PhIP in this study. The
N-sulfooxy-PhIP (13.4 μg, 42 nmol, in 20 μL of 50% CH3CN),
N-acetoxy-PhIP (11.8 μg, 42 nmol, in 20 μL of CH3OH), or HONH-
PhIP (10.1 μg, 42 nmol, in 20 μL of C2H5OH) was added to a solution of
dG [1 mg/mL in 50 mM potassium phosphate buffer (pH 8.0)]. The
reaction mixture was agitated at 37 °C for 1 h. Reaction products were
applied to an Isolute C18 SPE column, followed by washing the resin
with 10% CH3OH, and eluting dG-C8-PhIP with 100% CH3OH.11
Modification of Human Serum Albumin and Plasma with
HONH-PhIP, N-Acetoxy-PhIP, and N-Sulfooxy-PhIP. Mixed
disulfides formed at Cys34 of albumin were reduced by treatment with
βME.33 Reduced serum albumin [1 nmol in 300 μL of 100 mM
potassium phosphate buffer (pH 7.4)] was reacted with HONH-PhIP
(3 nmol/10 μL of C2H5OH), N-acetoxy-PhIP (3 nmol/10 μL of
CH3OH), or N-sulfooxy-PhIP (3 nmol/20 μL of 50% CH3CN).
The mixtures were incubated at 37 °C for 12 h (HONH-PhIP) or 3 h
(N-acetoxy-PhIP and N-sulfooxy-PhIP) with agitation. The PhIP
products unbound to albumin were removed by solvent extraction
with a 2× volume of ethyl acetate, twice. The albumin solution
underwent vacuum centrifugation for 5 min to remove the residual ethyl
acetate, and the solution was subjected to a buffer exchange [450 μL of
100 mM potassium phosphate buffer (pH 7.4), two times] to remove
residual unbound PhIP using centrifugal filters (10000 molecular weight
cutoff).
provided by the supplier. The protein concentration was estimated
by measuring the UV absorption at 280 nm (extinction coefficient,
35218 M−1 cm−1).69
PhIP DNA and Albumin Adduct Formation in Human
Hepatocytes. Human liver samples were obtained from patients
undergoing liver resection for primary or secondary hepatomas through
́
the Centre de Ressources Biologiques (CRB)-Sante of Rennes (http://
www.crbsante-rennes.com, CHRU Pontchaillou, Rennes, France). The
research protocol was conducted under French legal guidelines and
the local institutional ethics committee. Hepatocytes were isolated by a
two-step collagenase perfusion procedure, and parenchymal cells were
seeded in Petri dishes at a density of 3 × 106 viable cells/19.5 cm2 dish in
3 mL of Williams’ modified medium, except that bovine serum albumin
was replaced with human albumin pre-reduced with βME (1 g/L).52
The cells were then incubated with PhIP (50 μM) or a PhIP/[2H5]PhIP
mixture (1/1, 50 μM) for 24 h. The cells were retrieved from the Petri
dishes and pelleted by centrifugation.
PhIP DNA Adduct Measurements in Human Hepatocytes by
UPLC−ESI/MS3. DNA was isolated by chloroform/phenol extraction,
and dG-C8-PhIP was quantitated by UPLC−ESI/MS3.11,52 [13C10]dG-
C8-PhIP was employed as an internal standard and spiked into DNA
prior to enzymatic digestion at a level of one adduct per 106 DNA bases.
The analyses were conducted with a Waters NanoAcquity UPLC system
(Waters Corp., New Milford, MA) equipped with a Waters Symmetry
trap column (180 μm × 20 mm, 5 μm particle size), a Michrom C18 AQ
column (0.3 mm × 150 mm, 3 μm particle size), and a Michrom
CaptiveSpray source interfaced with a linear quadrupole ion trap mass
spectrometer (LTQ Velos, Thermo Fisher, San Jose, CA), employing
chromatographic conditions and MS data acquisition parameters as
described previously.52
Albumin Isolation and Proteolytic Digestion. The albumin in
the cell culture media of hepatocytes was extracted twice with 2 volumes
of ethyl acetate to remove unmetabolized PhIP, and then the aqueous
phase was precipitated with 2 volumes of chilled ethanol. The pre-
cipitated albumin was centrifuged and resuspended in 0.3 mL of
100 mM potassium phosphate buffer (pH 7.4). The protein was then
isolated by the Albumin Depletion Kit as described above, followed by
desalting using Millipore centrifugal filters (10000 molecular weight
cutoff).
