Article

pubs.acs.org/jpr

Optimization of Enrichment Conditions on TiO2 Chromatography

Using Glycerol As an Additive Reagent for Eective
Phosphoproteomic Analysis
Isao Fukuda, Yoshino Hirabayashi-Ishioka, Ikue Sakikawa, Takeshi Ota, Mari Yokoyama,
Takaoki Uchiumi, and Atsushi Morita*
Shionogi Pharmaceutical Research Center, Shionogi & Co. Ltd., 3-1-1 Futaba-cho, Toyonaka-shi, Osaka 561-0825, Japan

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Publication Date (Web): November 21, 2013 | doi: 10.1021/pr400546u

S Supporting Information
*

ABSTRACT: Metal oxide anity chromatography (MOAC)

represented by titanium dioxide (TiO2) chromatography has
been used for phosphopeptide enrichment from cell lysate
digests prior to mass spectrometry. For in-depth phosphoproteomic analysis, it is important for MOAC to achieve high
phosphopeptide enrichment eciency by optimizing purication conditions. However, there are some dierences in
phosphopeptide selectivity and specicity enriched by various
TiO2 materials and procedures. Here, we report that binding/
wash buers containing polyhydric alcohols, such as glycerol,
markedly improve phosphopeptide selectivity from complex
peptide mixtures. In addition, the elution conditions combined
with secondary amines, such as bis-Tris propane, made it
possible to recover phosphopeptides with highly hydrophobic properties and/or longer peptide lengths. To assess the practical
applicability of our improved method, we conrmed using PC3 prostate cancer cells. By combining the hydrophilic interaction
chromatography (HILIC) with the optimized TiO2 enrichment method prior to LC-MS/MS analysis, over 8300 phosphorylation
sites and 2600 phosphoproteins were identied. Additionally, some dephosphorylations of those were identied by treatment
with dasatinib for a kinase inhibitor. These results indicate that our method is applicable to understanding the proling of kinase
inhibitors such as anticancer compounds, which will be useful for drug discovery and development.
KEYWORDS: phosphoproteomics, metal oxide anity chromatography, TiO2, phosphopeptide enrichment, mass spectrometry,
dasatinib, prostate cancer

INTRODUCTION
Protein phosphorylation is one of the most important posttranslational modications (PTM) with roles in regulating cell
growth, adhesion, proliferation, and dierentiation via a variety
of signaling pathways.13 In living cells, phosphorylation
precisely controls spatiotemporal forms of cytoskeletal
components and temporal activities of protein kinases.
Therefore, it is important to comprehensively investigate
activated or basal phosphorylation states of these proteins to
understand the biological processes and/or mechanisms of
diseases.
Over the past decade, accompanying progress in liquid
chromatography (LC) combined with mass spectrometry
(MS),4 many researchers have reported comprehensive
analyses focusing on phosphorylated proteins such as drug
kinase interactions,5,6 mechanisms of drug side eects,7 and
analyses of signaling pathways.810 In phosphoproteomic
analysis, enrichment of phosphopeptides prior to MS is the
key factor to success because it is dicult to detect
phosphopeptides that have a low degree of ionization by
their own negatively charged phosphate groups in the presence
2013 American Chemical Society

of other nonphosphopeptide contaminants. Although MS

analyses such as neutral loss scan1113 and substitution of
phosphate groups by chemical modication such as elimination coupled with the Michael addition reaction14
have also been reported, the more eective and simple solution
for the improvement of detection is considered to be
improvement of the purication steps to increase the
population of phosphopeptides as much as possible.
Many enrichment techniques have been utilized to
concentrate phosphopeptides prior to MS analysis.1518
Although immobilized metal ion anity chromatography
(IMAC) is a well-known method for concentrating phosphopeptides, metal oxide anity chromatography (MOAC) such as
titanium dioxide (TiO2) chromatography has also been utilized
due to its robustness and resistance against rigid washing
conditions, such as low pH, organic solvents, and detergents in
comparison to IMAC.19 These methods, however, have a
number of problems with regard to nonspecic binding of
Received: June 10, 2013
Published: November 18, 2013
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grade), phosphoric acid (H3PO4), dithiothreitol (DTT), and

iodoacetamide (IAA) were from Wako Pure Chemical
Industries (Osaka, Japan). RapiGest SF detergent was from
Waters (Milford, MA). DHB matrix was obtained from Bruker
Daltonics (Billerica, MA). Urea was purchased from MP
Biomedicals (Solon, OH). Ethylenediaminetetraacetic acid
(EDTA) was obtained from Kanto Chemical (Tokyo, Japan).
In addition, deionized distilled water was prepared with a MilliQ system (Millipore, Bedford, MA).

