Mapping serum albumin adducts of the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine by data-dependent tandem mass spectrometry

Article abstract of DOI:10.1021/tx300253j

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed during the cooking of meats. PhIP is a potential human carcinogen: it undergoes metabolic activation to form electrophilic metabolites that bind to DNA

Full text of DOI:10.1021/tx300253j

Article
pubs.acs.org/crt
Mapping Serum Albumin Adducts of the Food-Borne Carcinogen
2‑Amino-1-methyl-6-phenylimidazo[4,5‑b]pyridine by Data-
Dependent Tandem Mass Spectrometry
Lijuan Peng,† Surendra Dasari,‡ David L. Tabb,‡ and Robert J. Turesky*,†
†Division of Environmental Health Sciences, Wadsworth Center, New York State Department of Health, Albany, New York 12201,
United States
‡Department of Biomedical Informatics, Vanderbilt University, Nashville, Tennessee 37232-8575, United States
S
* Supporting Information
ABSTRACT: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP) is a heterocyclic aromatic amine that is formed during the
cooking of meats. PhIP is a potential human carcinogen: it undergoes
metabolic activation to form electrophilic metabolites that bind to
DNA and proteins, including serum albumin (SA). The structures of
PhIP-SA adducts formed in vivo are unknown and require elucidation
before PhIP protein adducts can be implemented as biomarkers in
human studies. We previously examined the reaction of genotoxic N-
oxidized metabolites of PhIP with human SA in vitro and identified
covalent adducts formed at cysteine34 (Cys34); however, other
adduction products were thought to occur. We have now identified
adducts of PhIP formed at multiple sites of SA reacted with isotopic
mixtures of electrophilic metabolites of PhIP and 2-amino-1-methyl-
6-[2H5]-phenylimidazo[4,5-b]pyridine ([2H5]-PhIP). The metabo-
lites used for study were 2-nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO2-PhIP), 2-hydroxyamino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (HONH-PhIP), or N-acetyloxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-acetoxy-
PhIP). Following proteolytic digestion, PhIP-adducted peptides were separated by ultra performance liquid chromatography and
characterized by ion trap mass spectrometry, employing isotopic data-dependent scanning. Analysis of the tryptic or tryptic/
chymotryptic digests of SA modified with NO2-PhIP revealed that adduction occurred at Cys34, Lys195, Lys199, Lys351, Lys541
,
Tyr138, Tyr150, Tyr401, and Tyr411, whereas the only site of HONH-PhIP adduction was detected at Cys34. N-Acetoxy-PhIP, a
penultimate metabolite of PhIP that reacts with DNA to form covalent adducts, did not appear to form stable adducts with SA;
instead, PhIP and 2-amino-1-methyl-6-(5-hydroxy)-phenylimidazo[4,5-b]pyridine, an aqueous reaction product of the proposed
nitrenium ion of PhIP, were recovered during the proteolysis of N-acetoxy-PhIP-modified SA. Some of these SA adduction
products of PhIP may be implemented in molecular epidemiology studies to assess the role of well-done cooked meat, PhIP, and
the risk of cancer.
meat and cancer risk has been difficult to establish. In the 11th
Report on Carcinogens, PhIP and several other HAAs were
classified as “reasonably anticipated to be human carcinogens”.7
However, there is a critical need to establish long-term
biomarkers of HAAs that can be implemented in molecular
epidemiology studies to firmly evaluate the health risk of these
genotoxicants.
Long-term biomarkers of carcinogens, such as DNA or
protein adducts, are a measure of the biologically effective dose
and have been used for human risk assessment of environ-
mental and dietary genotoxicants.8,9 Electrophilic genotoxic
metabolites of PhIP react with DNA and proteins.10 PhIP
undergoes N-oxidation by cytochrome P450 enzymes to form
INTRODUCTION
■
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a
carcinogenic heterocyclic aromatic amine (HAA) that is
formed, by reaction of creatinine and phenylalanine, during
the cooking of meats, poultry, and fish.1 The concentrations of
PhIP can reach up to 480 parts per billion in well-done cooked
poultry.2 PhIP comprises about 70% of the daily mean intake of
HAAs in the United States.3 Many epidemiological studies have
reported a positive association between the frequent con-
sumption of well-done cooked meats containing PhIP and an
increased risk of stomach, colon, pancreas, prostate, and breast
cancers, although some studies have failed to find associations
between well-done meat and cancer risk.4−6 A major limiting
factor in most epidemiological studies is the uncertainty in
quantitative estimates of chronic exposure to PhIP or other
HAAs, and thus, the association of HAAs formed in cooked
Received: June 6, 2012
© XXXX American Chemical Society
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2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(HNOH-PhIP), which can directly react with DNA, or
HONH-PhIP can undergo conjugation reactions with phase
II enzymes to form highly reactive esters that covalently bind to
DNA.10 PhIP-DNA adducts have been detected in human
tissues and fluids by immunohistochemistry methods,11,12
accelerator mass spectrometry,13 or liquid chromatography/
mass spectrometry.14 However, DNA adduct measurements are
often precluded by the unavailability of biopsy samples in large-
scale human studies. Moreover, DNA adducts are generally
repaired, and the adduct levels can be below the limit of
detection, even when measured by sensitive tandem mass
spectrometry techniques.
Hemoglobin (Hb) and serum albumin (SA) carcinogen
adducts have been used as an alternative to DNA adducts for
biomarkers of several different classes of carcinogens.9,15 Stable
carcinogen protein adducts are expected to accumulate and
follow the kinetics of the lifetime of Hb or half-life of SA,
during chronic exposure, and augment the sensitivity of adduct
detection.16 The N-hydroxylated metabolites of many aromatic
amines, carcinogens that are structurally related to HAAs, bind
to human Hb at appreciable levels.17 The arylhydroxylamines
penetrate the erythrocyte and undergo a co-oxidation reaction
with oxy-hemoglobin (oxy-Hb) to form the arylnitroso
intermediates and methemoglobin (met-Hb).18 The arylnitroso
compounds can react with the Cys93 residue of the human β-
Hb chain to form a sulfinamide adduct.9,19 However, the
covalent binding of HONH-PhIP and other N-hydroxylated-
HAAs to Hb in rodents and humans is very low, and the HAA-
Hb sulfinamide adduct does not appear to be a promising
biomarker to assess human exposure.10
Many genotoxicants and toxic electrophiles also bind
covalently to human SA.9,15,16,20 Among the 585 amino acids
of the mature SA sequence, the thiol group of Cys34, the
imidazole nitrogen atoms of histidine, the amino and guanidine
groups of the side chain of lysine and arginine, the carboxyl
groups of the side chain of aspartic and glutamic acid, and the
phenolic group of tyrosine are most frequently involved in the
formation of SA adducts.20 PhIP was reported to bind to
human SA in vivo at levels that may be sufficient to establish
mass spectrometry techniques for biomonitoring.13 At least one
of the PhIP-SA adducts undergoes hydrolysis under acidic pH
conditions to regenerate PhIP.21 However, the reactive species
responsible for adduct formation and the structures of the
intact PhIP-SA adduct(s) are unknown. N-Oxidation products
of PhIP covalently bind to rat or human SA in vitro.22−24 2-
Nitroso-1-methyl-6-phenylimidazo[4,5-b]pyridine (NO-PhIP)
reacted with SA in vitro to produce the N2-[cystein-S-yl-
PhIP]-S-oxide at Cys34.24 This sulfinamide adduct is labile
toward acid, and some portion of the acid-labile adduct(s) of
PhIP formed with SA in vivo may exist as the sulfinamide. The
Cys34 of SA also reacted with 2-nitro-1-methyl-6-
phenylimidazo[4,5-b]pyridine (NO2-PhIP) in vitro to form a
stable sulfur−carbon-linked adduct with PhIP.24 However,
other PhIP-SA adduction products are likely to have been
formed and remain to be characterized.
human SA with electrophilic N-oxidation products of PhIP in
vitro and to characterize the adduction products by mass
spectrometric techniques. We have exploited the isotopic data-
dependent scanning technique to map the sites of PhIP binding
to human SA modified with a mixture of unlabeled and [2H5]-
labeled N-oxidized metabolites of PhIP.
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, Canada). 2-Amino-1-methyl-6-
[2H5]-phenylimidazo[4,5-b]pyridine ([2H5]-PhIP, 99% isotopic pu-
rity) was a gift from Dr. Mark Knize and Dr. Kristen Kulp (Lawrence
Livermore National Laboratory, Livermore, CA). Human SA, cysteine,
tyrosine, lysine, tryptophan, β-mercaptoethanol, iodoacetamide,
dithiothreitol, N-ethylmaleimide (NEM), and 4-chloromercuribenzoic
acid (4-CMB) were obtained from Sigma (St. Louis, MO). The human
plasma was purchased from Bioreclamation LLC (Hicksville, NY).