Trypsin/Chymotrypsin Digestion. Albumin [50 μg in 200 μL of
50 mM ammonium bicarbonate buffer (pH 8.5)] was digested with
trypsin (1/50, w/w, protease/protein) and chymotrypsin (1/25, w/w,
protease/protein), which were dissolved in 1 mM HCl containing 2 mM
CaCl2. The mixture was incubated at 37 °C for 20 h. For the
denaturation of albumin, the protein (50 μg) was resuspended in 0.25 M
Tris buffer (pH 7.4) containing 8 M urea. Dithiothreitol (DTT)
(1.5 mg, 20 mM) was added to the solution, and the mixture was
incubated at 55 °C for 1 h, followed by addition of iodoacetamide (IAA)
(6 mg, 65 mM). The mixture was incubated in the dark at 22 °C for 1 h.
Excess DTT and IAA were removed using centrifugal filters. The
albumin was dissolved in 200 μL of 50 mM ammonium bicarbonate
buffer (pH 8.5) and subjected to proteolytic digestion as described
above. After digestion, the mixture was diluted with 10 volumes of
distilled water and applied to an Isolute C18 SPE column. Polar peptides
were removed with 10% CH3OH (2 mL), and adducts were eluted with
CH3OH (1 mL), followed by concentration to dryness by vacuum
centrifugation.
Trypsin Digestion. Denatured or nondenatured PhIP-modified
albumin [50 μg in 200 μL of 50 mM ammonium bicarbonate buffer
(pH 8.5)] was digested with trypsin (1/50, w/w, protease/protein). The
mixture was incubated at 37 °C for 20 h, followed by SPE purification as
described above.
Pronase E/Leucine Aminopeptidase/Prolidase Digestion. Unmodi-
fied or PhIP-modified albumin [50 μg in 200 μL of 50 mM ammonium
bicarbonate buffer (pH 8.5)] was digested with Pronase E (1/2, w/w,
protease/protein), leucine aminopeptidase (1/30, w/w, protease/
protein), and prolidase (1/8, w/w, protease/protein). The mixture
was incubated at 37 °C for 20 h, followed by SPE purification as
described above.
Human plasma (5 μL) was diluted with 300 μL of 100 mM potassium
phosphate buffer (pH 7.4), followed by addition of HONH-PhIP,
N-acetoxy-PhIP, or N-sulfooxy-PhIP at a molar ratio of 1/3 (albumin/
carcinogen). The mixture was incubated at 37 °C for 12 h, with
constant agitation. The albumin was processed as described above. The
albumin was purified with the Pierce Albumin Depletion Kit (Pierce
Biotechnology, Rockford, IL) employing chromatographic conditions
C
DOI: 10.1021/acs.chemrestox.5b00075
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Mass Spectral Measurements of PhIP Metabolites and
Ultraperformance Liquid Chromatography−Electrospray Ion-
ization Multistage Mass Spectrometry (UPLC−ESI/MSn) for
Albumin-PhIP Adducts. Mass spectral data of metabolites and
synthetic peptides LQQCPF (New England Inc., Gardner, MA) were
acquired by infusion with an Orbitrap EliteTM Hybrid Ion Trap Orbitrap
mass spectrometer (Thermo Fisher) and a Michrom Bioresource Inc.
(Auburn, CA) Advance CaptiveSpray source. Typical instrument tuning
parameters were as follows: capillary temperature, 270 °C; source spray
voltage, 2 kV for positive ion mode and 1.5 kV for negative ion mode; S-lens
RF level, 69%; skimmer offset voltage, 0 V; source fragmentation, 5 V.
Helium, set at 1 mTorr, was used as the collision and damping gas in the ion
trap. There was no sheath or auxiliary gas. All analyses were conducted in the
positive ionization mode except for the characterization of the N-sulfooxy-
PhIP. The isolation width was set at m/z 1 for the MS2 and MS3 scan modes;
the activation Q was set at 0.35, and the activation time was 10 ms.
digests, employing the same configurations. The IDPicker algorithm
version 3.0.634 filtered the identifications for each spectrum with a 2%
identification false discovery rate at the peptide spectrum match level.