acidic peptides containing aspartic acid and glutamic acid

residues because the structures of these carboxy groups are
similar to phosphate groups and interact with TiO2 surfaces by
forming chelating structures.16,17 Therefore, reduction of the
levels of nonspecic peptide contaminants as much as possible
is a critical factor in purication for successful identication of
phosphopeptides.
In previous studies, these problems of TiO2 columns have
been overcome by using acidic washing buers, such as
triuoroacetic acid (TFA)20 or heptauorobutyric acid
(HFBA),21 elution buers such as ammonium phosphate22 or
pyrrolidine,23 and several additives such as 2,5-dihydroxybenzoic acid (DHB),2426 glutamic acid,27,28 and hydroxy
acid.23,2932 In particular, these optimized additives used
under conditions of low pH can markedly prevent nonspecic
binding of acidic peptides to the TiO2 surface, which was
proposed to play a major role in successful enrichment. These
mechanisms, however, have not been completely claried,
although the carboxy groups of additives may be involved in the
interaction with titanium atoms.
The multikinase inhibitor dasatinib has been clinically
evaluated for solid tumors, such as prostate cancer,3335
although it was rst approved for the treatment of patients
with imatinib-resistant chronic myelogenous leukemia (CML)
and Philadelphia chromosome-positive acute lymphoblastic
leukemia (Ph+ ALL).36,37 Dasatinib markedly hampered the
phosphorylation of a variety of kinase targets, including BCRABL, SRC-family kinases (SRC, LCK, FYN, LYN, etc.), c-KIT,
platelet-derived growth factor receptors (PDGFRs), and ephrin
receptors (e.g., EPHA2).34,38 In addition, many phosphoproteomic approaches against dasatinib have been carried out to
investigate its mechanism of action.6,39 However, it is not
enough to understand the phosphorylation inhibitory actions of
dasatinib in prostate cancer cells, including the kinases
regulated by dasatinib. Hence, the exploration of dasatinib
targets using PC3 prostate cancer cells is considered to be
important to expand our understanding of the actions of
dasatinib, which will conrm the practical applicability of our
methodology to phosphoproteomic analysis.
In the present study, we investigated the eects of additive
reagents interacting with commercially available TiO2 columns
to improve selectivity and attempted to optimize elution
conditions to increase recovery. To assess our optimized
methodology, we analyzed the kinase targets of dasatinib in
PC3 cells. The results indicated the capability of our method to
understand the mechanisms of intercellular biological processes
and kinasedrug interactions, providing useful information for
future drug discovery and development.

Preparation of the Phosphopeptide Standard Mixture

To conrm the selectivity of the additive eect on the TiO2

column, a phosphopeptide standard mixture containing 10
chemically synthesized phosphopeptides (MS phosphomix-2
light; Sigma-Aldrich) and tryptic digests of bovine serum
albumin (BSA; Iwai Chemicals, Tokyo, Japan) was prepared.
One milligram of BSA in 1 mL of lysis buer (8 M urea, 100
mM NH4HCO3, pH 7.6, 0.1% RapiGest SF) was reduced and
alkylated by incubation at 37 C for 1 h with 150 L of 40 mg/
mL DTT, followed by further incubation at 37 C for 1 h in the
dark with 150 L of 100 mg/mL IAA. Then, the 8-fold diluted
BSA solution was digested by adding with 10 g of trypsin (MS
grade; Promega, Madison, WI), followed by incubation at 37
C overnight. After digestion, the solution was acidied with a
one-tenth volume of 10% TFA at 25 C for 30 min prior to
centrifugation at 15 000g for 5 min. The supernatant was
desalted by Sep-Pak C18-light (Waters) using wash buer
(0.1% TFA) and elution buer (0.1% TFA, 80% ACN) and
then dried on a savant SPD2010 SpeedVac system (Thermo
Fisher Scientic, Waltham, MA). For the phosphopeptide
standard mixture, 1 mg of BSA digest was dissolved in 1 mL of
50% ACN and added to MS phosphomix 2-light (10 pmol
each). The mixture was aliquoted in one-tenth volume and
stored at 80 C until use.
Phosphopeptide Enrichment by TiO2 Chromatography

TiO2-prepacked 200 L tips (10 m particles, 3 mg of TiO2)

supplied with a Titansphere Phos-TiO kit (GL Sciences,
Tokyo, Japan) were used in this study.
To conrm the selectivity by the additive eect using a
phosphopeptide standard mixture, we prepared wash buer A
(5% TFA, 80% ACN) and wash buer B (5% TFA, 60% ACN)
containing the following additives: 25% DL-lactic acid (Titansphere Phos-TiO kit accessory), 25% pyruvic acid, 25%
isopropanol, 25% propylene glycol, 25% trimethylene glycol,
or 5% glycerol. The sample (100 g) was made up to 300 L
with wash buer B and vortexed for 30 min. The tip was preequilibrated by adding 100 L of wash buer A, followed by
100 L of wash buer B with centrifugation at 3000g for 1 min
each. Then, the sample was applied to the tip and centrifuged at
1000g for 5 min. The ow-through fraction was passed through
the system again. Subsequently, the tip was washed three times
with 100 L of wash buer B with centrifugation at 3000g for 1
min, followed by three further washes with 300 L of wash
buer A with centrifugation at 3000g for 1 min each time.
Finally, the binding phosphopeptides were eluted with 100 L
of 5% NH4OH with centrifugation at 1000g for 5 min.
Immediately, the eluate was acidied with 200 L of 10% TFA
prior to desalting with a Monospin C18 spin column (GL
Sciences) using wash buer (0.1% TFA) and elution buer
(0.1% TFA, 80% ACN). The obtained phosphopeptide sample
was dried under vacuum conditions and stored at 20 C until
MALDI-TOF MS analysis.

MATERIALS AND METHODS

Reagents

TFA (HPLC grade), acetonitrile (ACN, HPLC grade),

glycerol, propylene glycol, trimethylene glycol, isopropanol,
ammonium hydroxide (NH4OH), triethylamine (TEA), tris(hydroxymethyl)aminomethane (Tris), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bis-Tris), 1,3-bis(tris(hydroxymethyl)methylamino)propane (bis-Tris propane),
sodium hydroxide (NaOH), ammonium bicarbonate
(NH4HCO3), and dimethyl sulfoxide (DMSO) were purchased
from Nacalai Tesque (Kyoto, Japan). Pyruvic acid and
triethylammonium bicarbonate (TEAB) were obtained from
Sigma-Aldrich (St. Louis, MO). Formic acid (FA, HPLC
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cells were washed twice with 15 mL of ice-cold D-PBS, and cell

lysis was performed by adding 1.5 mL of lysis buer (8 M urea,
100 mM TEAB, pH 8.6, 5 mM EDTA, 0.1% RapiGest SF)
containing PhosSTOP phosphatase inhibitor cocktail (Roche
Diagnostics, Basel, Switzerland). After sonication, the samples
were centrifuged at 15 000g and 4 C for 10 min, and the
supernatant was obtained as the whole-cell lysate. After
determining protein concentration, all lysates were stored at
80 C until use.

To optimize the elution conditions using cell lysate digests,

wash buer A (5% TFA, 80% ACN) and wash buer B (5%
TFA, 60% ACN, 5% glycerol) were used. The procedures were
almost the same as described above except the following elution
buers were used: 5% NH4OH, 5% TEA, 1 M NH4HCO3, 1 M
NaOH, 1 M Tris, 1 M bis-Tris, or 1 M bis-Tris propane. The
obtained phosphopeptide sample was divided into one-third
volume, dried under vacuum conditions, and stored at 20 C
until LC-MS/MS analysis.
To assess the combination of additive and elution conditions
using cell lysate digests, we prepared wash buer A (5% TFA,
80% ACN) and wash buer B (5% TFA, 60% ACN) containing
25% DL-lactic acid or 5% glycerol. In addition, elution buers
were prepared as follows: 5% NH4OH or a combination with
5% NH4OH and 1 M bis-Tris propane (two-step elution). All
procedures were performed as described above except two-step
elution, which was performed as follows: the rst elution was
performed by adding 100 L of 5% NH 4 OH with
centrifugation at 1000g for 5 min. The second elution was
then performed by further adding 100 L of 1 M bis-Tris
propane with centrifugation at 1000g for 5 min. These eluates
were mixed and acidied immediately by adding 400 L of 10%
TFA, followed by desalting. The obtained phosphopeptide
sample was divided into one-third volume, dried under vacuum
conditions, and stored at 20 C until LC-MS/MS analysis.
To perform the feasibility study under the optimized
conditions using cell lysate digests, we prepared wash buer
A (5% TFA, 80% ACN), wash buer B (5% TFA, 60% ACN,
5% glycerol), and the elution buer (combination with 5%
NH4OH and 1 M bis-Tris propane for two-step elution). All
procedures were performed as described above except two-step
elution. The obtained phosphopeptide sample was divided into
one-third volume, dried under vacuum conditions, and stored
at 20 C until LC-MS/MS analysis.

