Analysis of phosphorylated peptides by double pseudoneutral loss extraction coupled with derivatization using N-(4-bromobenzoyl)aminoethanethiol

Article abstract of DOI:10.1021/ac9017988

The analysis of post-translational phosphorylation is a crucial step in understanding the mechanisms of many physiological events. Numerous approaches for the development of analytical methods aimed at the detection and quantification of phosphorylated pr

Full text of DOI:10.1021/ac9017988

Anal. Chem. 2009, 81, 9395–9401
Analysis of Phosphorylated Peptides by Double
Pseudoneutral Loss Extraction Coupled with
Derivatization Using
N-(4-Bromobenzoyl)aminoethanethiol
Nariyasu Mano,*,†,‡ Sayaka Aoki,§ Takuma Yamazaki,§ Yoko Nagaya,§ Masaru Mori,‡
Kohei Abe,†,‡ Miki Shimada,†,‡ Hiroaki Yamaguchi,†,‡ Takaaki Goto,§ and Junichi Goto†
Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan,
Laboratory of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Tohoku University, 1-1 Seiryo-machi,
Aoba-ku, Sendai 980-8574, Japan, and Laboratory of Bioanalytical Chemistry, Graduate School of Pharmaceutical
Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan
The analysis of post-translational phosphorylation is a
crucial step in understanding the mechanisms of many
physiological events. Numerous approaches for the de-
velopment of analytical methods aimed at the detection
and quantification of phosphorylated proteins by mass
spectrometry have been reported in the literature. In this
paper, we report a new strategy for the identification of
phosphorylated serine and threonine residues in phos-
phoproteins. This method consists of selective derivati-
zation of phosphoproteins coupled with double pseudo-
neutral loss extraction after nanoLC/ESI-MS/MS analysis.
First, we designed and synthesized a new derivatization
reagent, N-(4-bromobenzoyl)aminoethanethiol, which can
selectively react with r,ꢀ-unsaturated ketones produced
by ꢀ-elimination of a phosphoryl group from phosphory-
lated serine or threonine residues. The mass spectrum
of the derivatized peptide shows a product ion with a
characteristic isotopic pattern. After derivatization, frag-
ment ions of peptides with phosphoserine or phospho-
threonine have twin peaks with an intensity ratio of
approximately 1:1 and a difference of two mass units,
while product ions from peptides without phosphoserine
or phosphothreonine have normal isotopic patterns. There-
fore, the neutral loss of the derivatized residue in the
product ion mass spectrum includes two difference losses
caused by 79Br and 81Br. The extraction of the product
scan mass spectrum with double pseudoneutral losses
is very effective for identification of the derivatized
peptides produced from phosphorylated peptides. The
new strategy represents a useful tool for the analysis
of phosphorylated serine and threonine residues in
phosphorylated proteins.
tion, and apoptosis.1 Reversible phosphorylation and dephospho-
rylation are strictly controlled by the activity modulation of kinase
and phosphatase.2 To understand the molecular mechanisms of
the physiological events regulated by post-translational phospho-
rylation, a reliable method for the analysis of phosphorylation of
proteins is required. The detection of phosphorylated peptides in
a complex peptide mixture is generally difficult because of the
low concentration of phosphorylated peptides in the mixture. The
selective concentration of phosphorylated peptides is one useful
approach to overcoming this problem. An antiphosphorylated
tyrosine antibody has been used for affinity extraction of phos-
phorylated tyrosine-containing peptide fragments in complicated
peptides,3,4 but the generation of high-quality antibodies with
sufficient affinity and selectivity for phosphorylated serine- and
threonine-containing peptides has been difficult. In immobilized
metal affinity chromatography, bound metal ions on a chelating
support capture phosphoryl groups of phosphopeptides by
complexation,5-7 but the process is not completely selective, and
the undesirable complexation of acidic amino acid-containing
peptides frequently occurs as well.8 Ishihama et al. have reported
an effective method for phosphopeptide enrichment using metal
oxide chromatography combined with aliphatic hydroxy acids, in
which the added hydroxy acids enhance selectivity by preventing
the incorporation of acidic amino acid-containing peptides.9 On
the other hand, Oda et al. developed a process involving the
Michael addition of biotin to R,ꢀ-unsaturated ketones derived from
the ꢀ-elimination of phosphoryl groups in phosphorylated resi-
(1) Pawson, T.; Scott, J. D. Science 1997, 278, 2075–2080
(2) Hunter, T. Cell 2000, 100, 113–127
(3) Pandey, A.; Podtelejnikov, A. V.; Blagoev, B.; Bustelo, X. R.; Mann, M.;
Lodish, H. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 179–184
.
.
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(4) Salomon, A. R.; Ficarro, S. B.; Brill, L. M.; Brinker, A.; Phung, Q. T.; Ericson,
C.; Sauer, K.; Brock, A.; Horn, D. M.; Schultz, P. G.; Peters, E. C. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 443–448
(5) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883–2892
(6) Shi, X.; Belton, R. J., Jr.; Burkin, H. R.; Vieira, A. P.; Miller, D. J. Anal.
Biochem. 2004, 329, 289–292
(7) Ueda, E. K. M.; Gout, P. W.; Morganti, L. J. Chromatogr. A 2003, 988,
1–23
(8) Stensballe, A.; Andersen, S.; Jensen, O. N. Proteomics 2001, 1, 207–222
(9) Sugiyama, N.; Masuda, T.; Shinoda, K.; Nakamura, A.; Tomita, M.; Ishihama,
Y. Mol. Cell. Proteomics 2007, 6, 1103–1109
.
Post-translational phosphorylation of proteins regulates many
physiological events, such as cell growth, proliferation, differentia-
.
.
* Corresponding author. Tel: +81-22-717-7525. Fax: +81-22-717-7545. E-mail:
n-mano@mail.tains.tohoku.ac.jp.
.
† Tohoku University Hospital.
.
‡ Laboratory of Clinical Pharmacy, Tohoku University.
§ Laboratory of Bioanalytical Chemistry, Tohoku University.
.
10.1021/ac9017988 CCC: $40.75  2009 American Chemical Society
Published on Web 10/21/2009
Analytical Chemistry, Vol. 81, No. 22, November 15, 2009 9395

dues.10 The biotin-modified peptides were then selectively isolated
using the avidin-biotin binding property.
Mass spectrometry (MS) is a powerful tool useful for analyzing
phosphorylated peptides. Because phosphorylated peptides pos-
sess strong negative charges and therefore have a low ionization
efficiency in the positive ion detection mode on the matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry (MAL-
DI-TOF/MS),11 postsource decay has been used for the identifica-
tion of phosphorylated amino acid residues.12,13 In electrospray
ionization (ESI)-MS, common positive ion detection analysis can
detect phosphorylated peptide ions as well as other nonphospho-
rylated peptide ions. However, the elimination of H3PO4 from
phosphorylated peptides is more frequent than backbone
cleavages on the low-energy collision-induced dissociation
(CID), making the analysis of phosphorylated peptides difficult
with ESI-MS.14 Precursor ion scan analysis can effectively
identify the precursor ion of a phosphorylated peptide by
Figure 1. Synthesis of N-(4-bromobenzoyl)aminoethanethiol (A) and
derivatization of phosphoserine and phosphothreonine residues (B).
-
monitoring the PO3 fragment at m/z 79 in the negative ion
bromobenzoyl)aminoethanethiol, which includes one bromine
atom and a thiol group, and its structure and synthesis were
shown in Figure 1A. After Michael addition of the thiol group to
R,ꢀ-unsaturated ketones (6) produced by the ꢀ-elimination of a
phosphoryl group from phosphoserine and phosphothreonine (5)
(Figure 1B), the bromine-containing thioether products (7) are
easily detected by MS. This approach represents a useful analytical
method for the detection of phosphorylated proteins.
detection mode, and the process is facilitated by the high
sensitivity to PO3 caused by lower background levels under
-
low-pH conditions.15 In the positive ion detection mode, the
method for the detection of the phosphotyrosine immonium
ion at m/z 216 has been used to easily identify phosphotyrosine-
containing peptides,16 and a neutral loss scan of H3PO4 has
also been used to detect a phosphorylated peptide with CID.17
Because MS is effective in the separation of isotopes from each
other, a characteristic isotopic pattern facilitates the selective
identification of target compounds. For example, when proteins
are enzymatically digested in a 1:1 mixture of H218O and H216O,
the 18O atom is introduced into the C-terminus with a 50%
probability.18 The C-terminus peptides are easily distinguish-
able from other peptides because only the C-terminus from
the precursor protein have natural isotopic patterns.19 Bromine
has two natural nuclear species with mass numbers of 79 and
81. Therefore, a compound containing one bromine atom has
a characteristic isotopic pattern on MS of two peaks with almost
the same intensity and a difference of two mass units. Taking
advantage of bromine’s signature MS pattern, Miyagi et al.