RESULTS
■
Characterization of HONH-PhIP, N-Acetoxy-PhIP, and
N-Sulfooxy-PhIP. The UV spectra of PhIP, HONH-PhIP, and
N-acetoxy-PhIP are provided in Figure S-1 of the Supporting
Information. Because of its instability, the N-sulfooxy-PhIP could
not be isolated and characterized by UV or online UPLC−MS
analysis. Therefore, the yield of synthesis could not be
determined. N-Sulfooxy-PhIP underwent rapid hydrolysis once
it was dissolved in an aqueous solution, and only HONH-PhIP
was detected by HPLC. The sulfate ester of N-hydroxy-N-acetyl-
2-aminofluorene was also reported to be highly unstable in an
aqueous solution.51 In contrast, N-acetoxy-PhIP had sufficient
stability to be assayed by UPLC−MS and UV measurement.38
The full scan mass spectra of HONH-PhIP, N-acetoxy-PhIP, and
N-sulfooxy-PhIP, acquired by infusion, are shown in panels
A−C of Figure 2, respectively. The protonated ions [M + H]+
are observed for HONH-PhIP (observed at m/z 241.1081 vs
calculated at m/z 241.1084) and N-acetoxy-PhIP (observed at
m/z 283.1192 vs calculated at m/z 283.1190); however, the
N-sulfooxy-PhIP underwent extensive hydrolysis, and the
protonated ion was present at extremely low abundance
([M + H]+ at m/z 321.0647 vs calculated at m/z 321.0652).
The base peak of N-sulfooxy-PhIP was observed at m/z 223.0979
(calculated at m/z 223.0978) in the full scan mass spectrum
(Figure 2A) and assigned as the nitrenium ion ([M + H −
H2SO4]+). The relative abundance of the ion at m/z 223.0979 in
Figure 2A was considerably greater than that observed in the
full scan mass spectra of N-acetoxy-PhIP and HONH-PhIP
(Figure 2B,C), an observation consistent with the labile nature
of the N-sulfooxy linkage of N-sulfooxy-PhIP. The minor ion
present in the full scan spectrum of N-sulfooxy-PhIP at m/z
208.0866 (calculated at m/z 208.0869) is consistent with in-
source fragmentation and loss of hydroxylamine-O-sulfonic
acid [NH2OSO3H]. Further fragmentation of the nitrenium ion
(m/z 223.0979) produces the ions at m/z 196.0866 (calculated
at m/z 196.0869) [loss of HCN] and m/z 179.0601 (calculated
at m/z 179.0604) [loss of HCN + NH3] (Figure 2A).
The UPLC−ESI/MSn system consists of a Dionex Ultimate 3000 LC
instrument (Thermo Scientific, Waltham, MA) and an Orbitrap
EliteTM Hybrid Ion Trap Orbitrap mass spectrometer. The peptides of
the albumin digest were resolved with a Magic C18AQ column
(0.3 mm × 150 mm, Michrom Bioresource Inc.) with a 25 min (targeted
MS/MS) or 60 min (data-dependent scanning) gradient starting from
100% solvent A (5% CH3CN with 0.01% HCO2H) to 100% solvent B
(95% CH3CN with 0.01% HCO2H) at a flow rate of 5 μL/min.
The Chromeleon 7.2 Chromatography Data System was used for the
HPLC management. The Advance CaptiveSpray was employed as the
ion source, and parameters were set as follows: capillary temperature,
270 °C; ionization voltage, 2 kV for positive ion mode; in-source
fragmentation, 5 V; one microscan; maximal injection time, 10 ms for
MS and 50 ms for MSn; MS fragmentation, a normalized collision energy
of 35%; no auxiliary and sheath gases used. The isolation width was set at
m/z 1 for both MS2 and MS3 scan modes, and the activation Q was set at
0.35. AGC (automated gain control) was set 30000 for ion trap (IT) MS
and 10000 for IT MSn, 106 for Orbitrap (FT) MS, and 50000 for FT.