Digestion of Cell Lysate

Whole-cell lysates (untreated or treated with 300 nM dasatinib)

were diluted to 1 mg/mL with dilution buer (8 M urea, 100
mM TEAB, pH 8.6, 5 mM EDTA). These lysates (1 mg each)
were reduced and alkylated by incubation at 37 C for 1 h with
150 L of 40 mg/mL DTT, followed by further incubation at
37 C for 1 h in the dark with 150 L of 100 mg/mL IAA.
Then, the lysate solution was diluted 2-fold with 100 mM
TEAB and digested by adding 20 g of Lys-C endopeptidase
(sequence grade; Wako Pure Chemical Industries), followed by
incubation at 37 C overnight. Next, the digest solution was
diluted 4-fold with 100 mM TEAB and further digested at 37
C for 6 h with 20 g of trypsin (MS grade; Promega).
Subsequently, the peptide solutions were acidied with 1 mL of
10% TFA at 25 C for 30 min, followed by centrifugation at 15
000g for 5 min. The supernatant was desalted using Sep-Pak
C18-light (Waters) prior to drying under vacuum conditions.
The obtained digests of whole-cell lysates were stored at 20
C until following fractionation.
Hydrophilic Interaction Chromatography (HILIC)

For peptide separation, an A KTAexplorer 10S system (GE

Healthcare, Uppsala, Sweden) was used. One milligram of each
peptide sample (untreated or treated with 300 nM dasatinib)
was dissolved in 1 mL of buer A (0.013% TFA, 80% ACN)
and vortexed for 30 min, followed by centrifugation at 15 000g
for 10 min. The supernatant was loaded onto a ZIC-HILIC
column (4.6 mm i.d. 250 mm, 5 m particles, 100 pore
size; Merck, Darmstadt, Germany) at a ow rate of 0.5 mL/min
with buer A and eluted with buer B (0.013% TFA, 5% ACN)
according to the following time courses and linear gradient B%:
040 min, 040%; 4043 min, 40100%; 4350 min, 100%.
The fractions were dried under vacuum conditions and stored
at 20 C until phosphopeptide enrichment by TiO 2
chromatography.

MALDI-TOF MS Analysis

For analysis of phosphopeptides enriched by TiO2 chromatography with each additive, we utilized an Ultraex II MALDITOF/TOF mass spectrometer (Bruker Daltonics). After the
TiO2-enriched samples were dissolved in 10 L of matrix
solution (10 mg/mL DHB, 0.1% TFA, 30% ACN, 1% H3PO4),
1 L was spotted onto an AnchorChip target plate (Bruker
Daltonics), followed by drying prior to MALDI-TOF MS
analysis. MS spectrum acquisition was carried out with the
following parameters: ion mode, positive-reectron; laser
power, 15% constant; accumulated sample positions, 20
positions; total laser shot number, 1000 shots; mass range,
m/z 8005000. To evaluate the selectivity of the TiO2 column
with each additive, the obtained MS spectra were cut o below
both the 5.0% maximal peak area intensity and a signal-to-noise
ratio of 5.0. All measurements were performed in triplicate.

LC-MS/MS Analysis

For optimization of elution conditions, the combination of

additives and elution conditions, and feasibility study of
exploring dasatinib-target kinases by TiO2 chromatography,
an online nanoLC-MS/MS system was used, which consisted of
an Easy-nLC 1000 system (Thermo Fisher Scientic) and a Qexactive hybrid quadrupole orbitrap mass spectrometer
(Thermo Fisher Scientic). Phosphopeptide samples were
dissolved in 5 L of 0.1% TFA containing 2.5% ACN and
loaded onto a reverse-phase C18 analytical column (50 m i.d.
150 mm, 2 m particles, 100 pore size; Thermo Fisher
Scientic) at a ow rate of 300 nL/min with buer A (0.1%
FA) and buer B (0.1% FA, 99.9% ACN) with an Easy-nLC
system with the following linear gradient B%: 060 min, 0
35%; 6062 min, 3590%; 6270 min, 90%. Eluted
phosphopeptides were sprayed directly into a Q-exactive
orbitrap MS applying a nanoelectrospray ion source at a
spray voltage of 1.7 kV. In addition, MS and MS/MS spectra

Cell Culture and Lysis

PC3 prostate cancer cells were maintained in RPMI-1640 (Life

Technologies, Carlsbad, CA) supplemented with 10% fetal
bovine serum (Life Technologies), 1% nonessential amino acid
solution (Life Technologies), and 1% penicillinstreptomycin
solution (Life Technologies) and cultured at 37 C in a
humidied atmosphere with 5% CO2. For stimulation, 8090%
conuent cells in 15 cm dishes were used in all experiments.
The cells were treated by adding 25 mL of the culture medium
without antibiotics in the presence or absence of 300 nM
dasatinib (BioVision, Milpitas, CA) in 0.05% DMSO, followed
by incubation at 37 C and 5% CO2 for 1 h. Subsequently, the
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substrate (GE Healthcare) and a LAS-3000 image analysis

system (Fuji Film, Tokyo, Japan). All procedures were similar
for antitotal SRC rabbit monoclonal antibody (Cell Signaling
Technology) and antitotal FAK rabbit monoclonal antibody
(Cell Signaling Technology) as primary antibodies except
blocking was performed with 5% skimmed milk instead of BSA,
and these antibodies were used at 1:1000 dilution.

were automatically acquired for 60 min in positive ion mode

against the range of full MS scan (m/z 3001650) at a
resolution of R = 70 000 and a subsequent data-dependent MS/
MS scan (top 10 highest peaks of +2, +3, or +4 charged ions) at
a resolution of R = 17 500. In addition, ions chosen in the datadependent MS/MS scan were excluded for 60 s due to
fragmentation of other peaks. All measurements were
performed in triplicate except for the feasibility study on
identication of the target kinases of dasatinib.

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MS Data Analysis

RESULTS AND DISCUSSION

Phosphopeptide Selectivity on TiO2 Column by Additive

Eect

To determine the optimal elution conditions and the

combination of additives and elution conditions for enrichment
using TiO2 chromatography, peptide identication of obtained
product ion spectra was conducted by Proteome Discoverer
software, version 1.3 (Thermo Fisher Scientic) applying the
MASCOT search engine, version 2.4 (Matrix Science, London,
UK) against the International Protein Index (IPI)-human
database, version 3.87 (91 464 sequences, September, 2011)
with the following allowance conditions: enzymes, Lys-C and
trypsin; missed cleavage, 2; xed modications, carbamidomethylation of Cys residues; variable modications, oxidation of
Met residues and phosphorylation of Tyr, Ser, and Thr
residues; MS tolerance, 3 ppm; MS/MS tolerance, 0.02 Da.
Phosphopeptides with condence interval (CI) > 95.0% were
extracted with a MASCOT peptide ion score cuto >25 as the
identity threshold. In addition, the other phosphopeptides with
false discovery rate (FDR) < 1.0% calculated with a decoy
database were also considered to be identied with high
reliability.
When we analyzed the dasatinib-treated PC3 cell digests, all
of the MS/MS spectra generated from each phosphopeptide
fraction were merged and identied as described above except
the application of the SEQUEST search engine (Thermo Fisher
Scientic). For the quantication, representative MS peak area
intensities of identied phosphopeptide were calculated using
Xcalibur software (Thermo Fisher Scientic).

To understand the properties of the additives, we investigated

phosphopeptide selectivity on a TiO2 column by adding several
compounds with a propane backbone and a few hydroxy/
carbonyl groups. Phosphopeptides among excess BSA-digested
peptides were specically enriched by TiO2 column with each
additive, and the eluate was evaluated by counting the number
of phosphopeptides and by measuring their peak area
intensities using MALDI-TOF MS. The representative MS
spectra were shown in Figure S1 in the Supporting Information.
The enrichment eciency on each additive was estimated by
calculating the ratio of total peak area intensities of
phosphopeptides to those of all detected peptides. MALDITOF MS analysis revealed that the phosphopeptide selectivity
was markedly improved in the addition of glycerol as well as
lactic acid; in the best case, the enrichment eciency of glycerol
additive was 80.6 4.7% (mean SD, Table 1). Propylene
Table 1. The Enrichment Eciency of Phosphopeptides
Using Phosphopeptide Standard Mixture by TiO2
Chromatography with Various Additivesa

Western Blotting Analysis

Aliquots of 20 g of dasatinib-treated (0, 100, and 300 nM at

37 C for 1 h) PC3 whole-cell lysate in SDS sample buer were
denatured at 70 C for 10 min, followed by 412% SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Separated
proteins were transferred onto PVDF membranes, which were
blocked with blocking buer (5% BSA in TBS containing 0.1%
Tween 20) at 25 C for 1 h. Subsequently, the membranes
were incubated at 4 C overnight with the following primary
antibodies: anti-pY419-SRC rabbit monoclonal antibody
(1:1000; Cell Signaling Technology, Danvers, MA), antipY772-EPHA2 rabbit monoclonal antibody (1:1000; Cell
Signaling Technology), anti-pY577-FAK rabbit polyclonal
antibody (1 g/mL; Thermo Fisher Scientic), anti-pY827ACK1 rabbit polyclonal antibody (1 g/mL; Biorbyt Ltd.,
Cambridge, UK), antitotal EPHA2 rabbit monoclonal antibody
(1:1000; Cell Signaling Technology), and antitotal ACK1
mouse monoclonal antibody (1 g/mL; Santa Cruz Biotechnology, Dallas, TX). On the following day, the membranes
were washed three times with TBST for 5 min each time and
incubated with HRP-conjugated donkey antirabbit IgG
secondary antibody (1:10 000; GE Healthcare) for rabbit
primary antibodies or HRP-conjugated sheep antimouse IgG
secondary antibody for mouse primary antibody at 25 C for 1
h. After washing three times with TBST for 5 min each time,
the protein bands were detected with ECL-plus or ECL-prime

additive

phosphopeptides

nonphosphopeptides

input (before
purication)
no additive
lactic acid
pyruvic acid
glycerol
propylene glycol
trimethylene glycol
isopropanol

NDc

38.3 1.5d

1.3
6.7
2.0
6.3
6.3
6.3
0.7

0.6
0.6
0.0
0.6
0.6
0.6
0.6

38.0
8.3
14.0
6.0
15.7
21.7
30.0

2.6
3.1
1.0
3.6
2.1
1.5
3.6

eciencyb
(%)

1.1
76.1
7.4
80.6
34.4
19.6
0.6

0.2
1.7
0.5
4.7
1.5
2.3
0.6

The analysis was performed by MALDI-TOF MS instrument.

Calculated from the ratio of the sum of peak area intensities of
phosphopeptides to that of total peptides. cNot detected. dMean SD
(n = 3).

glycol and trimethylene glycol additives also observed high

eciency on phosphopeptide enrichment, but many nonphosphopeptides were also recovered together, resulting in
poor improvement of phosphopeptide selectivity. A previous
study indicated that the addition of lactic acid improved
phosphopeptide selectivity.29 In addition, it has been reported
that lactic acid is a better reagent for MS analysis because it is
easy to desalt using a reverse phase column prior to MS unlike
adsorptive DHB. Similar to this previous report, lactic acid
showed high selectivity in the present study.
Interestingly, glycerol, which is categorized as an alcohol, a
neutral compound, also improved the phosphopeptide
selectivity, although the conventional additives are carboxylic
acid compounds possessing carboxy groups as the common
intermolecular structure. Glycerol has been reported as a
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lysates. The phosphopeptides bound to the TiO2 column were

eluted by each alkaline solution and identied by LC-MS/MS
analysis. Among these elution solutions, bis-Tris propane could
elute 1162.3 117.6 (mean SD) phosphopeptides from the
TiO2 column, which showed the largest recovery rate of all of
those examined (Table 2). The next largest recovery, 1005.0

condensing agent to synthesize TiO2 particles in the solgel

method4042 or as a cross-linking agent in the condensation of
TiO2 particles.43,44 However, this is the rst report that
polyhydric alcohols such as glycerol could bring about the gain
of function as high phosphopeptide selectivity for TiO2
particles.
The binding mode of alcohol additives is not known, but
previous infrared spectroscopy studies indicated that carboxy
groups of carboxylic acids, such as benzoic acid or salicylic acid,
interact with Ti atoms by bidentate chelation.45,46 Therefore,
the trihydroxy groups of glycerol may also make it possible to
extensively adsorb on functional TiO2 surfaces by forming
titaniumglycerol complexes coordinated via three CO bonds
of the trihydroxy groups, i.e., tridentate chelation or bridging.
This capping eect for Ti atoms by multiple hydroxy groups
may play an important role in preblocking of heterogeneous
adsorption sites, such as H2O-reacted titanium-hydroxo clusters
(TiOH) or titanium-oxo bridges (TiOTi). This was
strongly supported by the observation that the isopropanol
additive consisting of a monohydroxy group showed no
improvement of phosphopeptide selectivity and that the
enrichment eciency was stronger in the order of the number
of hydroxy groups on the propane backbone, such as glycerol
(1,2,3-triol) > propylene glycol (1,2-diol) > trimethylene glycol
(1,3-diol) > isopropanol (2-ol). In addition, it is considered that
glycerol could recognize the same adsorption sites in which
carboxy groups of acidic peptides show nonspecic binding,
interacting competitively with them, which contributed to
disruption of carboxy group-based bidentate chelation, and
reducing the binding to nonphosphopeptides.
On the other hand, lactic acid as an additive showed higher
selectivity than pyruvic acid, suggesting that the -hydroxy
group of lactic acid is more important than the -carbonyl
group of pyruvic acid for the highly selective interaction with
TiO2 surfaces. On the basis of the common 1,2-diol structure
between lactic acid and glycerol, this is the rst key structure
identied for high phosphopeptide selectivity. However, simple
1,2-diols such as propylene glycol were not sucient to
improve selectivity. These results suggest that the further
oxygen atom neighboring 1,2-diol structure, derived from the
hydroxy group (CH2OH) at the 3-carbon position of
glycerol and the carbonyl group (>CO) at the 1-carbon
position of lactic acid, are also essential as second key structures
for high selectivity. Thus, titanium additive chelation
coordinated via at least three oxygen atoms of these additives
may be required to achieve marked improvement of
phosphopeptide selectivity.
Consequently, our ndings suggest that the key structure for
higher phosphopeptide selectivity consists of the following
moieties: (i) 1,2-diol (dihydroxy groups) and (ii) one or more
additional hydroxy/carbonyl groups neighboring 1,2-diol. In
particular, alcohol additives are required to possess more than
two hydroxy groups to maintain stable adsorption between the
TiO2 surface and additives coordinated via multidentate
chelation/bridging. By preparing the binding conditions,
phosphate groups of phosphopeptides could dominantly
recognize their adsorption sites on TiO2 surfaces, which is
thought to contribute the greatest gain of highly specic
selectivity to phosphopeptides.

Table 2. Numbers of Phosphopeptides Obtained from PC3

Cell Lysate Digests by TiO2 Chromatography with Various
Alkaline Elution Buers

elution buer

pH

5% NH4OH
5% TEA
1 M Tris
1 M bis-Tris propane
1 M bis-Tris
1 M NH4HCO3
1 M NaOH

12.1
12.3
10.9
11.3
9.5
8.6
13.7

identied phosphopeptides
1005.0
661.3
549.0
1162.3
80.0
259.0
495.0

113.9a
20.2
207.9
117.6
14.1
87.7
166.3

Mean SD (n = 3).

113.9 phosphopeptides, was observed with conventional

NH4OH elution. On the other hand, NaOH elution buer
had the highest pH, but the number of phosphopeptides
obtained was not signicant. Furthermore, NH4HCO3 elution
buer, which had the lowest pH, did not show the minimum
recovery. These results indicated that there was no pH
dependency of phosphopeptide elution, suggesting that the
structure of the elution solvent molecule is a more important
factor for phosphopeptide recovery as compared to the degree
of alkaline pH.
In general, NH4OH is widely used as the rst-choice elution
buer. However, a previous study indicated that cyclic
secondary amines, such as pyrrolidine and piperidine, showed
greater phosphopeptide recovery rates than basic inorganic salts
such as NaOH and disodium hydrogen phosphate.23 Even
acyclic secondary amines such as bis-Tris propane would be
expected to show high recovery. Thus, the number of eluted
phosphopeptides tended to increase in the order amine
nucleophilicity, such as bis-Tris propane (secondary), Tris
(primary), and bis-Tris (tertiary). In particular, the phosphopeptide recovery by bis-Tris elution was extremely poor, which
was considered to be the reason for the low accessibility of its
nitrogen atom against Ti atoms due to steric hindrance by its
side chains. These ndings suggest that eective elution of
phosphopeptide caused by amine groups is subject to not only
negative electrostatic repulsion between adsorbing phosphopeptides and TiO2 surfaces by conversion to basic pH but also
nucleophilicity against Ti atoms by their moieties.
With regard to the pH dependence of the phosphate
molecule and TiO2 surfaces, a previous study indicates that
bound phosphate could begin to be desorbed from TiO2
surfaces at pH 5.06.0 and detached completely when the
solution basicity reached over pH 11.0.47 Thus, higher pH
elution could be advantageous in higher recovery. However, our
results indicated that there was little correlation between the
maximal phosphopeptide recovery and the solvent pH. Unlike
elution of phosphate molecules, other factors mentioned above
(e.g., amine nucleophilicity, etc.) are also considered to be
related to phosphopeptide elution. Therefore, we investigated
the elution capability of each buer to determine which
parameters made major contributions to the eective elution of
phosphopeptides.