developed a straightforward amino acid sequence analysis
method in which the amino termini of peptides were derivatized
using N-bromonicotinic acid N-hydroxysuccinimidyl ester.20 We
proposed the application of the signature MS pattern of
bromine for the identification of phosphorylated peptides. In
this paper, we developed a new derivatization reagent, N-(4-
EXPERIMENTAL SECTION
Reagents and Apparatus. R-Cyano-4-hydroxycinnamic acid
(CHCA), the matrix for MALDI-TOF/MS, was supplied by Aldrich
Chemical Co. (Milwaukee, WI). Sequence-grade modified trypsin
was obtained from Promega (Madison, WI). Dithiothreitol, io-
doacetamide, cystamine hydrochloride, p-bromobenzoic acid, and
p-nitrophenol were purchased from Nacalai Tesque Inc. (Kyoto,
Japan). ꢀ-Casein, bovine serum albumin, immunoglobulin G,
R-chymotrypsinogen A, catalase, carbonic anhydrase, transferrin,
plasmin, myoglobin, and lysozyme were purchased from Sigma-
Aldrich Inc. (St. Louis, MO). A model phosphorylated peptide
(FAGSSYApSFK) was synthesized by Fmoc solid-phase chemical
synthesis using 4-hydroxymethylphenoxymethyl-co-polystyrene-
1% divinylbenzene resin. ZipTip C18 cartridges were purchased
from Millipore (Milford, MA). Ultrapure water was prepared
using a PURELAB ultra-apparatus (Organo Co., Ltd., Tokyo,
Japan). All other reagents were analytical grade and solvents
were HPLC or LC/MS grade.
MALDI-TOF/MS was performed using a Voyager DE-STR
spectrometer (Applied Biosystems, Framingham, MA) equipped
with an N2 laser (337 nm). In positive ion mode, a matrix
solution of fresh saturated CHCA in water/acetonitrile/trifluo-
roacetic acid (TFA) (50:50:0.1, v/v/v) was used. Peptide
samples dissolved in 0.5 µL of water/acetonitrile/TFA (25:75:
0.1, v/v/v) were mixed with 0.5 µL of the matrix solution on
the MALDI plate. Spectra were calibrated internally using the
monoisotopic protonated molecules of two peptide standards,
angiotensin I and an adrenocorticotropic hormone 7-38
(American Peptide Co., Inc., Sunnyvale, CA), in the matrix
solution.
(10) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379–382
(11) Janek, K.; Wenschuh, H.; Bienert, M.; Krause, E. Rapid Commun. Mass
Spectrom. 2001, 15, 1593–1599
(12) Metzger, S.; Hoffmann, R. J. Mass Spectrom. 2000, 35, 1165–1177
(13) Annan, R. S.; Carr, S. A. Anal. Chem. 1996, 68, 3413–3421
(14) Edelson-Averbukh, M.; Pipkorn, R.; Lehmann, W. D. Anal. Chem. 2007,
79, 3476–3486
(15) Tholey, A.; Reed, J.; Lehmann, W. D. J. Mass Spectrom. 1999, 34, 117–
123
(16) Steen, H.; Ku¨ster, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal. Chem.
2001, 73, 1440–1448
(17) Schlosser, A.; Pipkorn, R.; Bossemeyer, D.; Lehmann, W. D. Anal. Chem.
2001, 73, 170–176
(18) Rose, K.; Simona, M. G.; Offord, R. E.; Prior, C. P.; Otto, B.; Thatcher,
D. R. Biochem. J. 1983, 215, 273–277
(19) Kosaka, T.; Takazawa, T.; Nakamura, T. Anal. Chem. 2000, 72, 1179–1185
(20) Miyagi, M.; Nakao, M.; Nakazawa, T.; Kato, I.; Tsunasawa, S. Rapid
Commun. Mass Spectrom. 1998, 12, 603–608
.
.
.
.
.
.
.
.
.
.
An HPLC-isolated fraction of a derivatized peptide in 100 µL
of water/methanol/formic acid (50:50:1, v/v/v) was infused at a
.
9396 Analytical Chemistry, Vol. 81, No. 22, November 15, 2009

separation was performed at a flow rate of 200 nL/min using a
linear gradient elution method of 5%-80% mobile phase B over
60 min: mobile phase A, water/methanol/formic acid (98:2:0.1,
v/v/v); mobile phase B, water/methanol/formic acid (2:98:0.1,
v/v/v). NanoFrontier nLC was controlled by NanoLC Control
Panel (ver. 1.0.0.1) software, and NanoFrontier LD was controlled
by Control Panel (ver. 1.0.0.0) software. Those two types of
software worked under the control of Auto Sequence (ver. 0.0.0.1)
software. The measurements were performed by IBA (information
based acquisition) mode. The spray potential, Ex potential, and
AP2 potential were 2000 V, 105 V, and 45 V, respectively. Both
AP1 and AP2 temperatures were 140 °C, and the flow rate of
curtain gas (nitrogen) was 0.8 L/min. Spectrum acquisition
settings were as follows: scan range, 100-2000 amu; microse-
quence, 81; accumulation time, 20.0 ms; MS/MS isolation time,
10.0 ms; MS/MS isolation width, 5 amu; CID time, 10.0 ms; CID
gain, auto. The software chose up to three target peaks for MS/
MS scan by MS scan. Data analysis was performed by using
NanoFrontier LD Data Processing (ver. 1.0.0.0), which was
specially modified to equip double pseudoneutral loss extraction
function.
Synthesis of N-(4-Bromobenzoyl)aminoethanethiol. The
synthesis route is shown in Figure 1A. To a solution of p-
bromobenzoic acid (1, 500 mg, 2.5 mmol) in dioxane (10 mL)
were added 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hy-
drochloride (500 mg, 2.6 mmol) and p-nitrophenol (300 mg, 2.2
mmol). The reaction mixture was stirred at room temperature
overnight and then evaporated in vacuo. Ethyl acetate (300 mL)
and saturated aqueous sodium hydrogen carbonate (100 mL) were
added to the resultant residue, and the organic layer was
evaporated in vacuo to afford p-bromobenzoic acid p-nitrophenyl
ester (2).
Figure 2. MALDI-TOF mass spectra of a phosphorylated peptide
fragment (amino acids 33-48) from ꢀ-casein (A), its ꢀ-eliminated
intermediate (B), and the final derivatized product (C). MS condi-
tions: instrument, Voyager DE-STR (reflector mode); matrix, CHCA;
accelerating voltage, 25 kV; grid voltage, 18 kV; guide wire voltage,
50 V.
flow rate of 200 nL/min into a QSTARXL mass spectrometer
(Applied Biosystems) equipped with a handmade nanoESI
probe attached to a metal nanosprayer (GL Sciences, Tokyo,
Japan). The product ion scan was performed utilizing an
ionspray voltage of 3000 V, a declustering potential of 60 V, a
collision energy of 50 eV, and a collision gas (nitrogen) of 3
units.
Nanoscale-liquid chromatography/electrospray ionization tan-
dem mass spectrometry (nanoLC/ESI-MS/MS) was performed
using a NanoFrontier LD (Hitachi High-Technologies Co., Tokyo)
equipped with NanoFrontier nLC (Hitachi High-Technologies
Co.). The prepared samples were injected into the system with
an injection volume of 1.0 µL, and peptides were concentrated
for 3 min (10 µL/min) on a MONOLITH TRAP C18 trapping
column (25 mm length × 50 µm I.D., Hitachi High-Technologies)
using water/methanol/acetic acid (98:2:0.1, v/v/v). The peptides
were transferred from the trapping column to the analytical
column, a PicoFrit C18 column (100 mm length × 75 µm i.d., New
Objective, Woburn, MA) by switching the flow channel. The
A solution of the obtained activated ester (2) in tetrahydrofuran
(50 mL) was added to a solution of cystamine dihydrochloride
(480 mg, 2.1 mmol) in water/tetrahydrofuran/triethylamine (60
mL, 40:20:1.2, v/v/v). After stirring at room temperature for 1 h,
the reaction mixture was concentrated in vacuo to remove the
organic solvent, and ethyl acetate (250 mL) was added. The
organic layer was washed with 5% aqueous hydrogen chloride (3
× 100 mL) to remove unreacted cystamine and then concentrated
in vacuo. The residue was dissolved in a mixture of tetrahydro-
furan (50 mL) and 1% aqueous sodium hydroxide (50 mL) for
hydrolysis of unreacted p-nitrophenyl ester and stirred at room
Figure 3. Time course of the derivatization reaction at reaction temperatures of 20 °C (A), 37 °C (B), and 50 °C (C).
Analytical Chemistry, Vol. 81, No. 22, November 15, 2009 9397

159.2 mg, 0.6 mmol). The total yield was approximately 56%, and
any impurity spots were not observed on TLC (n-hexane/ethyl
acetate, 1:1). HR-MS (FAB): m/z calculated for C9H11ONSBr [M
+ H]+ 259.9745, found 259.9729. 1H NMR (500 MHz,
CD3COCD3): δ 8.09 (br s, 1H), 7.83 (d, J ) 8.5 Hz, 2H), 7.63
(d, J ) 8.0 Hz, 2H), 3.54 (q, J ) 6.3 Hz, 2H), 2.74 (q, J ) 7.5
Hz, 2H). 7.83 (t, J ) 8.4 Hz, 1H).
Derivatization of Protein. The protein (5 nmol) was dissolved
in 30 µL of 1.65 mol/L Tris-HCl buffer (pH 8.6) containing EDTA
(0.03 mol/L) and guanidine hydrochloride (7.3 mol/L). After the
addition of dithiothreitol in the same buffer (38 µg, 1 mg/mL),
the mixture was incubated at 50 °C for 3 h under a nitrogen
atmosphere. Iodoacetamide in the same buffer (23 µL, 10 mg/
mL) was added and the reaction mixture was incubated at room
temperature for 30 min under a nitrogen atmosphere in the dark.
The reduced and alkylated protein was lyophilized after dialysis
using a mini dialysis unit (PIERCE, Rockford, IL).
To a solution of the reduced and alkylated protein (100 pmol)
in water (20 µL) were added a solution of N-(4-bromobenzoy-
l)aminoethanethiol (100 nmol) in dimethyl sulfoxide (10 µL) and
saturated aqueous barium hydroxide (20 µL), and the mixture
was incubated at 37 °C for 2 h. The reaction was terminated by
adding 10% aqueous TFA (10 µL), and the mixture was diluted
with 200 µL of 50 mmol/L Tris-HCl buffer (pH 8.0) containing 1
mmol/L EDTA. The solution was applied onto a Thiopropyl
Sepharose 6B column (33 mg, Amercham Biosciences, Uppsala,
Sweden), and then 250 µL of the same buffer was added onto the
column. The eluant was collected and sequentially dialyzed against
50 mmol/L ammonium bicarbonate/ethanol (9:1, v/v).
Figure 4. Product ion mass spectrum [M + 2H]2+ of derivatized
peptide fragment (amino acids 33-48) from ꢀ-casein. MS conditions:
instrument, QSTARXL; flow rate, 200 nL/min; solvent, water/methanol/
formic acid (50:50:1, v/v/v); ion spray voltage, 3000 V; declustering
potential, 60 V; collision energy, 50 eV; collision gas (N2), 3 units.
temperature for 30 min. After evaporation of the organic solvent,
diethyl ether (600 mL) and 1% aqueous sodium hydroxide (200
mL) were added to the residue to extract the disulfide dimer (3),
and this step was repeated four times. The residue obtained from
evaporation of the combined organic layers was recrystallyzed
from ethyl acetate to afford the disulfide dimer as white crystals
(3, 727 mg).
A solution of tris(2-carboxyethyl)phosphine (345 mg, 1.2 mmol)
in water (3 mL) was added to a solution of the disulfide dimer
(3, 150 mg, 0.3 mmol) in tetrahydrofuran (12 mL). The reaction
mixture was stirred at room temperature overnight and then
concentrated to remove tetrahydrofuran. The aqueous layer was
extracted with ethyl acetate and the extract was concentrated to
afford N-(4-bromobenzoyl)aminoethanethiol as white crystals (4,
A solution of trypsin in 50 mmol/L aqueous ammonium
bicarbonate (2 pmol/10 µL) was added to the protein solution
and the mixture was incubated at 37 °C for 24 h. The reaction
Figure 5. Strategy for double pseudoneutral loss extraction.
9398 Analytical Chemistry, Vol. 81, No. 22, November 15, 2009

Figure 6. Analytical results for a mixture of a derivatized synthetic
phosphorylated peptide (FAGSSYApSFK) and excess trypsin-
digested BSA. (A) Total ion chromatogram for MS. (B) Total ion
chromatogram for MS/MS. (C) Double neutral loss extraction chro-
matogram for 327.98 and 329.98 mass units. (D) Product ion mass
spectrum of [M + 2H]2+ of derivatized synthetic phosphorylated
peptide.
was terminated by adding 10% aqueous TFA (100 µL), and then
the sample was dissolved in water/methanol/formic acid (98:2:
0.1, v/v/v) and injected into the nanoLC/ESI-MS/MS.
RESULTS AND DISCUSSION
Figure 7. Analytical results for the tryptic digest of a mixture of 10
different derivatized proteins including ꢀ-casein. (A) Total ion chro-
matogram for MS. (B) Total ion chromatogram for MS/MS. (C) Double
neutral loss extraction chromatogram for 327.98 and 329.98 mass
units. Product ion mass spectrum of [M + 2H]2+ of an extracted peak
at the retention time of (D) 57.5 min and (E) 59.7 min.
The derivatization agent N-(4-bromobenzoyl)aminoethanethiol
was prepared according to the synthetic route shown in Figure
1A. Since the free mercapto group is highly reactive, we chose to
generate the desired thiol by reductive cleavage of the disulfide
bond in the relatively stable homodimer. The homodimer (3) was
produced by slow addition of cystamine hydrochloride to an
excess of p-bromobenzoic acid activated ester (2) in the presence
of triethylamine. The structure of the homodimer was confirmed
by the fast atom bombardment mass spectrum, which showed a
characteristic isotopic pattern of three peaks with an intensity ratio
of approximately 1:2:1 and a peak-to-peak separation of two mass
units (data not shown). After reductive cleavage of the disulfide
bond in the homodimer (3), the new derivatization reagent (4)
was obtained. The fast atom bombardment mass spectrum of the
product included the characteristic signals for bromine, with the
Analytical Chemistry, Vol. 81, No. 22, November 15, 2009 9399

79Br-containing monoisotopic peak observed at m/z 260 and
(less than 1%), but it was detected at a level of approximately 10%
in the 20 °C experiment. The amount of final product (7) increased
proportionately with the decrease of the starting material at all
tested temperatures, but full conversion to the final product (7)
did not occur at 20 °C after 180 min. In contrast, nearly complete
conversion of the phosphopeptide to the final product was
observed after 60 min at 37 °C or after 20 min at 50 °C.
the 81Br-containing monoisotopic peak of similar intensity at
1
m/z 262 (data not shown). H NMR data also confirmed the
structure of this derivatization reagent.
Derivatization of peptides with N-(4-bromobenzoyl)aminoet-
hanethiol proceeds in two steps, as shown in Figure 1B. A
phosphorylated peptide derived from ꢀ-casein (amino acids 33-48,
FQpSEEQQQTEDELQDK) was selected as a model peptide. The
fragment was isolated by HPLC fractionation after tryptic digestion
of ꢀ-casein. As a test reaction, a mixture of the model peptide (1
nmol) in water (20 µL) and saturated aqueous barium hydroxide
(20 µL) was incubated at 50 °C for 2 h with or without the addition
of a solution of N-(4-bromobenzoyl)aminoethanethiol (9.75 µmol)
in ethanol (7.5 µL). The reaction mixture was injected into an
HPLC system, and the product peaks were collected and analyzed
by MS.
MALDI-TOF mass spectra of the starting material (5), the
ꢀ-eliminated product (6), and the final product (7) are provided
in Figure 2. The ꢀ-eliminated product peak was clearly observed
at m/z 1963.88, and the 98 mass unit decrease from the starting
material (5) corresponded to a loss of H3PO4 (Figure 2A,B). The
mass difference between the ꢀ-eliminated intermediate (6) and
the final product (7) was consistent with the expected increase
resulting from the Michael addition of N-(4-bromobenzoyl)ami-
noethanethiol to the ꢀ-eliminated intermediate (6) (Figure 2B,C).
The results from the test reactions with the model peptide
provided evidence that the derivatization of a phosphorylated
peptide using N-(4-bromobenzoyl)aminoethanethiol produced the
desired compound.
The derivatization procedure can be run in one pot because
the second step of the sequence proceeds very quickly. The
phosphorylated peptide (amino acids 33-48 of ꢀ-casein) used as
a model compound in the proof of concept study was also used
to optimize reaction conditions for both steps of the derivatization
procedure. The first step of the sequence, ꢀ-elimination of
phosphoryl groups, typically occurs under strongly basic condi-
tions. However, the use of a highly concentrated sodium hydroxide
solution has been shown to result in ꢀ-elimination of not only the
phosphoryl group but also of O-linked sugar chains.21 Byford et
al. reported that the use of barium hydroxide solution results in
rapid and specific ꢀ-elimination of phosphoryl groups, without the
nonspecific eliminations of other groups observed with sodium
hydroxide solution.22 Therefore, saturated barium hydroxide
solution was selected as the base for ꢀ-elimination of phosphoryl
group in the first step of the derivatization sequence. The barium
hydroxide solution was generally added into the peptide solution
to give a final concentration of Ba(OH)2 of approximately 0.15
mol/L. With the optimal base for efficient ꢀ-elimination
selected, we turned to optimization of the reaction temperature.