The Orbitrap was routinely calibrated in positive and negative ion
modes using Pierce LTQ Velos ESI Positive Ion Calibration Solution
(2 μg/mL caffeine, 1 μg/mL MRFA, 0.001% Ultramark 1621, and
0.00005% n-butylamine) and Pierce ESI Negative Ion Calibration
Solution (2.9 μg/mL sodium dodecyl sulfate, 5.4 μg/mL sodium
taurocholate, and 0.001% Ultramark 1621) (Pierce Biotechnology).
For the mass tag-triggered data-dependent acquisition method, a full
MS scan (mass range, 100−1800 Da) was obtained, followed by five data
dependent scans (mass range, 100−1800 Da), which were triggered by
a pattern of the unlabeled PhIP to [2H5]PhIP adducts at a partner
intensity ratio of 65−100%, by enabling the mass tags option (m/z 5,
2.5, or 1.67 for singly, doubly, or triply charged ions, respectively).38
The MS/MS spectra were recorded using dynamic exclusion of previously
analyzed precursors for 180 s with a repeat of 3 and a repeat duration of
60 s. CID was selected for ion fragmentation with a normalized collision
energy of 35%. The maximal injection time of MS/MS was set at 50 ms,
and the isolation width was set as m/z 2.
Data Analysis. Xcalibur version 2.2 was employed for data acquisi-
tion and data processing. Peptide adducts were identified manually and
facilitated by MyriMatch (version 2.1.140)53,54 using a 31-protein subset
database containing albumin from an initial search against the RefSeq
human protein database, version 37.3. MyriMatch was configured to
have Cys to contain carbamidomethyl (+57.0 Da) for IAA treatment or
(+32.0 or +48.0 Da) for oxidation of Cys to the sulfinic or sulfonic acids,
and to allow for the possibility of oxidation (+16.0 Da) on methionines
and deamidation (−17.0 Da) of N-terminal glutamines. Peptides
modified with HONH-PhIP and HONH-[2H5]PhIP were allowed to
have dynamic modifications at [C,K,Y,S,T,W,H] of 222.1 (HONH-
PhIP − H2O) and 227.1 Da (HONH-[2H5]PhIP − H2O), or dynamic
modifications at [C] of 238.1 and 243.1 Da for adduction with nitroso-
PhIP (sulfinamide adducts) and 254.1 and 259.1 Da (sulfonamide
adducts). Candidate peptides were allowed to have trypsin cleavages or
protein termini at one or both termini (semitryptic search), and up to two
missed cleavages were permitted. The precursor error was set at m/z 1.25,
but fragment ions were required to match within m/z 0.5. Analyses
of modified albumin were also performed with trypsin/chymotrypsin
The product ion spectra of N-acetoxy-PhIP and HONH-PhIP
are consistent with the spectral data in the literature.38
N-Acetoxy-PhIP (observed at m/z 283.1192) undergoes CID
with losses of C2H3O2 (observed at m/z 224.1057 vs calculated at
m/z 224.1057) to form the PhIP radical cation, and C2H4O2
(observed at m/z 223.0981 vs calculated at m/z 223.0978) to
form PhIP nitrenium ion (Figure S-2A of the Supporting
Information). CID studies with HONH-PhIP and 1-[2H3C]-
HONH-PhIP have shown that nitrenium ion formation in
the gas phase occurs primarily by homolytic cleavages at the
hydrogen atom bonded to the C1 methyl atom and the
HO-N2 bond of HONH-PhIP.55 This species may rearrange
to form a pyrazine ring,56 which undergoes fragmentation to
produce ions at m/z 206.0713, 196.0870, and 179.0605 as
described in the Supporting Information. These fragment ions
are observed in the product ion spectra of N-acetoxy-PhIP
(Figure S-2B of the Supporting Information) and HONH-PhIP
(data not shown).
The deprotonated ion of N-sulfooxy-PhIP [M − H]− was
observed in negative ion mode at m/z 319.0499 (calculated at
m/z 319.0506) (Figure 2D) and undergoes fragmentation to
form the HONH-PhIP [M − H − SO3]− ion at m/z 239.0939
D
DOI: 10.1021/acs.chemrestox.5b00075
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX