Five Tyrosines and Two Serines in Human Albumin Are Labeled by
the Organophosphorus Agent FP-Biotin
Shi-Jian Ding,†John Carr,†James E. Carlson,‡Larry Tong,§Weihua Xue,§Yifeng Li,†
Lawrence M. Schopfer,§Bin Li,§Florian Nachon,|Oluwatoyin Asojo,†
Charles M. Thompson,⊥Steven H. Hinrichs,†Patrick Masson,|and Oksana Lockridge*,§
Department of Pathology and Microbiology, UniVersity of Nebraska Medical Center, Omaha, Nebraska 68198,
Applied Biosystems, Framingham, Massachusetts 01701, Eppley Institute, UniVersity of Nebraska Medical
Center, Omaha, Nebraska 68198, Centre de Recherches d SerVice de Sante ´ des Arme ´es, Unite ´ d’Enzymologie,
BP87, 38702 La Tronche Cedex, France, and Department of Biomedical and Pharmaceutical Sciences,
UniVersity of Montana, Missoula, Montana 59812
ReceiVed April 23, 2008
Tyrosine 411 of human albumin is an established site for covalent attachment of 10-fluoroethoxyphosphinyl-
N-biotinamidopentyldecanamide (FP-biotin), diisopropylfluorophosphate, chlorpyrifos oxon, soman, sarin, and
dichlorvos. This work investigated the hypothesis that other residues in albumin could be modified by
organophosphorus agents (OP). Human plasma was aggressively treated with FP-biotin; plasma proteins were
separated into high and low abundant portions using a proteome partitioning antibody kit, and the proteins
were digested with trypsin. The FP-biotinylated tryptic peptides were isolated by binding to monomeric avidin
beads. The major sites of covalent attachment identified by mass spectrometry were Y138, Y148, Y401, Y411,
Y452, S232, and S287 of human albumin. Prolonged treatment of pure human albumin with chlorpyrifos
oxon labeled Y138, Y150, Y161, Y401, Y411, and Y452. To identify the most reactive residue, albumin was
treated for 2 h with DFP, FP-biotin, chlorpyrifos oxon, or soman, digested with trypsin or pepsin, and analyzed
by mass spectrometry. The most reactive residue was always Tyr 411. Diethoxyphosphate-labeled Tyr 411
was stable for months at pH 7.4. These results will be useful in the development of specific antibodies to
detect OP exposure and to engineer albumin for use as an OP scavenger.
Organophosphorus agents are used in agriculture as pesticides
and are stocked by the military as chemical warfare agents.
These chemicals are toxic to insects, fish, birds, and mammals.
Seizures, respiratory arrest, and death are explained by a cascade
of reactions that begins with inhibition of acetylcholinesterase.
Although acetylcholinesterase in red blood cells and butyryl-
cholinesterase in plasma are established biomarkers of organo-
phosphorus ester (OP)1exposure, additional biomarkers are
being sought. Albumin has the potential to serve as a new
biomarker of OP exposure (1, 2). Albumin has been reported
to covalently bind diisopropylfluorophosphate (DFP), sarin,
soman, cyclosarin, tabun, 10-fluoroethoxyphosphinyl-N-bioti-
namido pentyldecanamide (FP-biotin), chlorpyrifos oxon (CPO),
and dichlorvos (2-6) and to hydrolyze CPO, paraoxon (7, 8),
and O-hexyl O-2,5-dichlorophenyl phosphoramidate (9). Mass
spectrometry has identified tyrosine 411 of human albumin as
the site for covalent attachment of OP nerve agents and OP
pesticides (6). A second site for covalent attachment of soman
was suggested by experiments that found more fluoride ion
released than could be accounted for by one site in albumin
(3). Pretreatment of albumin with decanoate, a lipid that binds
to the Tyr 411 subdomain, inhibited incorporation of 91% of
3H-DFP, leaving open the possibility that 9% of the3H-DFP
bound to other sites (10). Crystallization trials of CPO-labeled
human albumin yielded gelatinous soft amorphous crystals,
further suggesting the likelihood that more than one site was
labeled and that labeling was not uniform. The goal of this study
was to determine if sites in addition to Tyr 411 could make a
covalent bond with OP and to identify the labeled residues.
Our starting premise was that OP bound exclusively to Tyr
411 of human albumin. For certain studies, we wanted 100%
labeling on Tyr 411. Therefore, we treated albumin with excess
CPO. The sample was checked by mass spectrometry to confirm
the site of OP labeling, and to our surprise, we found several
Our studies with human plasma were initiated with the goal of
to identify several FP-biotin-labeled proteins. However, we found
only FP-biotin-labeled albumin. The albumin was covalently
modified on five tyrosines and two serines. FP-biotin was used
for studies with plasma because biotinylated peptides are readily
purified by binding to immobilized avidin beads (1, 11).
Materials. FP-biotin (MW 592.32) was custom synthesized in
the laboratory of Dr. Charles M. Thompson at the University of
Montana (Missoula, MT) (12). FP-biotin was dissolved in methanol
and stored at -80 °C. CPO (ChemService Inc. West Chester, PA;
* To whom correspondence should be addressed. Tel: 402-559-6032.
Fax: 402-559-4651. E-mail: email@example.com.
†Department of Pathology and Microbiology, University of Nebraska
§Eppley Institute, University of Nebraska Medical Center.
|Unite ´ d’Enzymologie.
⊥University of Montana.
1Abbreviations: CPO, chlorpyrifos oxon; DFP, diisopropylfluorophos-
phate; FP-biotin, 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecana-
mide; OP, organophosphorus ester; LC/MS/MS, liquid chromatography
tandem mass spectrometry; MALDI-TOF-TOF, matrix-assisted laser de-
sorption tandem time-of-flight mass spectrometry.