The effect of reaction temperature on the derivatization
procedure was investigated, and the results are shown in Figure
3. The reaction course was evaluated at 20, 37, and 50 °C. The
starting material gradually decreased over time under every
temperature condition, but higher temperatures resulted in a more
rapid reaction progress. The reaction intermediate, the ꢀ-elimi-
nated peptide (6), was barely detected in the 50 °C experiment
The product ion mass spectrum of the final product ([M +
2H]2+) derived from derivatization of a phosphorylated peptide
(amino acids 33-48 of ꢀ-casein) is shown in Figure 4. The y2
,
y3, y4, y5, y6, y7, and y8 ions and b2, b3, and b4 ions confirmed
the structure of the derivatized peptide. The b3 and b4 ions,
which include the labeled serine residue, had a characteristic
isotopic pattern with an intensity ratio of approximately 1:1 and
a two mass unit difference between peaks, as shown in the
expansion of the b3 peaks in Figure 4. However, the b2 ion,
which does not include the labeled serine residue, showed a
natural isotopic pattern. The characteristic pattern, indicating
the presence of bromine from the derivatization agent, is very
useful for identifying phosphoserine and phosphothreonine
residues in peptides.
In addition, the characteristic isotopic pattern can be used for
the detection of derivatized peptides in a complex peptide mixture.
The MS/MS chromatogram of an enzymatically digested protein
mixture is usually complicated because of the presence of a wide
variety of peptides. The common single pseudoneutral loss
extraction on a hybrid tandem mass spectrometer with standard
resolution mode was not enough for the selection of target
peptides23 because of the existence of many nonspecific peaks.
Fortunately, in the case of the present derivatization approach,
the product ions possessing derivatized serine or threonine
residues show a characteristic isotopic pattern, and the residue-
free product ions have a normal isotopic pattern as described
above. Therefore, in the selection of the derivatized peptide,
double pseudoneutral loss extractions can be performed. For the
phosphorylated serine residue, the pseudoneutral loss extractions
are 79Br-Ser at 327.98 and 81Br-Ser at 329.98, and for the
phosphorylated threonine residue, the pseudoneutral loss
extractions are 79Br-Thr at 342.00 and 81Br-Thr at 344.00. The
extraction of the product scan mass spectrum by targeting
double pseudoneutral losses can significantly increase selectiv-
ity (Figure 5).
To validate the utility of this strategy for the specific extraction
of a derivatized peptide, a derivative produced from a synthetic
peptide (FAGSSYApSFK) was mixed with a 10-fold molar excess
of bovine serum albumin tryptic digest, and the mixture was
analyzed by nanoLC/ESI-MS/MS. The MS/MS chromatogram
(Figure 6B) included many intense peaks corresponding to peaks
on the MS chromatogram (Figure 6A), and the derivatized
synthetic peptide was difficult to detect. On the other hand, the
extracted chromatogram (Figure 6C), which was generated by
double pseudoneutral loss extraction, showed only two peaks at
retention times of 44.3 and 44.5 min. Both peaks had very similar
product ion mass spectra because nucleophilic attack to R,ꢀ-
unsaturated ketone creates two diastereoisomers.24 The peak
(23) Bateman, R. H.; Carruthers, R.; Hoyes, J. B.; Jones, C.; Langridge, J. I.;
Millar, A.; Vissers, J. P. C. J. Am. Soc. Mass Spectrom. 2002, 13, 792–803
(24) Tinette, S.; Feyereisen, R.; Robichon, A. J. Cell. Biochem. 2007, 100, 875–
882
.
(21) Mega, T.; Nakamura, N.; Ikenaka, T. J. Biochem. 1990, 107, 68–72
(22) Byford, M. F. Biochem. J. 1991, 280, 261–265
.
.
.
9400 Analytical Chemistry, Vol. 81, No. 22, November 15, 2009

intensity was less than two hundredth of the most intense peak
on the MS/MS chromatogram, and the result suggested that the
double pseudoneutral loss extraction strategy effectively selected
minor peaks of interest from a complicated chromatogram. The
product ion mass spectrum of the doubly charged protonated
molecule is shown in Figure 6D. The y3 ion consists of two peaks
with nearly equal intensity and a two mass unit difference, and
the mass difference between the y2 and y3 ions is consistent
with a derivatized serine residue. Although cysteine is some-
times modified to dehydroalanine by heat and alkali treat-
ments,25 any conversion of cysteine residues in BSA molecule
did not appear under the present reaction conditions.
of the product ion mass spectrum agrees completely with amino
acids 101-109, AISRLGLAK, suggesting that Ser103 is phosho-
rylated, although the reason for commingling of this protein
with the protein mixture is unclear. On the other hand, the
double pseudoneutral loss extraction for derivatized threonine
residues gave us a simpler chromatogram, which included only
one peak of the retention time at 57.5 min (data not shown).