Trypsin gold, sequencing grade trypsin, and chymotrypsin were
purchased from Promega (Madison, WI). Pronase E, leucine
aminopeptidase, and prolidase were purchased from Sigma. All
solvents were high-purity B & J Brand from Honeywell Burdick and
Jackson (Muskegon, MI). ACS reagent-grade formic acid (88%) was
purchased from J. T. Baker (Phillipsburg, NJ). Isolute C18 solid-phase
extraction (SPE) column (25 mg) was from Biotage (Charlotte, NC).
HiTrap Blue affinity and GE P10 columns were obtained from GE
Healthcare (Piscataway, NJ). Spire PEP tips were a gift from Thermo
(Bellefonte, PA). All other chemical reagents were ACS grade and
purchased from Sigma-Aldrich.
Synthesis of N-Oxidized Metabolites of PhIP. Unlabeled PhIP
and [2H5]-PhIP were mixed at a molar ratio of 1:1. NO2-PhIP and
NO2-[2H5]-PhIP were synthesized by diazotization of PhIP with
NaNO2, as described previously.27 Subsequently, the NO2-PhIP
analogues were reduced with hydrazine, using Pd/C as a catalyst, to
produce HONH-PhIP and HONH-[2H5]-PhIP.27 The method of
synthesis of N-(acetyloxy)-2-amino-1-methyl-6-phenylimidazo[4,5-b]-
pyridine (N-acetoxy-PhIP) is a modification of procedures reported in
the literature.28,29 An equimolar mixture of HONH-PhIP and HONH-
[2H5]-PhIP (20 μg, 83 nmol) in C2H5OH (50 μL) at −5 °C was
reacted with acetic anhydride in acetic acid (10% v/v, 2 μL) for 16
min. The solution was then diluted with chilled, deionized H2O (500
μL) and applied to the Spire PEP tip (12 mg) under a gentle vacuum.
The resin was washed with chilled, deionized H2O (500 μL), and the
mixture of N-acetoxy-PhIP and N-acetoxy-[2H5]-PhIP was eluted with
chilled C2H5OH (∼100 μL). The product was characterized by ultra
performance liquid chromatography (UPLC)-ESI/MS2 with a triple
stage quadrupole mass spectrometer (vide infra). The N-acetoxy-PhIP
derivatives were prepared immediately prior to their reaction with
peptides or SA.
2-Hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (2-HO-PhIP)
was prepared by incubation of NO2-PhIP (1.7 μg) in 90 mM NH4OH
(500 μL) at 40 °C for 1 h. 2-Amino-1-methyl-6-(5-hydroxy)-
phenylimidazo[4,5-b]pyridine (5-HO-PhIP) was obtained by the
solvolysis of N-acetoxy-PhIP as described previously.22,30 The
identities of 2-HO-PhIP and 5-HO-PhIP were confirmed by their
characteristic product ion spectra.24,30
Amino acid adducts of NO2-PhIP were synthesized by the reaction
of cysteine, tryptophan, tyrosine, or lysine (0.1 μmol) with NO2-PhIP
(0.1 μmol) in 50 mM ammonium bicarbonate buffer, pH 8.5 (1 mL),
at 37 °C for 18 h. The adducts were isolated by enrichment with C18
SPE resins that were prewashed with CH3OH and H2O. The reaction
products were applied to the resin, and the resin was washed with H2O
(1 mL). The adducts were eluted with CH3OH (2 mL) and
concentrated to dryness by vacuum centrifugation. The modified
amino acids were purified by HPLC with Agilent 1100 HPLC system
(Palo Alto, CA) and an Agilent Eclipse XDB-C18 column (4.6 mm ×
250 mm). A linear gradient was employed, starting from 100% A
solvent (0.1% HCO2H) and reaching 100% B solvent (95% CH3CN
Isotope pattern-dependent mass spectrometric scanning
methods have been employed to analyze the formation of
reactive metabolites from a number of toxicants.25 Isotopic
data-dependent scanning has also been used to identify proteins
in liver microsomal preparations chemically modified with
unlabeled and 14C-radiolabeled furan-containing drugs.26 The
goal of our current study was to investigate the reactivity of
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containing 4.9% H2O and 0.1% HCO2H) at 20 min, at a flow rate of 1
mL/min. The wavelength was monitored at 210 and 320 nm. The
structures of the PhIP-modified amino acids were determined by LC-
ESI/MSn with a linear quadrupole ion trap MS (LTQ MS, Thermo
Fisher, San Jose, CA). The approximate yields of the NO2-PhIP-
modified cysteine and tyrosine adducts were ∼50%, and the yield of
the NO2-PhIP-modified lysine adduct was ∼5% on the basis of UV
measurements. NO2-PhIP did not form an adduct with tryptophan
under these reaction conditions.
Modification of Human SA and Plasma with N-Oxidized
Metabolites of PhIP. Commerical human SA was pretreated with β-
mercaptoethanol to reduce mixed disulfides formed at Cys34.31 For
some studies, the Cys34 of SA was blocked by titration of the thiol
residue to its end point with 4-CMB31,32 or by reaction with a 5-fold
mol excess of NEM for 5 h at room temperature, followed by gel
filtration. The sulfhydryl content of SA was determined using Ellman's
reagent.33 The SA reduced with β-mercaptoethanol contained a
sulfhydryl content of 0.95 mol -SH/mol SA, whereas the sulfhydryl
content of 4-CMB- or NEM-modified SA was 0.02 mol -SH/mol SA.
A solution of NO2-PhIP and NO2-[2H5]-PhIP (90 nmol in 6.6 μL of
DMSO), or HONH-PhIP and HONH-[2H5]-PhIP (90 nmol in 13 μL
of C2H5OH), or N-acetoxy-PhIP and N-acetoxy-[2H5]-PhIP mixture
(90 nmol in 40 μL of C2H5OH) was reacted with reduced SA (2 mg,
30 nmol) in 1 mL of 10 mM potassium phosphate buffer (pH 7.4).
The reactions of NO2-PhIP and HONH-PhIP 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. Unbound PhIP
derivatives were removed from SA by solvent extraction with ethyl
acetate (3 mL, three times), followed by gel filtration chromatography
of the SA with the PD-10 column containing 10 mM potassium
phosphate buffer (pH 7.4).
Human plasma was diluted with PBS by 5-fold. A 1 mL solution of
diluted plasma was then treated with a 1:1 isotopic mixture of NO2-
PhIP and NO2-[2H5]-PhIP (450 nmol in 33 μL of DMSO) at 37 °C
for 18 h. The purification of SA from plasma matrix was done with a
HiTrap Blue affinity column.24 In brief, the treated plasma was diluted
with buffer A (50 mM KH2PO4, pH 7.0) and centrifuged to remove
particulates, before it was applied to a HiTrap Blue affinity column.
Buffer B [50 mM KH2PO4 buffer (pH 7.0) containing 1.5 M KCl (3
mL)] was used to elute SA from the affinity column. Thereafter, any
remaining unbound PhIP derivatives were removed from SA by
solvent extraction, followed by gel filtration chromatography of the SA
in 10 mM potassium phosphate buffer as described above.
Trypsin Digestion. Enzymes were prepared as described
previously.24 Three different proteolytic conditions were employed
to digest SA adducts modified by N-oxidized PhIP derivatives.
Trypsin Digestion. PhIP-modified SA (0.5 mg) was concentrated to
dryness by vacuum centrifugation and dissolved in 0.25 M Tris buffer
containing 6 M guanidine−HCl (pH 7.4, 0.5 mL). Dithiothreitol (1.6
mg, 20 mM) was added to the solution, and the mixture was incubated
at 55 °C for 1 h. After the solution was cooled to room temperature,
excess iodoacetamide (6 mg, 65 mM) was added, and the mixture was
incubated in the dark for 30 min. The denatured and alkylated SA was
then subjected to gel filtration chromatography with the PD-10
column in 50 mM ammonium bicarbonate buffer (pH 8.5) to remove
excess iodoacetamide. The SA solution (6 μg in 35 μL) was mixed
with CaCl2 (1 μL of 50 mM stock solution), followed by the addition
of trypsin at a protease:SA ratio of 1:50 (w/w). The digestion was
performed for 20 h at 37 °C. Thereafter, the protein digest was diluted
with H2O (1 mL) and applied to a C18 SPE. The CH3OH eluents
were concentrated to dryness by vacuum centrifugation and
resuspended in 1:1 H2O:DMSO for UPLC-ESI/MSn analysis.
Trypsin/Chymotrypsin Digestion. The denatured and alkylated
PhIP-modified SA (5 μg, 75 pmol, in 35 μL of 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:SA ratio of 1:12.5 (w/w). The digestion
was performed at 37 °C for 20 h, followed by the same SPE
purification procedure described above.
Pronase E/Leucine Aminopeptidase/Prolidase Digestion. This
enzymatic digestion was performed as described previously.24 In brief,
the PhIP-modified SA (5 μg, 75 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:SA ratio of 1:8 (w/w). The digested was
conducted at 37 °C for 20 h, followed by the same SPE purification
procedure as described above.
Mass Spectrometry Methods. Product ion spectra of N-oxidized
PhIP derivatives were acquired by infusion with a Finnigan Quantum
Ultra triple stage quadrupole mass spectrometer (TSQ MS, Thermo
Fisher, San Jose, CA) interfaced with a CaptiveSpray source from
Michrom Bioresource Inc. (Auburn, CA). Typical instrument tuning
parameters were as follows: capillary temperature, 200 °C; source
spray voltage, 1.4 kV; tube lens offset, 95 V; capillary offset, 35 V; and
in-source fragmentation, 5 V. Argon, set at 1.5 mTorr, was used as the
collision gas, and the collision energy was variable and set between 15
and 35 eV. In some instances, metabolites were characterized online
with a Waters NanoAcquity UPLC (New Milford, MA) interfaced
with the TSQ MS. LC separation of peptides was performed with a
Magic C18AQ column (0.3 mm × 150 mm) (Michrom Bioresource
Inc.). Metabolites 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.
Accurate mass measurements of the synthesized amino acid and
peptide adducts of NO2-PhIP were acquired on the LTQ Orbitrap XL
(Thermo Fisher Scientific, Bremen, Germany) at the Proteomics Core
Facility in the Center for Biotechnology and Interdisciplinary Studies,
Rensselaer Polytechnic Institute (Troy, NY). The MS was interfaced
with the Michrom CaptiveSpray ion source. Full scan mass spectra
were acquired from a scan range of m/z 100 to 700 at a resolution of R
= 60000 at m/z 400. The injection time was 500 ms, and data were
acquired with a μscan count of 1. The detected ions were recalibrated
on the fly using phthalate as the lock mass at m/z 149.02332. The
spray voltage was 2.2 kV, the capillary temperature was 200 °C, and
the tube lens was 100 V. The isolation width was 2 Da, and the
collision-induced dissociation (CID) in the linear ion trap was set at a
normalized collision energy of 35%.
UPLC-ESI/MSn. The separation of peptides was performed with a
NanoAcquity UPLC system (Waters Corp., Milford, MA) equipped
with a Magic C18AQ column (0.3 mm × 150 mm) from Michrom
Bioresource Inc. The digests (3 μL) were injected, and 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 60 min, at a flow rate of 5 μL/min. MS
spectra were acquired with a LTQ MS (Thermo Fisher, San Jose, CA).
Xcalibur version 2.07 software was used for data manipulations. All
analyses were conducted in the positive ionization mode and
employed an Advance CaptiveSpray source from Michrom Bio-
resource 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. Helium was used as the collision
and damping gas in the ion trap and was set at a pressure of 1 mTorr.
One μscan was used for data acquisition. The automatic gain control
(AGC) settings were full MS target 30000 and MSn target 10000, and
the maximum injection time was 10 ms.
Data-Dependent MS/MS. A full scan was obtained for eluting
peptides in the range of 300−2000 Da, followed by three data-
dependent MS/MS scans. 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. MS/MS spectra were
generated by CID of the peptide ions at a normalized collision energy
of 35% to generate a series of b and y ions as major fragments. For
isotopically labeled experiments, the mass spectrometer was
programmed to switch from the full survey scan MS to the MS/MS
scan mode, which was triggered by the characteristic isotopic pattern
of unlabeled PhIP/[2H5]-PhIP at a partner intensity ratio of 65−100%,
employing the mass tags scanning option. The mass to charge
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354.2) and its isotopic labeled species ([M + H]+ at m/z 359.2)
are shown in Figure 1. The masses are consistent with the
differences were set at m/z 5.00, 2.50, or 1.67, respectively, for singly,
doubly, or triply charged peptide species. The mass tag of singly
charged species (m/z 5) was employed to scan for amino acid adducts
recovered from SA digested with Pronase E/leucine aminopeptidase/
prolidase, whereas the mass tags of doubly and triply charged ions (m/
z 2.50 and 1.67) were employed to scan for SA peptide adducts
recovered from tryptic or tryptic/chymotryptic digests. The four most
abundant ions above 1000 counts that displayed the isotopic pattern
were selected for CID with a normalized collision energy of 35%,
employing an isolation width of 2 Da.
Data Analysis. The mass spectral data PhIP- and [2H5]-PhIP-
modified amino acid and peptide adducts were acquired by mass tags
with the Xcalibur software. Scan filters were employed to extract the
protonated ions and MS/MS spectral data of the isotopic pairs. 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). Fully automated
data analysis on peptide adducts was conducted by converting RAW
data to mz5 format in the ProteoWizard msConvert tool.34 Spectra
were identified by the MyriMatch algorithm,35 version 2.1.111, using a
31 protein subset database containing SA from an initial search against
the RefSeq human protein database, version 37.3. Searching for
modifications in a protein database containing only a handful of
sequences can inappropriately force spectra to match to modified
peptides. Therefore, we generated a database of 31 proteins by a fully
tryptic search of the LC-MS/MS data generated here in combination
with a separate LC-MS/MS experiment from unrelated major proteins
frequently observed in pull-downs with immunoprecipitation to add
protein distractors to the database. The sequence database was
reversed so that each protein sequence appeared in both normal and
reversed orientations (totaling 62 sequences), enabling false discovery
rate estimation. MyriMatch was configured to have cysteines to
contain carbamidomethyl (+57.021 Da) as a dynamic modification and
to allow for the possibility of oxidation (+15.996 Da) on methionines
and deamidation (−17.03 Da) of N-terminal glutamines. Peptides
modified with NO2-PhIP and NO2-[2H5]-PhIP were searched with
[C,K,Y] allowed to have dynamic modifications of 207.1 and 212.1 Da;
peptides modified with HONH-PhIP and HONH-[2H5]-PhIP or their
N-acetoxy derivatives were allowed to have dynamic modifications at
[C,K,Y,S,T,W,H] of 222.1 and 227.1 Da, for adduction with PhIP, or
dynamic modifications at [C] of 238.1 and 243.1 Da for adduction
with NO-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 1.25 m/z, but fragment ions were required to
match within 0.5 m/z. Analyses of modified SA were also performed
with trypsin/chymotrypsin digests, employing the same configurations.
The IDPicker algorithm36 v3.0.420 filtered the identifications for each
spectrum with a 5% identification false discovery rate at the peptide-
spectrum match level.
Figure 1. Product ion mass spectra of desamino-PhIP-K ([M + H]+,
m/z 354.2) recovered from NO2-PhIP-modified SA (upper panel) and
desamino-[2H5]-PhIP-K ([M + H]+, m/z 359.2) recovered from NO2-
[2H5]-PhIP-modified SA (bottom panel), following digestion of with
Pronase E, leucine aminopeptidase, and prolidase.
molecular weight of a reaction product formed between lysine
(146.2 Da) and NO2-PhIP or NO2-[2H5]-PhIP (254.1 or 259.1
Da), where the amine group of the side chain of lysine
displaced the nitro moiety of PhIP. The product ion spectra of
desamino-PhIP-K ([M + H]+ at m/z 354.2) and [2H5]-
desamino-PhIP-K ([M + H]+ at m/z 359.2) exhibited a “twin”
pattern of ions that differ by 5 Da. The product ion spectrum of
desamino-PhIP-K contained three major fragment ions at m/z
337.2, 309.2, and 291.2, corresponding to the losses of NH3,
NH3 + CO, and NH3 + HCO2H, respectively. The peaks
observed at m/z 225.1 and 230.1, respectively, in the spectra of
desamino-PhIP-K and desamino-[2H5]-PhIP-K are attributed to
protonated PhIP and [2H5]-PhIP, which were produced by
cleavage of the bond between the ε-carbon and the side chain
amine group of Lys. The product ion spectra acquired at the
MS3 scan stage on m/z 225.1 and 230.1 are identical to the
spectra of PhIP and [2H5]-PhIP (data not shown). The
synthetic desamino-PhIP-K adducts exhibit the identical tR and
product ion spectra (data not shown). The assignments of
these fragment ions were supported by exact mass measure-
ments (Table S-1 in the Supporting Information).
RESULTS
■
Mapping of Human SA Modified with NO2-PhIP and
NO2-[2H5]-PhIP. Data-Dependent Analysis of Human SA
Modified with NO2-PhIP Followed by Digestion with Pronase
E, Leucine Aminopeptidase, and Prolidase. This three enzyme
mixture efficiently digests many proteins to amino acids.37,38
The full scan mode was monitored from the mass range of
300−600 Da, a range that encompasses the molecular masses
[M + H]+ of all possible amino acid adducts formed with NO2-
PhIP. The mass chromatograms of the enzymatic digests of
unmodified and NO2-PhIP-modified SA acquired by the data-
dependent MS/MS with mass tags enabled are presented in
Figure S-1 in the Supporting Information.
The product ion spectra of adducts ([M + H]+ at m/z 329.2
and 334.2) of the second pair of adducts (tR = 13.9 min) are
identical to the spectra that we reported previously for
desamino-PhIP-C adduct, where the thiol group of Cys34
displaced the nitro moiety of PhIP.24
The most abundant amino acid adducts (tR = 17.8 min) have
molecular masses of 388.2 and 393.2 Da; these masses are
consistent with masses of adducts formed between tyrosine
(181.1 Da) and 209.1 (desamino-PhIP) or 214.1 Da
(desamino-[2H5]-PhIP), less two protons. Major fragment
ions are observed in the product ion spectrum of desamino-
PhIP-modified tyrosine ([M + H]+ at m/z 389.3) (Figure 2A)
at m/z 344.2 [M + H−NH3−CO]+, 343.2 [M + H−HCO2H]+
(tyrosine immonium ion plus desamino-PhIP), 328.2 [M + H−
Three pairs of adducts were detected (tR = 6.7, 13.9, and 17.8
min). The first adduct was characterized as desamino-PhIP-K.
The product ion spectra of the adduct ([M + H]+ at m/z
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formation between NO2-PhIP and tyrosine (Figures 2C−E).
• +
The radical cation at m/z 315.2 [M + H−C2H4NO2 ] (m/z =
320.2 for desamino-[2H5]-PhIP-Y) is proposed to arise by
homolytic cleavage of the bond between the α- and the β-
carbon atoms of tyrosine; this is an uncommon mechanism of
fragmentation of amino acids by ESI-MS under low-energy
CID conditions (Scheme 1).39 The second generation product
Scheme 1. Proposed Mechanisms of Fragmentation of
Desamino-PhIP-Y
ion spectrum (MS3) of the ion at m/z 315.2 (m/z 320.2 for
desamino-[2H5]-PhIP-Y) contains the ion at m/z 209.2 (m/z
214.2 for desamino-[2H5]-PhIP-Y) as the base peak (Figure
2C). This ion is proposed to occur by cleavage of the bond of
the C-2 atom of the imidazole moiety of PhIP and the phenolic
oxygen of tyrosine, to produce the radical cation of desamino-
PhIP and the quinone methide (106.1 Da) as a neutral species
(Scheme 1). The second generation product ion spectrum
(MS3) of the m/z 343.2 ([M + H−HCO2H]+ (tyrosine
immonium ion containing desamino-PhIP) (Figure 3D)
contains a major fragment at m/z 226.2, which is the m/z of
the protonated 2-HO-PhIP. The third generation product ion
spectrum (MS4) of m/z 226.2 produced ions at m/z 211.2,
198.2, and 183.2; these ions correspond to, respectively, the
•
loss of the CH3 , CO, and the losses of CH3• and CO from 2-
HO-PhIP (Figure 2E). The mass spectrum is in excellent
agreement to the spectrum of synthetic 2-HO-PhIP (data not
shown). The mass spectral data demonstrate that bond
formation adduct occurred between the 4-HO group of
tyrosine and the C-2 imidazole atom of PhIP. The assignments
of these fragment ions were supported by exact mass
measurements (Table S-1 in the Supporting Information).
Data-Dependent Analysis of Human SA Modified with
NO2-PhIP Following Protein Digestion with Trypsin. Data
mining of the peptide chemical features by MyriMatch revealed
that the sequence coverage of unmodified SA exceeded 80% in
the data-dependent MS/MS mode (data not shown). Cys34 is
the only unpaired cysteine residue in SA that is available for
reaction with carcinogenic metabolites and toxic electro-
philes.16,20 However, human SA contains 59 lysine and 18
tyrosine residues40 that can potentially react with NO2-PhIP to
form adducts. The specific lysine and tyrosine residues within
Figure 2. Product ion mass spectra of (A) desamino-PhIP-Y ([M +
H]+ at m/z 389.2) recovered from NO2-PhIP-modified SA; (B)
desamino-[2H5]-PhIP-Y ([M + H]+ at m/z 394.2) recovered from
NO2-[2H5]-PhIP-modified SA, following digestion with Pronase E,
leucine aminopeptidase, and prolidase; (C) second generation product
ion spectrum of the ion at m/z 315.2; (D) second generation product
ion spectrum of ion at m/z 343.2; and (E) third generation product
ion spectrum of m/z 226.2 (2-HO-PhIP) from desamino-PhIP-Y.
• +
NH3−CO2]+, m/z 315.2 [M + H−C2H4NO2 ] , and at m/z
• +
209.2 [M + H−C9H10NO3 ] , which is attributed to the radical
cation of desamino-PhIP. The product ion spectrum of the
desamino-[2H5]-PhIP-Y ([M + H]+ at m/z 394.1) displays the
corresponding fragment ions at m/z 349.2, 348.2, 333.2, 320.2,
and 214.2 (Figure 2B). Consecutive reaction monitoring was
performed at the MS3 and MS4 scan stages for several fragment
ions of desamino-PhIP-Y to determine the site of bond
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spectra of representative peptide adducts are presented Figure
4.
The first pair of modified peptides (tR = 4.1 min) were
present as doubly charged ions [M + 2H]2+ at m/z 477.8 and
480.3. This molecular mass corresponds to the mass of the
amino acid sequence 191−197 of SA (ASSAKQR, [M + H]+ at
m/z 747.4) plus desamino-PhIP or desamino-[2H5]-PhIP,
respectively (addition of 207 or 212 Da). The product ion
spectra of the proposed ASSAK*QR adducts ([M + 2H]2+ at
m/z 477.8 and 480.3) are shown in Figure 4A,B. The ions
observed at m/z 291.1 and m/z 296.1, respectively, in the
spectrum of NO2-PhIP- and NO2-[2H5]-PhIP-modified AS-
SAKQR are at the same m/z observed in the spectra of
desamino-PhIP-K ([K*-NH3-HCO2H]+ (Figure 1), suggesting
that NO2-PhIP had bound to lysine. The peptide fragment ions
(y2, y2-NH3, b3, and b4) were detected, respectively, at m/z
303.1, 286.2, 246.2, and 317.2 for both NO2-PhIP- and NO2-
[2H5]-PhIP-modified peptides, as would be expected for
fragment ions in the unmodified peptide ASSAKQR. The
peptide fragment ions (y3*, y4*, y5*, y6*, b5*, and b6*) were
shifted by 207 and 212 Da, respectively, for the NO2-PhIP- and
the NO2-[2H5]-PhIP-modified peptides.
Figure 3. Data-dependent MS/MS scanning with mass tags of NO2-
PhIP and NO2-[2H5]-PhIP-modified SA following trypsin digestion,
either unmodified (upper panel) or SA modified with a 3 mol excess of
an equimolar mixture of NO2-PhIP and NO2-[2H5]-PhIP modified
(bottom panel). The chromatograms of the MS/MS data were
acquired on the ions exhibiting a difference of m/z 2.5 in doubly
charged form and a difference of m/z 1.67 in triply charged form,
employing the mass tags scanning option with a partner intensity ratio
of 65−100% for PhIP/[2H5]-PhIP. The ion intensities are normalized
to the same scale for untreated and NO2-PhIP-modified SA.
Fraction 2 was found to contain another adducted peptide of
NO2-PhIP at a lysine residue (tR = 5.7 min). The sequence was
identified as ATK*EQLK, which covers the amino acid
sequence 539−545 of SA. The product ion spectra of the
NO2-PhIP- and NO2-[2H5]-PhIP-adducted ATK*EQLK are
presented in Figure S-2 in the Supporting Information.
Two more adducted peptides containing NO2-PhIP-modified
lysine residues were detected in fraction 3 (tR = 5.9 min) and
fraction 4 (tR = 8.3 min) at relatively low abundance, which
encompasses the amino acid sequences 198−205
(LK*CASLQK) and 349−359 (LAK*TYETTLEK) of SA,
respectively (Table 1).
SA that reacted with NO2-PhIP were identified by tandem MS
sequencing of the proteolytic digests of the modified SA.41
The mass chromatograms of unmodified SA peptides and
NO2-PhIP- and NO2-[2H5]-PhIP adducted peptides of SA are
shown in Figure 3. A deuterium isotope effect caused the [2H5]-
PhIP-peptide adducts to elute about 2 s earlier than the
unlabeled adducts. These slight differences in tR values skewed
the 1/1 ratio of the unlabeled PhIP/[2H5]-PhIP adduct signals.
Therefore, the partner intensity ratio for PhIP/[2H5]-PhIP was
expanded to 65−100%, to efficiently trigger the acquisition of
MS/MS spectra on both unlabeled and labeled peptide adducts.
This “relaxed” ratio resulted in elevated background signals of
some peptides in nonmodified SA. Ten NO2-PhIP-modified
peptides were identified (Table 1), and several product ion
The fifth NO2-PhIP-modified peptide displayed a strong
signal (tR = 9.7 min), and both singly charged [M + H]+ (m/z
618.4 and 623.4) and doubly charged species [M + 2H]2+ (m/z
Table 1. Assignment of NO2-PhIP-Modified Amino Acid Residues from SA That Were Identified by Data-Dependent Analyses
enzyme(s)
fraction
position
peptide sequence
N/A
modified site
Pronase E/leucine aminopediase/prolidase
1
2
3
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
N/A
lysine
cysteine
tyrosine
Lys195
Lys541
Lys199
Lys351
Tyr411
Cys34
N/A
N/A
N/A
N/A
trypsin
191−197
539−545
198−205
349−359
411−413
30−41
ASSAK*QR
ATK*EQLK
LK*CASLQK
LAK*TYETTLEK
Y*TK
YLQQC*PFEDHVK
AQYLQQC*PFEDHVK
Y*LYEIAR
QNCELFEQLGEY*K
FY*APELLFFAK
ASSAK*QR
ATK*EQLK
Y*TK
LQQC*PFEDHVK
LQQC*PF
FY*APELL
FY*APELLF
FY*APELLFF
28−41
Cys34
138−144
390−402
149−159
191−197
539−545
411−413
29−41
Tyr138
Tyr401
Tyr150
Lys195
Lys541
Tyr411
Cys34
trypsin/chymotrypsin
31−36
Cys34
149−155
149−156
149−157
Tyr150
Tyr150
Tyr150
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Figure 4. Product ion mass spectra of (A) fraction 1, ASSAK*QR (desamino-PhIP-K) adduct ([M + 2H]2+ at m/z 477.8) recovered from NO2-
PhIP-modified SA; (B) ASSAK*QR (desamino-[2H5]-PhIP-K) adduct ([M + 2H]2+ at m/z 480.3) recovered from NO2-[2H5]-PhIP-modified SA;
(C) fraction 5, Y*TK (desamino-PhIP-Y) adduct ([M + 2H]2+ at m/z 309.9) recovered from NO2-PhIP-modified; (D) Y*TK (desamino-[2H5]-
PhIP-Y) adduct ([M + 2H]2+ at m/z 312.4); (E) fraction 8, Y*LYEIAR (desamino-PhIP-Y) adduct ([M + 2H]2+ at m/z 568.1) recovered from
NO2-PhIP-modified SA; and (F) Y*LYEIAR (desamino-[2H5]-PhIP-Y) adduct ([M + 2H]2+ at m/z 570.6) recovered from NO2-[2H5]-PhIP-
modified SA by trypsin digestion.
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309.9 and 312.4) were observed. This modified peptide was
assigned to the amino acid sequence 411−413 of SA (YTK ([M
+ H]+ at m/z 411.2) and contained a mass increment of 207
and 212 Da, consistent with adduction by desamino-PhIP and
desamino-[2H5]-PhIP. Either Tyr411 or Lys413 of SA could be
the site of modification. The product ion spectra of the
modified YTK peptide contained several prominent fragment
ions (Figure 4C,D). The y1 ion observed at m/z 147.1
corresponds to lysine, and the fragment ions observed at m/z
230.1 and 248.2 (y2 and y2-H2O) in the spectra for both NO2-
PhIP- and NO2-[2H5]-PhIP-modified peptides are also seen in
the spectrum of YTK (data not shown). The fragment ions
(a2*, b2*, and b2*-H2O) contain mass increments of desamino-
PhIP (207 Da) and desamino-[2H5]-PhIP (212 Da),
respectively. The b2*-CO2 ion (C25H26N5O2) at m/z 428.3
and 433.3 for unlabeled and desamino-[2H5]-PhIP adducts is
proposed to occur by a rearrangement involving the HO group
of the threonine side chain to the electrophilic carbonyl moiety,
followed by loss of CO2.42 The ions at m/z 343.2 and 348.2
(y*) for desamino-PhIP- and desamino-[2H5]-PhIP adducts are
assigned to the desamino-PhIP-Y immonium ions. These
spectral data prove that adduction of NO2-PhIP occurred at
tyrosine and not lysine. The second generation product ion
spectrum of the ion at m/z 343.2 for desamino-PhIP-Y*TK and
at m/z 348.2 for desamino[2H5]-PhIP-Y*TK ([M + H−
HCO2H]+) contained the prominent fragment ion at m/z
226.1, attributed to 2-HO-PhIP (m/z 231.1 for 2-HO-[2H5]-
PhIP), as was observed in the spectra of desamino-PhIP-Y
(Figure 1). Accurate mass measurement of the monoisotopic
elemental mass of synthetic Y*TK and its product ion spectra
confirmed the assignment (Table S-1 in the Supporting
Information). Thus, the structure of the modified peptide in
fraction 5 is assigned Y*TK with the site of adduction occurring
between the 4-HO-tyrosine group and the C-2 imidazole atom
of NO2-PhIP.
Three more fractions containing peptides modified with
NO2-PhIP at tyrosine residues were observed in fraction 8 (tR
=14.9 min), fraction 9 (tR = 16.8 min), and fraction 10 (tR =
21.6 min) (Table S-1 in the Supporting Information). The
modified peptides in fraction 8 were doubly charged species
([M + 2H]2+ at m/z 568.1 and 570.6), corresponding to the
peptide containing amino acid residues 138−144 of SA
(Y*LYEIAR [M + H]+ at m/z 927.5) plus the desamino-
PhIP and desamino-[2H5]-PhIP moieties, respectively. The
product ion spectra of this pair of modified peptides contained
almost the complete series of b and y ions and the immonium
ions for the desamino-PhIP modified tyrosine (Figure 4E,F),
which confirmed that Tyr138 was the amino acid modified in
this sequence. The peptide adduct observed in fraction 9
corresponded to the amino acid residues 390−412 of SA
(QNCELFEQLGEY*K, [M + 2H]2+at m/z 933.2) with NO2-
PhIP adducted to Y, based on the interpretation of the product
ion spectra (Table S-1 and Figure S-3 in the Supporting
Information).
and 574.0 (Figure S-4A,B in the Supporting Information). The
peptide sequence is ascribed to amino acid residues 30−41 of
SA, with adduct formation occurring at Cys34 (YLQQC*P-
FEDHVK, 1505.7 Da). The ions observed at m/z 242.1 in the
mass spectrum of NO2-PhIP-modified peptide and at m/z
247.1 in the spectrum of NO2-[2H5]-PhIP-modified peptide
were identified as the protonated 2-thioimdazole derivatives of
PhIP and [2H5]-PhIP, by their spectra at the MS3 scan stage as
reported previously.24 The y and b ion series supported the
proposed assignment of the adduct structure. The b2 (m/z
277.1), b3 (m/z 404.6), b4 (m/z 533.2), b5 (m/z 843.1), b6 (m/
z 940.4), y2 (m/z 246.2), y3 (m/z 383.3), y4 (m/z 498.3), y5
(m/z 627.3), y6 (m/z 774.6), and y7 (m/z 871.3) observed in
both product ion spectra are consistent with predicted
sequence for unmodified YLQQCPFEDHVK. A number of
ions display the characteristic isotopic pattern with a mass
difference of 5 Da and encompass the modified amino acid
residue, b6*-NH3, y9*-NH3, y9*-NH3-HS-PhIP, y10*-NH3, y10*-
NH3-HS-PhIP, and y11*.
A pair of PhIP- and [2H5]-PhIP-labeled peptide adducts are
observed as triply charged species [M + 3H]3+ at m/z at 638.5
and 640.2 in fraction 7. The product ion spectrum displayed
many of the peptide sequence series of y and b ions in common
to that observed for YLQQC*PFEDHVK but also contained
ions attributed to three additional amino acid residues. The
peptide was identified as AQYLQQC*PFEDHVK (1704.8 Da)
modified by desamino-PhIP (207 Da) or desamino-[2H5]-PhIP
(212 Da); this pair of peptide adducts occurred at amino acid
residues 28−41 of SA (Figure S-4C,D in the Supporting
Information).
Data-Dependent Analysis Human SA Modified with NO2-
PhIP Following Protein Digestion with Trypsin/Chymotryp-
sin. Data-dependent scanning of modified SA digested with
trypsin/chymotrypsin resulted in the discovery of six pairs of
modified peptide adducts (Figure S-5 in the Supporting
Information and Table 1). Consistent with the trypsin
digestion, Y*TK adducts derived from trypsin/chymotrypsin
of modified SA were identified as doubly charged ion [M +
2H]2+ at m/z 309.9 and 312.4. The NO2-PhIP-modified
LQQC*PF adducts (amino acid residues 31−36 of SA) were
observed as doubly charged species [M + 2H]2+ (m/z 472.0
and 474.4) (tR = 17.5 min). The product ion spectra were
consistent with results from our previous study that showed the
formation of LQQC*PF (C-desamino-PhIP or C-desamino-
[2H5]-PhIP) adducts.24
Peptide adducts observed at 11.4 min displayed triply
charged ions [M + 3H]3+ at m/z 517.8 and 519.7, which are
an addition mass of 207 and 212 Da to the protonated peptide
LQQCPFEDHVK (m/z 1343.6, residues 31−41 of SA) with a
missed cleavage at C-terminal of phenylalanine. The appear-
ance of b2 (m/z 242.1), b2-NH3 (m/z 225.1), y3 (m/z 383.2),
b4*(m/z 340.8 or 343.1, doubly charged), y7 (m/z 871.3),
y8*(m/z 591.4 or 594.1, doubly charged), y9*(m/z 655.5 or
657.9, doubly charged), y10*-NH3 (m/z 710.7 or 713.2, doubly
charged), as well as protonated HS-PhIP and HS-[2H5]-PhIP
ions (m/z 242.1 and 247.1) provided the evidence for PhIP
adduction at LQQC*PFEDHVK (Figure S-6 in the Supporting
Information).
The pair of peptide adducts detected in fraction 10 were
consistent with the amino acid residues 149−159 of SA
(FY*APELLFFAK, [M + 2H]2+at m/z 777.1 and 779.6) having
adductions with desamino-PhIP and desamino-[2H5]-PhIP
moieties at Y, respectively.
Another site of NO2-PhP modification of human SA was
identified at Tyr150. Three peptides containing the FY*AP, due
to missed cleavages with chymotrypsin, were detected at tR
22.8, 25.6, and 28.0 min (Table 1 and Figure S-7 in the
Supporting Information).
NO2-PhIP-modified peptides of the Cys34 residue were
detected in fractions 6 and 7 (tR = 11.9 and 12.2 min). Fraction
6 contained triply charged desamino-PhIP- and desamino-
[2H5]-PhIP-labeled peptide adducts [M + 3H]3+ at m/z 572.4
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Figure 5. UPLC-ESI/MS2 total ion current chromatograms of adducts of NO2-PhIP-modified SA formed by reaction of the carcinogen with human
SA at a molar ratio of 3:1 (left) and 0.3:1 (right); the SA was digested with trypsin. Targeted UPLC-ESI/MS2 was done on the following modified
peptides: fraction 1, ASSAK*QR as doubly charged species ([M + 2H]2+ at m/z 477.8); fraction 2, ATK*EQLK ([M + 2H]2+ at m/z 512.9);
fraction 3, LK*CASLQK ([M + 2H]2+ at m/z 577.8); fraction 4, LAK*TYETTLEK ([M + 3H]3+ at m/z 501.9, extracted ions: m/z 378.8, 660.6, and
720.4); fraction 5, Y*TK ([M + 2H]2+ at m/z 309.2); fraction 7, AQYLQQC*PFEDHVK ([M + 3H]3+ at m/z 638.5); fraction 8, Y*LYEIAR ([M +
2H]2+ at m/z 568.1); and fraction 9, QNCELFEQLGEY*K ([M + 2H]2+ at m/z 933.2).
Data-Dependent Analyses of SA in Human Plasma
Reacted with a Mixture of NO2-PhIP and NO2-[2H5]-PhIP.
The formation of PhIP-SA adduction products was also
examined in human plasma treated with NO2-PhIP and NO2-
[2H5]-PhIP, because plasma proteins, fatty acids, drugs, and
other organic compounds that are associated with SA can
induce structural changes in the conformation of SA and affect
the reactivity of the amino acid residues.40,43 Data-dependent
scanning of the proteolytic digest with trypsin or trypsin/
chymotrypsin revealed that NO2-PhIP-modified peptides at
Cys34, Lys195, Lys199, Lys351, Lys541, Tyr401, Tyr411, and Tyr150 of
SA results consistent with the adduction products of NO2-PhIP
formed with commercial purified human SA (Figure S-8 in the
Supporting Information).
about 99% of the total ion counts of SA adducts (Figure 5).
Thus, Tyr411 is a primary binding site of NO2-PhIP.
Mass Spectrometric Characterization of Human SA
Modified with HNOH-PhIP. Data-Dependent Analysis of
Human SA Modified with HONH-PhIP Following Protein
Digestion with Pronase E, Leucine Aminopeptidase, and
Prolidase. The reaction of human SA with HONH-PhIP in
vitro produced the N2-cysteinesulfinyl-PhIP; this sulfinamide
linkage accounted for greater than 95% of the HONH-PhIP
bound to SA.24 The enzymatic digestion of HONH-PhIP- and
HONH-[2H5]-PhIP-modified SA with Pronase E, leucine
aminopeptidase, and prolidase resulted in extensive hydrolysis
of the sulfinamide linkage, and targeted MS/MS analyses of the
protein digest showed that PhIP was recovered in high yields.24
The amino acid adduct N2-cysteinesulfinyl-PhIP was not
detected in the three-enzyme digest; however, data-dependent
scanning identified two pairs of HONH-PhIP-tripeptide
adducts (tR = 17.8 and 19.3 min) (Figure S-9 in the Supporting
Information and Table 2). The first pair of adducts was
detected as singly charged species [M + H]+ at m/z 604.2 (m/z
= 609.2 for [2H5]-PhIP-peptide). The product ion spectra
(Figure 6A,B) were in excellent agreement with the mass
spectra previously reported for the N2-cysteinesulfinyl-PhIP
tripeptide C*PF (C-[SO]-PhIP) and (C-[SO]-[2H5]-
PhIP) adducts.24 The second set of adducts (tR = 19.3 min)
Targeted UPLC-ESI/MS2 Analysis of Human SA Modified
with NO2-PhIP Following Protein Digestion with Trypsin. The
sites of NO2-PhIP binding to human SA reported above were
characterized with SA that had been modified with a 3 mol
excess of NO2-PhIP. However, when the reaction of NO2-PhIP
with SA was performed with 10-fold less carcinogen (0.3 mol
NO2-PhIP: 1 mol SA), targeted UPLC-ESI/MS2 analysis
revealed that the desamino-PhIP adducts were hardly formed
at Lys195, Lys199, Lys541, Cys34, Tyr138, and Tyr401, and
desamino-PhIP adduct formation at Tyr411 accounted for
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PhIP) and (C-[SO2]-[2H5]-PhIP). The assignment of the
structure is supported by the product ion spectra (Figure
6C,D). The fragment ion observed at m/z 396.2 in the spectra
of both modified peptides is attributed to the loss of PhIP
(224.1 Da) and [2H5]-PhIP (229.1 Da). Thus, the two oxygen
atoms are associated with the C*PF peptide and not the PhIP
moiety. The mass difference of 30 Da observed between the
fragment ion at m/z 396.2 and the protonated CPF ([M + H]+
at m/z 366.1) is consistent with the presence two sulfur−
oxygen double bonds on Cys34. The ions at m/z 263.2, 313.2,
330.1, 358.1, 427.1, and 455.1 correspond to, respectively, y2,
the C* immonium ion-NH3, the C* immonium ion, and the
b1*, a2*, and b2* ions.
Table 2. Assignment of HONH-PhIP-Modified Amino Acid
Residues from SA That Were Identified by Data-Dependent
Analyses
modified
site
enzyme(s)
fraction position
peptide sequence
Pronase E/leucine
aminopediase/
prolidase
1
2
34−36 C*PF (sulfinamide)
34−36 C*PF (sulfonamide)
Cys34
Cys34
trypsin/
chymotrypsin
1
2
3
31−41 LQQC*PFEDHVK
(sulfinamide)
Cys34
Cys34
Cys34
31−41 LQQC*PFEDHVK
(sulfonamide)
31−36 LQQC*PF
sulfinamide
Data-Dependent Analysis of Human SA Modified with
HONH-PhIP Following Protein Digestion with Trypsin/
Chymotrypsin. The PhIP sulfinamide linkage at Cys34 of SA
undergoes hydrolysis to produce PhIP, by heat and treatment
with DTT during the denaturation of SA (Peng, L.
Unpublished observations). However, the hexapeptide N2-
cysteinesulfinyl-PhIP LQQC*PF (C-[SO]-PhIP, tR = 16.8
min, [M + 2H]2+ m/z 487.4 and 489.7 for PhIP and [2H5]-
PhIP-modified peptides) was recovered from HONH-PhIP-
modified SA, following digestion with trypsin/chymotrypsin
without heat denaturation and DTT treatment. The missed
cleavage adducts LQQC*PFEDHVK (C-[SO]-PhIP) and
LQQC*PFEDHVK (C-[SO2]-PhIP) were also observed,
respectively, at tR = 11.3 and 13.4 min, occurring as triply
protonated species [M + 3H]3+ (Figures S-10 and S-11 in the
Supporting Information).
Mass Spectrometric Characterization of N-Acetoxy-
PhIP and Its Adduction Products with Human SA. N-
Acetoxy-PhIP is a penultimate metabolite of PhIP that reacts
with DNA to form stable covalent adducts.28,29,44 The
conditions of synthesis of N-acetoxy-PhIP were optimized to
produce this reactive intermediate in high yield with minimal
side-reaction products (Figure 7A). The amount of N-acetoxy-
PhIP was about 100-fold greater than the amount of its inactive
isomer, the hydroxamic acid, N-hydroxy-N-(1-methyl-6-
phenylimidazo[4,5-b]pyridin-2-yl)-acetamide (N-hydroxy-N-
acetyl-PhIP), assuming comparable ionization efficiencies of
the compounds by ESI/MS. The structures of these acetylated
isomers of HONH-PhIP ([M + H]+ at m/z 283.1) are readily
distinguished by their product ion spectra (Figure 7B,C). The
product ion spectrum of N-acetoxy-PhIP contains major
fragment ions at m/z 223.0 and 224.0, corresponding,
•
respectively, to losses of CH3CO2 and CH3CO2H, whereas
the product ion spectrum of N-hydroxy-N-acetyl-PhIP contains
a base peak ion at m/z 241.1, which is attributed to the loss of
ketene (CH2CO).
Figure 6. Product ion mass spectra of (A) C*PF (C-[SO]-PhIP)
sulfinamide adduct ([M + H]+ at m/z 604.2) recovered from HONH-
PhIP-modified SA; (B) C*PF (C-[SO]-[2H5]-PhIP) sulfinamide
adduct ([M + H]+ at m/z 609.2) recovered from HONH-[2H5]-PhIP-
modified SA; (C) C*PF (C-[SO2]-PhIP) sulfonamide adduct ([M +
H]+ at m/z 620.2) recovered from HONH-PhIP-modified SA and (D)
C*PF (C-[SO2]-[2H5]-PhIP) sulfonamide adduct ([M + H]+ at m/z
625.2) recovered from HONH-[2H5]-PhIP-modified SA digested with
Pronase E, leucine aminopeptidase, and prolidase.
Data-Dependent Analysis of Human SA Modified with N-
Acetoxy-PhIP Following Protein Digestion with Pronase E,
Leucine Aminopeptidase, and Prolidase. The UV spectrum of
human SA modified with N-acetoxy-PhIP displays a maximum
at 320−325 nm, whereas nonmodified SA does not possess a
chromophore around these wavelengths. The level of chemical
modification of SA by N-acetoxy-PhIP was estimated at ∼170
pmol PhIP/nmol SA, based on the assumption that N-acetoxy-
PhIP-SA adducts possess molar absorption coefficients that are
comparable to that of PhIP [315 nm; ε (M−1 cm−1) at 22220].
However, the isotopic data-dependent scanning was not
successful in detecting SA adducts of N-acetoxy-PhIP,
irrespective of the enzymatic digestion conditions. Nucleophilic
amino acids of SA were expected to react with N-acetoxy-PhIP
was detected as singly charged species [M + H]+ at m/z 620.2
and 625.2, respectively, for the HONH-PhIP- and HONH-
[2H5]-PhIP-modified peptides. The mass of this adduct pair is
16 Da greater than the molecular mass of the C*PF (C-[S
O]-PhIP and C-[SO]-[2H5]-PhIP). The structure of this
adduct pair is proposed to be the sulfonamide C*PF (C-[SO2]-
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Figure 8. Product ion mass spectra of (A) 5-HO-PhIP ([M + H]+ at
m/z 241.2) recovered from N-acetoxy-PhIP-modified SA (upper
panel) and 5-HO-[2H5]-PhIP ([M + H]+ at m/z 246.2) recovered
from N-acetoxy-[2H5]-PhIP-modified SA (bottom panel) digested
with Pronase E, leucine aminopeptidase, and prolidase. The ions
observed at m/z 227.2 and m/z 226.2 in the product ion spectrum of
5-HO-[2H5]-PhIP are attributed to losses of HDO and D2O.
Figure 7. (A) UPLC-ESI/MS analysis of reaction products formed by
reaction of HONH-PhIP with acetic anhydride, following SPE. The
peaks in the chromatogram are PhIP (tR = 10.0), 5-HO-PhIP (tR
=
domains, and each domain contains two subdomains.47 Studies
have shown that microenvironments of pH within the different
domains of SA can lower the pKa values of some nucleophilic
side chain groups of amino acids and enhance their reactivity
with electrophiles.43,48 Moreover, the tertiary structure of SA
can exert an influence on noncovalent protein−electrophile
interactions and direct ensuing covalent adduct formation
between specific amino acids and electrophiles.49 Thus, we had
anticipated that nucleophilic amino acids of SA would display
different degrees of reactivity toward these distinct electrophilic
metabolites of PhIP, resulting in an array of covalent adducts of
PhIP within different sequence locations of SA.
We employed a two tier approach to map the sites of human
SA modification with NO2-PhIP, HONH-PhIP, and N-acetoxy-
PhIP. In the first approach, SA samples modified with
metabolites of PhIP were enzymatically digested with a mixture
of Pronase E, leucine aminopeptidase, and prolidase to produce
amino acid containing adducts.37,38 Data-dependent scanning
of NO2-PhIP-modified SA revealed that adducts were formed
with Cys, Lys, and Tyr. This three-enzyme mixture did not
generate amino acid adducts of SA modified with HONH-PhIP
but produced tripeptide adducts of PhIP formed at Cys34. The
Cys34 residue of human SA is known to react with many
genotoxicants and toxic electrophiles to form adducts with
acrylamide,50 nitrogen mustard,51 α,β-unsaturated aldehydes,52
the neurotoxin brevetoxin B,53 acetaminophen,54 benzene,55 2-
amino-3-methylimidazo[4,5-f ]quinoline (IQ),31 and PhIP.22−24
Many of these adducts do not undergo digestion beyond the
tripeptide stage with Pronase. Thus, the efficacy of proteoloytic
digestion of chemically modified SA is highly dependent upon
structure of the toxicant and the nature of the bond formed
between the toxicant and the sulfhydryl group of Cys34.
10.2), N-hydroxy-N-acetyl-PhIP (tR = 10.9), and N-acetoxy-PhIP (tR =
13.7). Product ion spectra were acquired on acetylated isomers of
HONH-PhIP ([M + H]+ at m/z 283.1): (B) product ion spectrum of
N-acetoxy-PhIP and (C) product ion spectrum of N-hydroxy-N-acetyl-
PhIP.
or the proposed nitrenium ion45 to form adducts with amino
acids or peptides with mass increments of 223 or 228 Da,
attributed, respectively, to reactive PhIP or [2H5]-PhIP species,
less one proton from the amino acid residue. Although data-
dependent scanning analyses failed to detect stable covalent
adducts, targeted UPLC-ESI/MS2 analysis of the N-acetoxy-
PhIP-modified SA digested with the Pronase E, amino leucine
peptidase and prolidase revealed the presence of high levels of
PhIP and 5-HO-PhIP. The identities of PhIP (data not shown)
and 5-HO-PhIP and 5-HO-[2H5]-PhIP were corroborated by
their product ion spectra (Figure 8). Pretreatment of SA with
the selective thiol reagents 4-CMB or NEM prior to reaction
with N-acetoxy-PhIP decreased the amounts of PhIP and 5-
HO-PhIP recovered from the proteolytic digest by 3-fold. This
finding suggests that a substantial portion of the N-acetoxy-
PhIP had bound to Cys34 as unstable adducts that underwent
hydrolysis to form PhIP and 5-HO-PhIP, during proteolytic
digestion.
DISCUSSION
■
NO2-PhIP, HONH-PhIP, and N-acetoxy-PhIP are reactive
species that contribute to the deleterious biological effects of
PhIP through DNA and protein adduct formation.10,46
Metabolites of PhIP are known to bind to human SA in
vivo;13 however, the structures of the adducts remain to be
elucidated. Human SA is comprised of three homologous
K
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Scheme 2. Proposed Mechanisms of Hydrolysis of Cys34 PhIP-Sulfenamide of SA To Produce 5-HO-PhIP and PhIP
With knowledge about the primary amino acids that had
formed adducts with PhIP metabolites, the sequence locations
of PhIP adducts were mapped in tryptic or tryptic/chymotrypic
digests obtained from SA modified with a 3-fold excess of an
equimolar mixture of unlabeled and [2H5]-labeled PhIP
metabolites. The dual isotope label provided a characteristic
fingerprint for the unambiguous identification of PhIP-modified
peptides. Data-dependent scanning of NO2-PhIP-modified SA
enabled us to identify adducts at nine sequence locations:
Cys34, Lys195, Lys199, Lys351, Lys541, Tyr138, Tyr150, Tyr401, and
Tyr411. The identification of SA modifications by N-oxidized
derivatives of PhIP was greatly accelerated, by the MyriMatch
algorithm over manual interpretation. However, the major
adduct formed at Tyr411, Y*TK, escaped detection by
MyriMatch, probably because there was an insufficient number
of y and b ions to identify the modified peptide by the
algorithm (Figure 3C). Similarly, the C*PF adducts formed
with NO-PhIP that were recovered from the three-enzyme
digest also escaped detection by MyriMatch. The employment
of the isotopic mixture of the N-oxidized PhIP metabolites was
critical for the successful identification of these tripeptide
adducts by manual inspection.
NO2-PhIP also formed adducts with SA at Lys195, Lys199, and
Lys541. The Lys195 and Lys199 residues occur at the interface of
subdomains IB and IIA and Lys541 resides within subdomain III
B.47 These lysine residues are known to form acyl-linked
adducts with glucuronide conjugates of tolmetin and
permethrin.61,62 Lys195 or Lys199 was also reported to form an
adduct with 2,4-dinitro-1-chlorobenzene,63 and both lysine
residues are reactive sites in SA and form adducts with 4-
hydroxy-trans-2-nonenal.52
Cys34 is another reactive site in human SA that forms adducts
with NO2-PhIP. The Cys34 resides in a shallow crevice of SA
and predominantly exists in the thiolate form due to its
interaction with three ionizable residues, Asp38, His39, and
Tyr84, which are within close vicinity because of the tertiary
structure of SA.43,48,64 As a result, the thiol of Cys34 has an
unusually low pKa value of 6.5 as compared to about 8.0−8.5 in
many other proteins or peptides at physiological pH.43,52 These
molecular and structural features of SA can help to explain the
high reactivity of Cys34 toward many low molecular weight
electrophiles of diverse structure.20,65
The sites of NO2-PhIP binding to human SA were
characterized with SA that had been modified with a 3 mol
excess of NO2-PhIP. However, when the reaction of NO2-PhIP
with SA was performed with a limiting amount of carcinogen,
desamino-PhIP-Y*TK adduct formation at Tyr411 accounted
for about 99% of the total ion counts of SA adducts. These
binding data indicate that Tyr411 is a primary binding site of
NO2-PhIP and a likely adduction site of NO2-PhIP in vivo.
The adduction of HONH-PhIP to SA was found to occur
only with Cys34; these data are in agreement with our previous
finding.24 The adduct exists as the sulfur−nitrogen-linked N2-
[cystein-S-yl-PhIP]-S-oxide. Therefore, the reactivity of
HONH-PhIP with SA was poor, and protein adduct formation
The major site of binding of NO2-PhIP to SA occurred at
Tyr411; this amino acid is present in the center of a binding
pocket with Arg410 in subdomain IIIA of human SA.48 The
phenolic HO group of tyrosine has a pKa of ∼10, whereas the
pKa of phenolic HO group of Tyr411 is lowered to 7.9 due to
the electrostatic interaction with guanidinium ω-NH of
56
Arg410
.
Tyr411 is a major site for covalent adduct with
nitrophenyl acetate,48 organophosphorous pesticides,57 and
nerve agents (tabun, sarin, cyclosarin, and VX); secondary
adduction sites with many of these toxicants occurs at Tyr148
Tyr150, and Tyr161 of SA.57−60
,
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only occurred following the oxidation of HONH-PhIP to NO-
PhIP. The sulfhydryl SH of Cys34 reacted with the NO bond
of NO-PhIP to form a semimercaptal, which underwent
rearrangement to the more stable sulfinamide structure.66 We
also identified the sulfonamide adduct of PhIP formed at Cys34;
this adduct was not previously reported, and its mechanism of
formation is uncertain.24 The reaction of HONH-PhIP with
oxygen results in the production of NO-PhIP and superoxide
anion.67 The superoxide anion may have oxidized a portion of
the Cys34 to the sulfinic acid prior to its reaction with N-
oxidized-PhIP, resulting in formation of the sulfonamide
adduct.68 Alternatively, the superoxide anion generated over
the time, by HONH-PhIP, may have oxidized a portion of the
N2-[cystein-S-yl-PhIP]-S-oxide linkage to the sulfonamide
structure; the oxidation of N2-[cystein-S-yl-PhIP]-S-oxide to
the sulfonamide adduct during enzymatic digestion also cannot
be excluded.69
The pathway by which NO2-PhIP is formed in hepatocytes or
in liver of rodents is not clear, but the findings indicate that
NO2-PhIP occurs during the N-oxidation of PhIP.
In summary, our mapping study of N-oxidized metabolites of
PhIP adducted to human SA point to the Tyr411 and Cys34
residues as major sites of binding, respectively, for NO2-PhIP
and NO-PhIP. Sensitive tandem MS methods are under
development to determine if these SA adducts of PhIP are
present in humans. Our goal is to implement stable covalent SA
protein adducts of PhIP as biomarkers in molecular
epidemiology studies to measure exposure and to assess the
role of this HAA in diet-related cancers.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional information as noted in the text (Table S-1 and
Figures S-1−S-11). This material is available free of charge via
the Internet at http://pubs.acs.org.
N-Acetoxy-PhIP, a penultimate metabolite of PhIP that
forms stable covalent adducts at appreciable levels with
DNA,29,44 was the least efficient of the N-oxidized metabolites
of PhIP to react with human SA and form stable covalent
adducts. Both HONH-PhIP and N-acetoxy-PhIP bind to DNA,
almost exclusively at dG.29,44,70 DNA adduct formation with N-
acetoxy-PhIP is believed to occur through the proposed
nitrenium ion45 via an SN1 or an intermediate SN2
mechanism.71 The acetate group of N-acetoxy-PhIP undergoes
solvolysis more facilely than HONH-PhIP, to produce the
nitrenium ion, and explains the superior reactivity of N-acetoxy-
PhIP toward DNA.45 Adduct formation of N-acetoxy-PhIP with
SA was expected to occur by a similar mechanism. Although
stable adducts of N-acetoxy-PhIP were not detected, appreci-
able levels of PhIP and 5-HO-PhIP were recovered from the N-
acetoxy-PhIP-treated SA digested with Pronase E, leucine
aminopeptidase, and prolidase. The pretreatment of SA with
the selective thiol reagents 4-CMB or NEM decreased the
amounts of PhIP and 5-HO-PhIP recovered from the digests of
N-acetoxy-PhIP-modified SA by about 3-fold. These findings
point to the formation of unstable adducts of PhIP at Cys34. A
previous study reported that certain adducts formed between
N-acetoxy-PhIP and rat SA were unstable and underwent
hydrolysis to produce 5-HO-PhIP.22 The reaction of N-acetoxy-
PhIP with cysteine or glutathione also produced labile adducts,
tentatively assigned as sulfenamide conjugates, which under-
went hydrolysis to form 5-HO-PhIP.22 Thus, a large portion of
the PhIP and 5-HO-PhIP recovered from N-acetoxy-PhIP
bound to rat or human SA may be attributed to an unstable
sulfenamide linked adduct formed at Cys34 (Scheme 2).23
The Tyr411 adduct formed with NO2-PhIP and the Cys34
sulfinamide adduct formed with NO-PhIP are distinct
structures derived from different N-oxidized metabolites of
PhIP; however, both adducts are biomarkers of the genotoxic
metabolite, HONH-PhIP. The N-hydroxy metabolites of
aromatic amines and HAAs are well-known to undergo
oxidation, by enzymatic and nonenzymatic chemistry, to form
their corresponding nitroso derivatives.67,72,73 We have also
observed that NO2-PhIP is formed from HONH-PhIP, through
the NO-PhIP intermediate, under aerobic conditions in
phosphate-buffered saline (pH 7.4) (Turesky, R. Unpublished
observations). Moreover, NO2-PhIP is formed during the
metabolism of PhIP in hepatocytes of rats pretreated with
polychlorinated biphenyls, and the desamino-PhIP-gluathione
conjugate, glutathionyl-1-methyl-6-phenylimidazo[4,5-b]-
pyridine, has been detected in bile of rats exposed to PhIP.74
AUTHOR INFORMATION
Corresponding Author
*Tel: 518-474-4151. Fax: 518-473-2095. E-mail: Rturesky@
wadsworth.org.
■
Funding
This research was supported by Grants R01 CA122320 (L.P.
and R.J.T.) and R01 CA126479 (D.T. and S.D.) from the
National Cancer Institute.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Comments and discussion of the data with Dr. Paul Vouros,
Northeastern University; Dr. Paul Skipper, MIT; and Dr. Dan
Liebler, Vanderbilt University, are greatly appreciated.
ABBREVIATIONS
■
PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 5-
HO-PhIP, 2-amino-1-methyl-6-(5-hydroxy)-phenylimidazo-
[4,5-b]pyridine; HONH-PhIP, 2-hydroxyamino-1-methyl-6-
phenylimidazo[4,5-b]pyridine; 2-HO-PhIP, 2-hydroxy-1-meth-
yl-6-phenylimidazo[4,5-b]pyridine; NO2-PhIP, 2-nitro-1-meth-
yl-6-phenylimidazo[4,5-b]pyridine; NO-PhIP, 2-nitroso-1-
methyl-6-phenylimidazo[4,5-b]pyridine; N-acetoxy-PhIP, N-
(acetyloxy)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine;
N-hydroxy-N-acetyl-PhIP, N-hydroxy-N-(1-methyl-6-
phenylimidazo[4,5-b]pyridin-2-yl)-acetamide; IQ, 2-amino-3-
methylimidazo[4,5-f ]quinoline; 4-CMB, 4-chloromercuriben-
zoic acid; Hb, hemoglobin; HAA, heterocyclic aromatic amine;
LTQ MS, linear quadrupole ion trap MS; LC-ESI/MS/MS,
liquid chromatography−electrospray ionization/tandem mass
spectrometry; NEM, N-ethylmaleimide; SA, serum albumin;
SPE, solid-phase extraction; TSQ MS, triple stage quadrupole
mass spectrometer; UPLC, ultra performance liquid chroma-
tography
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