Chem. Res. Toxicol. 2008, 21, 1787–1794
10.1021/tx800144z CCC: $40.75
2008 American Chemical Society
Published on Web 08/16/2008
MET-674B) was dissolved in ethanol and stored at -80 °C. DFP,
a liquid with a concentration of 5.73 M, was from Sigma (D0879).
Soman from CEB (Vert-le-Petit, France) was dissolved in isopro-
panol. A Proteome Partitioning Kit, ProteomeLab IgY-12 High
Capacity in Spin Column format contained IgY antibodies directed
against the 12 most abundant proteins in human plasma (Beckman
Coulter #A24331 S0510903) including albumin, IgG, fibrinogen,
transferrin, IgA, IgM, HDL (apo A-I and apo A-II), haptoglobin,
R-1-antitrypsin, R-1-acid glycoprotein, and R-2-macroglobulin.
Cibacron blue 3GA agarose (Sigma, C1535) bound 10-20 mg of
human albumin per mL of gel. Porcine trypsin (Promega, Madison,
WI; V5113 sequencing grade modified trypsin) at a concentration
of 0.4 µg/µL in 50 mM acetic acid was stored at -80 °C. Pepsin
(Sigma, St. Louis, MO; P6887 from porcine gastric mucosa) was
dissolved in 10 mM HCl to make a 1 mg/mL solution and stored
at -80 °C. Monomeric avidin agarose beads (#20228) were from
Pierce Co. NeutrAvidin agarose beads (#29202) were from Thermo
Scientific (Rockford, IL). Human plasma (EDTA anticoagulant) was
from an adult male, who had fasted overnight before donating blood.
Fatty acid free human albumin (Fluka 05418) was from Sigma/
Procedures for FP-Biotin-Labeled Plasma. Separation of
Low and High Abundance Proteins in Human Plasma. Two
hundred microliters of human plasma were fractionated into low
and high abundance proteins by processing 20 µL of plasma at a
time on the Beckman Coulter Proteome IgY Spin column depletion
kit. The yield of high abundance proteins was 4800 µg in 400 µL.
Of this, 123 µL was labeled with FP-biotin, 123 µL was used as a
negative control, and the remainder was used for determination of
High Abundance Proteins Labeled with FP-Biotin, Digested
with Trypsin, and Purified on Monomeric Avidin. The high
abundance fraction of plasma had albumin as its major component.
A 123 µL aliquot of the high abundance fraction was treated with
1.25 µL of 20 mM FP-biotin for 48 h at 37 °C at pH 8.0. The final
FP-biotin concentration was 200 µM. Excess FP-biotin was removed
by dialysis against 2 × 4 L of 10 mM ammonium bicarbonate.
Proteins, in 8 M urea, were reduced with 5 mM dithiothreitol
and alkylated with 40 mM iodoacetamide. The samples were diluted
to reduce the concentration of urea to 2 M. Proteins were digested
with a 1:50 ratio of trypsin to protein at 37 °C overnight. The trypsin
was inactivated by heating the sample in a boiling water bath for
10 min. It was necessary to inactivate trypsin because trypsin could
have destroyed the avidin protein used in the next step. FP-
biotinylated peptides were purified by binding to 0.5 mL of
monomeric avidin beads. Nonspecifically bound peptides were
washed off with high salt buffers. The column was washed with
water to remove salts, and FP-biotinylated peptides were eluted
with 10% acetic acid. The eluate was dried in a vacuum centrifuge
in preparation for mass spectrometry. The negative control was
human plasma treated with everything except FP-biotin.
Depletion of Albumin on Cibacron Blue, Labeling with
FP-Biotin, Digestion with Trypsin, and Purification on
NeutrAvidin. An albumin-depleted plasma sample was prepared
by binding 0.6 mL of human plasma to 2 mL of Cibacron Blue
and collecting the protein that eluted in 10 mL of 10 mM TrisCl,
pH 8.0, containing 0.3 M NaCl. About 70% of the albumin was
removed from the plasma sample by this procedure. The protein
was desalted, concentrated to 0.5 mL, and labeled with 100 µM
FP-biotin at 37 °C for 16 h in 10 mM ammonium bicarbonate.
The labeled protein was denatured in 8 M urea, reduced with
dithiothreitol, carbamidomethylated with iodoacetamide, and de-
salted on a spin column. The yield was 2000 µg in 500 µL. The
entire sample was digested with 40 µg of trypsin (Promega) at 37
°C overnight. The FP-biotinylated tryptic peptides were bound to
0.1 mL of NeutrAvidin beads, washed with high salt buffers and
water, and eluted with 45% acetonitrile and 0.1% formic acid.
Mass Spectrometry on QSTAR Elite and QTRAP 2000. Five
micrograms of the high abundance FP-biotinylated peptides purified
with monomeric avidin beads was analyzed on the QSTAR elite
liquid chromatography tandem mass spectrometry (LC/MS/MS)
system with ProteinPilot 2.0 software at the Applied Biosystems
laboratories (Framingham, MA).
A second 5 µg aliquot from the same protein preparation, a
negative control sample, and the NeutrAvidin purified peptides were
analyzed by LC/MS/MS on the QTRAP 2000 mass spectrometer
(Applied Biosystems) at the University of Nebraska Medical Center
with Analyst 1.4.1 software. The digest was dried in a vacuum
centrifuge and dissolved in 5% acetonitrile and 0.1% formic acid
to make 0.5 µg/µL. A 10 µL aliquot was injected into the HPLC
nanocolumn (#218MS3.07515 Vydac C18 polymeric rev-phase, 75
µm i.d. × 150 mm long; P.J. Cobert Assoc, St. Louis, MO). Peptides
were separated with a 90 min linear gradient from 0 to 60%
acetonitrile at a flow rate of 0.3 µL/min and electrosprayed through
a nanospray fused silica emitter (360 µm o.d., 75 µm i.d., 15 µm
taper, New Objective) directly into the QTRAP 2000, a hybrid
quadrupole linear ion trap mass spectrometer. An ion spray voltage
of 1900 V was maintained between the emitter and the mass
spectrometer. Information-dependent acquisition was used to collect
MS, enhanced MS, and MS/MS spectra for the three most intense
peaks in each cycle, having a charge of +1 to +4, a mass between
400 and 1700 m/z, and an intensity >10000 counts per s. All spectra
were collected in the enhanced mode, that is, using the trap function.
Precursor ions were excluded for 30 s after one MS/MS spectrum
had been collected. The collision cell was pressurized to 40 µTorr
with pure nitrogen. Collision energies between 20 and 40 eV were
determined automatically by the software, based on the mass and
charge of the precursor ion. The mass spectrometer was calibrated
on selected fragments from the MS/MS spectrum of Glu-Fibrin-
opeptide B. MS/MS spectra were submitted to Mascot for identi-
fication of labeled peptides and amino acids (13). MASCOT
(score 14), and MPCAEDY*LSVVLNQLCVLHEK (score 15) but
none of the other FP-biotinylated peptides. The others were
identified by manually searching the MS/MS data files using the
Extracted Ion Chromatogram feature of the Analyst software. The
scoreswere low becausethe
nize the characteristic fragments of FP-biotin at 227, 312, and 329.
It also did not recognize the 591 ion of FP-biotin or the FP-biotin-
tyrosine immonium ions at 708 and 691 or fragments containing
dehydroalanine in place of serine. The ions that Mascot did not
recognize were often very intense.
The MASCOT modification file is an open source software called
UNIMOD. The FP-biotin modification on serine, threonine, and
tyrosine was introduced according to the instructions found on the
Web site http://www.unimod.org. Access to the modification is
freely available to all MASCOT users in the Variable Modifications
menu under the name FP-biotin. Fragments of FP-biotin are not
part of the MASCOT modification file. Peptides yielding FP-biotin
fragments at 227, 312, and 329 amu were identified using the
Extracted Ion Chromatogram feature of ABI’s Analyst software.
Neutral loss of fragments of FP-biotin were identified by manual
inspection of MS/MS spectra.
Mass Spectrometry by Matrix-Assisted Laser Desorption
Tandem Time-of-Flight Mass Spectrometry (MALDI-TOF-
TOF) 4800. A 0.5 µL aliquot of essentially salt-free samples was
spotted on a MALDI target plate, air-dried, and overlaid with 0.5
µL of 10 mg/mL R-cyano-4-hydroxy cinnamic acid in 50%
acetonitrile and 0.1% trifluoroacetic acid. MS spectra were acquired
using a MALDI-TOF-TOF 4800 (Applied Biosystems), with a laser
power of 3000 V, in positive reflector mode. Each spectrum was
the average of 500 laser shots. The mass spectrometer was calibrated
against des-Arg-Bradykinin (904.468 Da), angiotensin 1 (1296.685
Da), Glu-Fibrinopeptide B (1570.677 Da), and neurotensin (1672.918
Da) (Cal Mix 1 from Applied Biosystems).
Procedures for Pure Albumin. Percent OP-Labeled Tyr
411 Monitored by MALDI-TOF. A 5 µL aliquot of 10 mg/mL
albumin was diluted with 5 µL of 1% trifluoroacetic and digested
with 2 µL of 1 mg/mL porcine pepsin for 1-2 h at 37 °C. The
digest was diluted with 50% acetonitrile and 0.1% trifluoroacetic
acid to give a final protein concentration of about 0.5 mg/mL. A
0.5 µL aliquot was spotted on the MALDI target plate, dried, and
Chem. Res. Toxicol., Vol. 21, No. 9, 2008Ding et al.
overlaid with 0.5 µL of 10 mg/mL R-cyano-4-hydroxy cinnamic
acid. MS spectra were acquired with the laser set at 3000 V and
were saved to Data Explorer. When the saved spectrum was opened
in Data Explorer, the cluster areas appeared in an output window.
Percent OP-labeled Tyr 411 was calculated by dividing the cluster
area of the labeled peptide by the sum of the cluster areas for the
unlabeled and labeled peaks. The unlabeled peptides were409VRYT-
(1717.0 amu) and
VSTPTL423(1830.1 amu). After covalent bond formation with
CPO, these masses increased by 136 amu to become 1853.0 and
1966.1 amu. After covalent bond formation with FP-biotin, these
masses increased by 572.3 to become 2289.3 and 2402.4 amu.
Prolonged Treatment of Albumin with CPO. At the time that
we prepared CPO-labeled human albumin, we knew that Tyr 411
was labeled by CPO and had no reason to suspect that other residues
might also be labeled. CPO dissolved in ethanol was added to an
albumin solution in 10 mM ammonium bicarbonate, pH 8.3, and
0.01% sodium azide in six additions over a 1 month period.
The labeling efficiency was poor when the albumin concentration
was 500 mg/mL, so the albumin was diluted to 35 mg/mL, and
then to 5 mg/mL, and finally to 1 mg/mL. The final ratio was 7.7
µmol of albumin to 146 µmol of CPO. During the 1 month labeling
time, the decision to add more CPO was based on the percent Tyr
411 labeled. No further additions of CPO were made after 85% of
the Tyr 411 had been labeled. The labeled albumin was dialyzed
against 10 mM potassium phosphate, pH 7.0, and 0.01% azide and
processed for LC/MS/MS analysis in the QTRAP mass spectrometer.
Identification of the Most Reactive Residues. The conditions
reported to label 1 mol of albumin with 1 mol of DFP were used
(10). Human albumin (1.8 mg/mL) in 10 mM TrisCl, pH 8.0, was
treated with a 20-fold molar excess of DFP for 2 h at room
temperature. The reaction was stopped by the addition of solid urea
to 8 M and boiling for 10 min in the presence of 10 mM
dithiothreitol. Free sulfhydryl groups were alkylated with iodoac-
etamide. The carbamidomethylated albumin was dialyzed against
2 × 4 L of 10 mM ammonium bicarbonate and digested with
trypsin. Tryptic peptides were subjected to LC/MS/MS on the
The experiment was repeated with FP-biotin, soman, and CPO.
A 15 µM solution of albumin in 10 mM TrisCl pH 8.0 was treated
with 150 µM FP-biotin or 150 µM soman or 150 µM CPO for 2 h
at 22 °C. Samples with intact disulfides were digested with pepsin
and analyzed by MALDI-TOF. Carbamidomethylated tryptic pep-
tides were analyzed by LC/MS/MS. Tryptic peptides labeled with
FP-biotin were also purified on monomeric avidin beads eluted with
50% acetonitrile and 0.1% formic acid and analyzed by MALDI-
Stability of CPO-Labeled Tyr 411. The stability of CPO-labeled
Tyr 411 in human albumin was tested at pH 1.5, 7.4, and 8.3 after
incubation for up to 7 months at 22 and -80 °C. Albumin labeled
on 97% of Tyr 411 with diethoxyphosphate was prepared by
incubating 1 mg/mL human albumin (15.6 µM) in 10 mM TrisCl,
pH 8.0, and 0.01% sodium azide with 118 µM CPO for 2.5 days
at 22 °C. Excess CPO was removed by dialysis of the 8.5 mL
solution against 2 × 4 L of 10 mM ammonium bicarbonate, pH
8.3, and 0.01% azide.
pH 1.5. The pH of 2.6 mL of the dialyzed CPO-albumin was
adjusted to pH 1.5 by adding 2.6 mL of 1% trifluoroacetic acid.
Half of the sample was stored at room temperature, and half was
divided into 40 µL aliquots and stored at -80 °C.
pH 7.4. The pH was adjusted to pH 7.4 by dialyzing 3.3 mL of
the CPO-albumin preparation against 4.5 L of 10 mM potassium
phosphate, pH 7.4, and 0.01% azide. To avoid freeze-thaw
artifacts, samples intended for storage at -80 °C were divided into
20 µL aliquots so that each tube was thawed only once.
pH 8.3. The pH of 2.6 mL of CPO-albumin was brought to pH
8.3 by dialysis against 10 mM ammonium bicarbonate and 0.01%
sodium azide, pH 8.3. Samples to be stored at -80 °C were divided
into 65 tubes each containing 20 µL.
After various times, a tube was removed from -80 °C storage,
and the entire contents were digested with pepsin. Samples stored
at room temperature were also digested with pepsin. The digests
were analyzed by MALDI-TOF, and % labeled Tyr 411 was
calculated from cluster areas as described above.
The sample stored at -80 °C in pH 7.4 buffer was analyzed by
LC/MS/MS to determine whether sites in addition to Tyr 411 were
labeled. The CPO-albumin was denatured, reduced, carbamidom-
ethylated, and digested with trypsin in preparation for LC/MS/MS.
The diethoxyphosphate group was found on Tyr 411 and Tyr 138.
FP-Biotin Labeled Albumin in Human Plasma. The
structures of the organophosphorus agents are shown in Figure
1. Five tyrosines and two serines in human albumin were labeled
with FP-biotin including Tyr 138, Tyr 148, Tyr 401, Tyr 411,
Tyr 452, Ser 232, and Ser 287 (Table 1).
Supporting MS/MS spectra for these assignments are in
Figures 2-6. A peptide labeled with FP-biotin had ions at 227,
312, and 329 atomic mass units (amu) resulting from fragmen-
tation of FP-biotin (12). Two ions characteristic of covalent
binding of FP-biotin to the hydroxyl group of tyrosine are the
immonium ion of tyrosine-FP-biotin at 708 amu and its partner
ion at 691 amu, produced by loss of NH3. The 708 and 691
amu masses are prominent in Figure 2A,B but barely visible in
Figure 3A,B. An additional complexity in Figure 2A is the
presence of ions that had lost a 329 or 226 amu fragment from
The masses in Figure 2A are consistent with the sequence
YTK where the added mass of 572 amu from FP-biotin is on
Tyr. The complete y-ion series is present (y1, 147.0 amu, Lys;
Figure 1. Structures of organophosphorus agents. Covalent binding to
tyrosine or serine results in loss of the fluoride ion from soman, DFP,
and FP-biotin and of the aromatic ring from CPO, so that the added
mass is 162.2 for soman, 164.1 for DFP, 136.0 for CPO, and 572.3 for
FP-biotin. The arrows in FP-biotin indicate fragmentation sites. A 227
amu ion is produced by cleavage between carbon 16 and the adjacent
nitrogen. A 329 amu ion is produced by cleavage between carbon 10
and the adjacent nitrogen. The 312 amu ion is produced by loss of the
amine from the 329 ion.
CoValent Binding to Tyrosines in Albumin Chem. Res. Toxicol., Vol. 21, No. 9, 2008 1789
y2, 248.4 amu, LysThr; and the doubly charged, FP-biotinylated
parent ion). Peaks at 227.2, 312.4, 329.4, 691.3, and 708.5 amu
are indicative of the presence of FP-biotinylated tyrosine. The
remaining major peaks are consistent with various FP-biotiny-
lated tyrosine fragments missing pieces of the FP-biotin moiety.
Peptide YLYEIAR has two tyrosines. A y-ion series (y1-y6)
indicates that the FP-biotin label is on the N-terminal Tyr.
Additional evidence for labeling on Tyr 138 rather than on Tyr
140 was the presence of the a2 ion at 821.4 amu, the b2 ion at
849.2 amu, the a1+2ion at 354.8, and the a2+2ion at 411.4
m/z (Figure 2B). If the FP-biotin had been attached to Tyr 140,
the masses would have been a2 ) 249, b2 ) 277, a1+2) 68,
and a2+2) 125 amu. Peaks at 226.3, 312.4, and 329.3 are
fragments of FP-biotin. Masses at 708.2 and 691.3 amu for FP-
biotinylated tyrosine confirm the presence of FP-biotinylated
tyrosine in the peptide. Analysis of the missed cleavage peptide
KYLYEIAR supports labeling on Tyr 138 (data not shown).
Peptide HPYFYAPELLFFAK in Figure 3A also has two
tyrosines. Evidence for labeling on Tyr 148 rather than on Tyr
150 is the presence of the b4 ion at 1118.5 amu, the a4+2ion
at 545.4 amu, and the b4+2ion at 559.3 m/z. The total mass of
the b4 fragment (1117.6 amu) is equal to the fragment HPYF
Table 1. FP-Biotinylated Human Albumin Tryptic Peptides
Identified by LC/MS/MSa
146-159 2315.2 HPY*FYAPELLFFAK
226-233 1452.7 AEFAEVS*K
390-402 2230.0 QNCELFEQLGEY*K
aResidue numbers in accession # gi: 3212456 are for the mature
albumin protein and do not include the 24 amino acid signal peptide.
The added mass from FP-biotin is 572.3 amu. Cysteines were
carbamidomethylated, thus adding a mass of 57 amu.
Figure 2. MS/MS spectra of albumin peptides labeled with FP-biotin
on Tyrosine. (A) Tyr 411 in peptide YTK and (B) Tyr 138 in peptide
YLYEIAR are covalently modified by FP-biotin. The characteristic
fragments of FP-biotin at 227.2, 312.4, and 329.3 amu are present.
Ions characteristic of FP-biotin modification on tyrosine are the
immonium ion at 708 amu and the immonium ion minus 17 at 691
amu. Support for modification of the first tyrosine rather than the second
in YLYEIAR is the mass of ions a2, b2, and a2+2. The doubly charged
parent ion in panel A had a mass of 492.2 amu. The triply charged
parent ion in panel B had a mass of 500.9 m/z.
Figure 3. MS/MS spectra of albumin peptides labeled with FP-biotin
on Tyrosine. (A) Tyr 148 in peptide HPYFYAPELLFFAK is labeled
with FP-biotin. The quadruply charged parent ion has a mass of 579.7
m/z. The FP-biotin tyrosine immonium ion is at 708.5; after neutral
is covalently modified by FP-biotin. The triply charged parent ion in
B has a mass of 744.2 m/z.
Figure 4. MS/MS spectrum of albumin peptide labeled with FP-biotin
on tyrosine 452. The quadruply charged parent ion has a mass of 773.3
m/z. The carbamidomethylated cysteine is indicated as CAM. Internal
fragmentation at proline yielded the 458.2 ion for PC(CAM)AE and
the 573.2 ion for PC(CAM)AED. The characteristic fragments of FP-
biotin at 227.2, 312.2, and 329.4 are present. The FP-biotin-tyrosine
immonium ion is at 708.4 amu. After loss of an amine, the FP-biotin
tyrosine immonium ion has a mass of 691.4 amu.
Chem. Res. Toxicol., Vol. 21, No. 9, 2008Ding et al.
(545 amu) plus the added mass from FP-biotin (572 amu). Of
the four residues in the b4 fragment, Tyr 148 is the most
reasonable candidate for labeling. Fragment masses for b5 and
b6 also support labeling of Tyr 148 rather than Tyr 150. An
extensive y-ion series (y2-y8) supports the assignment of this
peptide. Masses at 227.3, 312.2, and 329.4 indicate the presence
of FP-biotin. Masses at 691.2 and 708.5 amu indicate the
presence of FP-biotinylated tyrosine. A similar analysis was
made for peptides RHPYFYAPELLFFAK and HPYFYAPELL-
FFAKR, which differ from HPYFYAPELLFFAK by virtue of
missed cleavages (data not shown).
Peptide QNCELFEQLGEYK in Figure 3B is FP-biotinylated
on Tyr 401 as demonstrated by the y2 ion at 882.5 amu, the y3
ion at 1011.5 amu, the y4 ion at 1068.5 amu, and the y5 ion at
1181.8 amu. The y2 mass is equal to the sum of Lys (147 amu),
Tyr (163 amu), and the added mass of FP-biotin (572 amu). A
variety of larger, multiply charged y-ion fragments support the
labeling assignment. Prominent b-ion fragments confirm the
identity of the peptide. Fragments at 227.2, 312.2, and 329.3
amu indicate the presence of FP-biotin in the peptide.
Peptide MPCAEDYLSVVLNQLCVLHEK in Figure 4 is FP-
biotinylated on Tyr 452. The best evidence in support of this
interpretation is the doubly charged mass at 720.4 m/z, which
is consistent with the b7 ion plus the added mass from FP-
biotin. The b7 ion consists of MPCAEDY. Of these residues,
only Tyr 452 is a reasonable candidate for FP-biotinylation. The
b8+2, b9+2, and b10+2ions support this interpretation. The
y-series (y3-y11) supports identification of this peptide. Masses
at 227.2, 312.2, and 329.4 indicate the presence of FP-biotin.
Masses at 691.4 and 708.4 amu indicate the presence of FP-
biotinylated tyrosine in this peptide. A missed cleavage form
of this peptide, RMPCAEDYLSVVLNQLCVLHEK, was also
analyzed, and the results support labeling of Tyr 452 (data not
Peptide SHCIAEVENDEMPADLPSLAADFVESK in Figure
5A is FP-biotinylated on Ser 287. Existence of an FP-
biotinylated serine is indicated by the major peak at 591.4 amu.
This is a characteristic mass from FP-biotin that appears as the
result of ?-type elimination of FP-biotin from a serine adduct
(Figure 5B), during collision-induced dissociation in the mass
spectrometer (12). The complementary peptide fragment arising
from this fragmentation contains a dehydroalanine in place of
serine. The masses of a b-series (∆b5-∆b12) containing a
dehydroalanine residue support the elimination of FP-biotin from
serine. Of the residues in the b5 fragment (SHCIA), serine at
position 287 is a candidate for FP-biotinylation. The cysteine
might have been considered a target for labeling, but the overall
mass of the fragment is consistent with carbamidomethylation
on the cysteine. A y-ion series (y4-y15) supports the identifica-
tion of the peptide. Additional support for the presence of FP-
biotin in the peptide comes from characteristic masses at 312.1
and 329.2 amu. The absence of the characteristic mass at 227
amu is common for FP-biotinylated serine.
The MS/MS spectrum for peptide AEFAEVSK labeled by
FP-biotin on Ser 232 is in Figure 6. The b- and y-ion masses
support the assigned sequence. Peaks not assigned by Protein
Pilot included six dehydroalanine fragments as well as the 591
amu ion of FP-biotin and the 227, 312, and 329 amu fragments
of FP-biotin. These additional peaks strongly support the
conclusion that Ser 232 of albumin was labeled by FP-biotin.
This labeled peptide was detected by the sensitive QSTAR elite
mass spectrometer but not by the QTRAP 2000 mass spec-
trometer. No FP-biotinylated peptides were found in the control
plasma that had not been treated with FP-biotin.
Search for Other FP-Biotin-Labeled Proteins in Human
Plasma. The present method identified 7 FP-biotin-labeled
albumin peptides but no FP-biotin-labeled peptides from any
other protein. A Western blot hybridized with Streptavidin
Alexafluor-680 showed many FP-biotinylated bands in human
plasma treated with FP-biotin under our conditions (data not
shown). One such protein is FP-biotinylated plasma butyryl-
cholinesterase (1, 14). However, the FP-biotinylated butyryl-
cholinesterase peptide was not found with the present methods.
FP-biotinylated peptides from proteins other than albumin are
difficult to detect in the presence of the overwhelmingly high
concentration of albumin. Even after depletion of albumin with
Cibacron Blue, the concentration of albumin was still too high
Figure 5. (A) MS/MS spectrum of albumin peptide labeled with FP-
biotin on Ser 287. The triply charged parent ion has a mass of 1183.8
m/z. The carbamidomethylated (CAM) peptide carried the FP-biotin
on Ser 287. The evidence for modification on serine is the presence of
a b-ion series for the dehydroalanine form of the peptide, designated
∆. The 591.4 amu ion is FP-biotin released from serine where the
fluoride ion has been replaced by a hydroxyl group. Release of the
entire OP accompanied by formation of dehydroalanine is a charac-
teristic of OP bound to serine. Internal fragmentation at proline yielded
masses at 284.0 for PAD, 369.1 for PSLA, and 397.2 for PADL. (B)
Scheme for ?-elimination of the OP label from serine. Fragmentation
in the mass spectrometer eliminates the OP from serine and simulta-
neously converts serine to dehydroalanine.
Figure 6. MS/MS spectrum of albumin peptide labeled with FP-biotin
on Ser 232. This spectrum was acquired on the QSTAR elite mass
spectrometer. The doubly charged parent ion is at 726.9 amu. The peak
at 591.3 is FP-biotin released from serine, carrying a hydroxyl group
in place of fluorine. Four y-ions (y2, y3, y4, and y5) carry FP-biotin
on serine, whereas six y-ions (∆y3-∆y8) have lost FP-biotin as well
as a molecule of water, thus converting serine to dehydroalanine.
CoValent Binding to Tyrosines in Albumin Chem. Res. Toxicol., Vol. 21, No. 9, 2008 1791
to allow detection of other FP-biotinylated peptides. Human
plasma contains 5 mg of butyrylcholinesterase and 50000 mg
albumin per L. In experiments not described in this report, we
found OP-labeled butyrylcholinesterase in human plasma only
after the butyrylcholinesterase had been purified by binding to
procainamide affinity gel, thus eliminating more than 95% of
Albumin Residues Labeled by CPO. Prolonged labeling of
pure human albumin with CPO resulted in labeling of six
tyrosines: Y138, Y150, Y161, Y401, Y411, and Y452 (Table
2). Four of these sites were also labeled by FP-biotin (Y138,
Y401, Y411, and Y452). The HPYFYAPELLFFAK peptide was
labeled on Tyr 150 by CPO, whereas it was labeled on Tyr 148
by FP-biotin. A new peptide YKAAFTECCQAADK was
labeled by CPO (Figure 7) and not by FP-biotin. Labeling on
tyrosine is supported by the b ion series. The identity of the
peptide is supported by the y2-y8 ions. Additional MS/MS
spectra for CPO-labeled peptides are in the Supporting Informa-
tion (Figures S1-S5).
Tyr 411 Reacts Most Readily with OP. The finding that
seven tyrosines and two serines make a covalent bond with OP
led to the question of which amino acid reacts most readily
Table 2. CPO-Labeled Human Albumin Peptidesa
146-159 1877.9 HPYFY*APELLFFAK
446-466 2653.2 MPCAEDY*LSVVLNQLCVLHEK
aPeptide KYLYEIAR was carbamylated on the N-terminal Lys by
degradation products in urea, adding a mass of 43. Chorpyrifos oxon
adds a mass of 136 to the labeled tyrosine.
Figure 7. MS/MS spectrum of albumin peptide labeled with CPO on
Tyr 161. The b2 and b3 ions support labeling on tyrosine.
Figure 8. MALDI-TOF spectrum of pepsin-digested albumin to show
labeling of Tyr 411 by FP-biotin. Pepsin digestion of albumin yields
two unlabeled peptides at 1717.2 (VRYTKKVPQVSTPTL) and 1830.3
(LVRYTKKVPQVSTPTL) amu, both containing Tyr 411. Both pep-
tides have a mass shift of 572.3 after reaction with FP-biotin, yielding
the peaks at 2289.6 and 2402.7 amu. The FP-biotin is covalently bound
to Tyr 411. About 50% of the Tyr 411 is labeled as calculated from
cluster areas of the labeled and unlabeled peaks.
Figure 9. Stability of the diethoxyphosphate adduct of human albumin
on Tyr 411. Albumin was treated with CPO to achieve 97% labeling
of Tyr 411. Excess CPO was removed by dialysis. The pH of the
dialyzed albumin was adjusted to 1.5, 7.4, and 8.3. CPO-albumin
samples were stored at 22 and -80 °C. After various times of storage,
samples were digested with pepsin and % labeling of Tyr 411 was
calculated from cluster areas of labeled and unlabeled peptides in the
MALDI-TOF mass spectrometer. CPO-labeled Tyr 411 was stable at
pH 1.5 and 7.4 when CPO-albumin was stored at 22 °C (top panel),
and it was stable at all pH values when CPO-albumin was stored at
-80 °C (bottom panel). Storage of CPO-albumin at pH 8.3 at 22 °C
resulted in release of half of the diethoxyphosphate group from Tyr
411 after 3.6 months.
Figure 10. Crystal structure of human albumin showing surface location
of Tyr 138, Tyr 148, Tyr 401, Tyr 411, Tyr 452, Ser 232, and Ser 287.
The residues are shown as space-filled structures. The picture was made
with PyMol software using the structure in PDB code 1bm0 (28).
Chem. Res. Toxicol., Vol. 21, No. 9, 2008Ding et al.
with OP. To answer this question, we duplicated the conditions
reported to label one molar equivalent of human albumin with
DFP (10). MALDI-TOF analysis of pepsin-digested, DFP-
treated human albumin suggested that 80% of Tyr 411 was
labeled with DFP. MS/MS analysis of a tryptic digest of
carbamidomethylated DFP-treated albumin confirmed that Tyr
411 in peptide Y*TK was labeled. In addition, less than 10%
of peptide EFNAETFTFHADICT*LS*EK was labeled (on
residues T515 and S517).
Albumin treated with FP-biotin for 2 h and digested with
pepsin had 52% of its Tyr 411 labeled in peptide VRY*TKKV-
PQVSTPTL as calculated by MALDI-TOF mass spectrometry
(Figure 8). The carbamidomethylated, trypsin-digested prepara-
tion analyzed by LC/MS/MS confirmed that Tyr 411 in peptide
Y*TK was labeled with FP-biotin. Peptide HPY*FYAPELLF-
FAK was labeled on Tyr 148 with FP-biotin but to less than
10%. A third method to identify FP-biotinylated peptides was
purification on monomeric avidin beads followed by MALDI-
TOF-TOF analysis. This method yielded only one FP-biotiny-
lated peptide, the Y*TK peptide labeled on Tyr 411.
Soman-treated albumin (150 µM soman for 2 h) analyzed
by MALDI-TOF and LC/MS/MS yielded only one labeled
peptide. The soman was on Tyr 411.
Albumin treated with CPO for 2 h before digestion with
pepsin or trypsin and analyzed by MALDI-TOF and LC/MS/
MS was labeled on Tyr 411. Approximately 30% of the Tyr
411 sites were labeled in peptides VRY*TKKVPQVSTPTL and
LVRY*TKKVPQVSTPTL. In addition, less than 5% of Thr
566 in peptide ET*CFAEEGKK and less than 5% of Thr 236
and Thr 239 in peptide LVT*DLT*KVHTECCHGDLLECADDR
were labeled. We conclude that Tyr 411 is the most OP reactive
residue in human albumin.
Support for the conclusion that Tyr 411 is the most OP
reactive residue in albumin comes from ref 2. Williams
incubated the albumin fraction of human plasma with radiola-
beled sarin, digested with trypsin, purified the radiolabeled
peptides by HPLC, and analyzed by LC/MS/MS. A single
radiolabeled peptide was isolated. Its sequence was YTK with
the isopropyl methylphosphonyl group on tyrosine.
Unstable OP-Ser and OP-Thr but Stable OP-Tyr. It was
noted that serine and threonine residues were labeled in addition
to tyrosine when samples had been incubated at pH 8.0-8.3
for 2-48 h but were not found in samples incubated at pH 8.3
for a month. In contrast, OP-labeled tyrosines were found even
after 1 month of incubation at pH 8.3. Our stability study of
CPO-labeled albumin confirmed that the Tyr 411 adduct was
stable (Figure 9). Incubation at pH 7.4 and 22 °C resulted in
almost no loss of the CPO label on Tyr 411 after 7 months. In
contrast, about half of the label was lost after 3.6 months at pH
8.3 and 22 °C. The CPO-labeled Tyr 411 was stable at pH 1.5
and 22 °C and was stable at all pH values when the labeled
albumin was stored at -80 °C. These results suggest that OP-
labeled serine and threonine adducts are unstable as compared
to OP-labeled tyrosine.
Surface Location of OP Reactive Residues. The crystal
structure in Figure 10 shows the five tyrosines and two serines
that become labeled by FP-biotin. These residues are located
on the surface of the albumin molecule where they are available
for reaction with OP.
Human albumin has 18 tyrosines and 24 serines but only five
tyrosines and two serines made a covalent bond with FP-biotin.
Their special reactivity may be explained by a nearby arginine
or lysine that stabilizes the ionized hydroxyl of tyrosine or
Many OP-Reactive Residues in Human Albumin. Although
Tyr 411 is the most OP reactive residue in human albumin, an
additional eight amino acids were labeled when the OP
concentration was high and the reaction time was prolonged.