However, the retention time of the peak was the same as the
derivative of amino acids 22-48 from ꢀ-casein, and the mass
difference between b3 (m/z 604 and 606) and y2 (m/z 262)
agreed accidentally with the theoretical double pseudoneutral
loss (342.00 and 344.00) of a derivatized threonine residue. The
results from this study suggest that the present strategy may
have applicability for the analysis of phosphorylated serine and
threonine in complex mixtures of phosphorylated proteins.
To further demonstrate the practicality of our approach, we
applied the derivatization method to the analysis of a phospho-
rylated protein in a complex protein mixture. Equimolar amounts
of ꢀ-casein, bovine serum albumin, immunoglobulin G, R-chymot-
rypsinogen, catalase, carbonic anhydrase, transferrin, plasmin,
myoglobin, and lysozyme were mixed and then reduced and
alkylated sequentially using dithiothreitol and iodoacetamide in
the presence of a high concentration of guanidine hydrochloride.
After removal of all reagents by dialysis, the mixture was incubated
with N-(4-bromobenzoyl)aminoethanethiol in the presence of ca.
0.15 mol/L barium hydroxide. To remove large excess amounts
of the derivatization reagent, the reaction mixture was applied onto
an activated thiol gel column, and the nonretained fraction was
collected, digested with trypsin, and analyzed by nanoLC/ESI-
MS/MS. Detection of the derivatized peptide was very difficult
because of the presence of many intense peaks on both the MS
(Figure 7A) and MS/MS (Figure 7B) chromatograms. The auto
MS/MS analysis contained 962 product ion mass spectra. As
shown in Figure 7C, application of the double pseudoneutral loss
method for derivatized serine residues extracted only a few weak
peaks with less than a 40th of the intensity of the most intense
peaks in the MS/MS chromatogram. Among them, two peaks at
retention times of 57.5 and 59.7 min were detected as relatively
intense peaks. The peak at 57.5 min had almost the same product
ion mass spectrum as that of the derivatized form of amino acids
33-48 from ꢀ-casein, including the expected characteristic isotopic
pattern for bromine (Figure 7D). In addition, the later peak (59.7
min) also contained the characteristic isotopic pattern of two peaks
with about 1:1 intensity ratio and two mass unit difference, and
the product ion mass spectrum suggested that the peptide
possesses pSRL(I)GL(I) as a partial sequence (Figure 7E). This
partial sequence is involved in hypothetical protein AN4196.2 in
Aspergillus nidulans FGSC A4 by NCBI database, and the result
CONCLUSIONS
In this paper, we described the development of a new
derivatization reagent, N-(4-bromobenzoyl)aminoethanethiol, use-
ful for the selective analysis of phosphorylated proteins. This
reagent reacts with R,ꢀ-unsaturated ketones produced by the
ꢀ-elimination of phosphoryl groups from phosphorylated serine
and threonine residues. The product ion mass spectra of the
derivatized thioether products possess a characteristic isotopic
pattern in which the derivatized residue-containing fragment ions
have twin peaks with an intensity ratio of approximately 1:1 and
a difference of two mass units. These characteristics permit the
effective identification of phosphorylated serine and threo-
nine residues in peptides. Moreover, derivatized peptides can be
detected in a complicated peptide mixture by use of the double
pseudoneutral loss extraction method and analysis of the extracted
peaks for the presence of the characteristic isotopic pattern. The
novel analytical method will be useful for the identification of
phosphorylated serine and threonine residues in proteins.
ACKNOWLEDGMENT
This work was supported by KAKENHI (Grant-in Aid for
Scientific Research (B): 20390009). The authors are grateful for
technical support and software modification for the NanoFron-
tierLD machine by Hitachi High-Technologies Corp. The authors
would like to thank Dr. Takashi Miura for his peptide synthesis
training.
Received for review August 10, 2009. Accepted October 9,
2009.
(25) Bar-Or, R.; Rael, L. T.; Bar-Or, D. Rapid Commun. Mass Spectrom. 2008,
22, 711–716
.
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