The reaction with FP-biotin at pH 8.0 resulted in the labeling
of five tyrosines and two serines in albumin. CPO labeled six
tyrosines (two of which were different from those labeled by
FP-biotin) and no serines. We agree with Means and Wu (10)
that about 90% of the label is on Tyr 411 and 10% is on other
The pKaof the tyrosine hydroxyl group is 10.1 and of the
serine hydroxyl group is approximately 16, based on comparison
to ethanol (15). In the absence of special activation, less than
1% of the tyrosines and less than 0.000001% of the serines
would be expected to be ionized at pH 8.0. Ionized forms react
preferentially with OP, so the reactivity of tyrosine and
especially of serine with OP would be expected to be poor at
pH 8. The special reactivity of Tyr 411 suggests that the pKa
of this particular tyrosine has been lowered. Means and Wu
identified an OP reactive residue in albumin that had a pKaof
8.3 (10). It is likely that Tyr 411 corresponds to that residue.
Albumin as an OP Scavenger. Our results show that albumin
is an OP scavenger, undergoing a covalent reaction with OP.
As such, albumin contributes to detoxication of OP. A significant
amount of OP can be bound by albumin because the concentra-
tion of albumin in serum is high (≈0.6 mM), even though the
rate of reaction with OP is slow (3).
Tyrosines with an abnormally low pKaare involved directly
or indirectly in the catalytic activity of numerous enzymes
including glutathione S-transferase (16), asparaginase (17),
?-lactamases (18), and old yellow enzyme (19). Lowering the
pKaof tyrosines in albumin by modifying their environment,
either by mutagenesis or by chemical modification of vicinal
residues, would increase the reactivity of albumin with OP.
Specific nitration of tyrosine by tetranitromethane was found
to lower the pKaof tyrosine to 6.8 (20). Another nitration reagent
of tyrosine, peroxynitrite, was found to increase the catalytic
activity of a few enzymes (21). Thus, specific nitration of
tyrosine residues in albumin could also lead to a gain in
reactivity of this protein, increasing its scavenging properties.
No Aging of OP-Tyrosine Adducts. When soman or DFP
are bound to acetylcholinesterase or butyrylcholinesterase, the
OP loses an alkyl group in a process called aging (22-25). An
aged soman-labeled peptide would have an added mass of 78
rather than 162; an aged DFP-labeled peptide would have an
added mass of 122 rather than 164; an aged CPO-labeled peptide
would have an added mass of 108 rather than 136. Masses
corresponding to aged OP-labeled peptides were not found in
MS scans. We conclude that albumin OP adducts on tyrosine
do not age.
Support for this conclusion comes from the work of others
(2, 26). Human albumin covalently labeled with soman or sarin
and treated with sodium fluoride to release the OP yielded intact
soman and sarin. Soman-tyrosine adducts isolated from nerve
agent-treated guinea pigs contained the pinacolyl group of
The absence of aging is a special advantage for OP-albumin
as a biomarker because it allows for a more precise identification
of the OP. In contrast, soman and sarin exposure cannot be
distinguished when the biomarker is cholinesterase, where aging
of OP adducts occurs rapidly.
OP Labeling of Albumin in Living Animals. Guinea pigs
treated with the nerve agents soman, sarin, cyclosarin, or tabun
CoValent Binding to Tyrosines in AlbuminChem. Res. Toxicol., Vol. 21, No. 9, 2008 1793
have nerve agent-labeled albumin in their blood (2). The OPs
are bound to tyrosine. The tabun-tyrosine and soman-tyrosine
adducts were detected in blood 7 days postexposure, indicating
that the adducts are stable. The adducts had not undergone aging
and had not been released from tyrosine by treatment of the
guinea pigs with oxime. These are characteristic features of OP
adducts on albumin. Mice treated with a nontoxic dose of FP-
biotin by intraperitoneal injection had FP-biotinylated albumin
in blood and muscle (1). These examples show that OP binds
covalently to albumin under physiological conditions and that
OP-albumin adducts could therefore be useful as biomarkers
of OP exposure (27). Low OP doses make a covalent bond with
Tyr 411 of albumin.
Significance. Our results suggest that OP exposure could be
monitored by mass spectrometry of OP-albumin adducts or with
antibodies against OP-albumin adducts. The surface location
of the OP-binding sites in albumin suggests that these epitopes
may be available for reaction with antibodies. This is in distinct
contrast with acetylcholinesterase and butyrylcholinesterase
where the OP binding site is buried deep within the molecule,
making it unavailable to antibodies. Antibodies to OP-albumin
would be primarily against OP-labeled Tyr 411 because Tyr
411 is the most reactive residue at low OP concentrations. The
common OP pesticides yield either diethoxyphosphate or
dimethoxyphosphate adducts. Therefore, only two antibodies
would be needed for detection of exposure to common OP
pesticides. The studies described here support investigation into
whether albumin could be engineered to become a more efficient
Acknowledgment. Mass spectra were obtained with the
support of the Mass Spectrometry & Proteomics core facility
at the University of Nebraska Medical Center. This work was
supported by U.S. Army Medical Research and Materiel
Command W81XWH-07-2-0034 (to O.L.), W81XWH-06-1-
0102 (to S.H.H.), NIH CounterACT U01 NS058056-02 (to
O.L.), NIH Eppley Cancer Center Grant P30CA36727, NIH
Grant U01 ES016102, and NIH CounterACT U44 NS058229
(to C.M.T.), DGA/PEA 08co501 (to F.N.), and DGA Grant
03co010-05/PEA01 08 7 (to P.M.).
Supporting Information Available: MS/MS spectra for
CPO-labeled peptides (Figures S1-S5). This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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