Optimization of Elution Conditions

To optimize the elution conditions, we investigated the elution

properties of several alkaline solutions using digests of PC3 cell
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Figure 1. The property distributions of phosphopeptides obtained from PC3 cell lysate digest by TiO2 chromatography with each elution buer. (A)
Molecular weight, (B) GRAVY score, and (C) relative ratio of the number of acidic amino acids (Asp and Glu) to the full-length of phosphopeptide.
Elution buers are represented as follows: () bis-Tris propane, () NH4OH, () TEA, () Tris, () NaOH, () NH4HCO3, and () bis-Tris.

particular 040% acidity) compared to NH4OH elution. This

result indicates that bis-Tris propane elution is suitable for
elution of highly acidic phosphopeptides, and this acidity is
assumed to be one of the parameters conferring hydrophilic
properties to phosphopeptides. In particular, this feature of bisTris propane elution may be advantageous for analyzing acidic
phosphorylation sites because phosphorylation substrate motifs
for some kinases, such as casein kinase 2 (CK2), are known to
contain multiple acidic residues.49
These results regarding the properties of bis-Tris propane
indicate that this elution buer could be greatly applicable to
recover longer phosphopeptides, in some cases, which
contained more hydrophilic and/or more acidic residues. In
addition, the intermolecular moiety consisting of hexahydroxy
groups of bis-Tris propane is also assumed to contribute to high
recovery of these phosphopeptides as well as its nucleophilicity.
At the time of elution, these hydroxy groups may competitively
interact with phosphopeptide-detached TiO2 adsorption sites
by forming titanium-bis-Tris propane complexes coordinated
via multiple CO bonds as well as glycerol additives, resulting
from prevention of rebinding of phosphate groups of
phosphopeptides. Therefore, we combined bis-Tris propane,
which showed maximal phosphopeptide elution, as the secondchoice elution buer after NH4OH elution to improve the
recovery and identication of longer and/or hydrophilic
phosphopeptides.

Figure 1A shows the distribution of molecular weight of

phosphopeptides obtained from TiO2 chromatography with
each elution buer using PC3 cell lysate digest. Although
NH4OH has generally been used as elution solution, it tended
to elute shorter phosphopeptides (10001500 Da). On the
other hand, bis-Tris propane eluted from shorter to longer
phosphopeptides (10004000 Da). These results suggest that
bis-Tris propane elution in combination with NH4OH elution
would make it possible to increase the phosphopeptide
identication by covering longer phosphopeptides that cannot
be eluted with NH4OH.
Subsequently, to investigate the key properties of bis-Tris
propane for elution of longer phosphopeptides, we compared
the distribution of peptide hydrophobicity (Figure 1B). The
results indicated that bis-Tris propane could elute phosphopeptides with lower GRAVY score48 (2.0 to 0), suggesting that
hydrophilic phosphopeptides could be recovered more
successfully than with NH4OH. On the other hand, hydrophobic phosphopeptides represented by a higher GRAVY score
(00.5) tended to be strongly eluted by TEA in comparison to
NH4OH and bis-Tris propane.
Moreover, we conrmed the acidic amino acid contents (Asp
and Glu) of phosphopeptides because these are the potentially
ionic residues in relation to peptide hydrophilicity (Figure 1C).
Bis-Tris propane could elute not only nonacidic phosphopeptides but also a wide range of acidic phosphopeptides (in
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Eects on the Optimized Method for Comprehensive

Phosphopeptide Analysis

Interestingly, recovery of monophosphorylated peptides by

glycerol additive was increased compared to that with a lactic
acid additive regardless of the elution conditions. The ratios of
the number of monophosphopeptides recovered by glycerol
additive to that of lactic acid additive was 1.3-fold (833.3 vs
654.7 with NH4OH elution) and 1.5-fold (986.0 vs. 650.7 with
two-step elution). However, there were no marked improvements in the number of multiphosphopeptides regardless of
whichever additive was used. Thus, monophosphopeptide
selectivity is specic only for the glycerol additive, and bisTris propane elution synergistically helps increase the recovery
amount of monophosphorylated peptides. These features were
considered to be due to the dierences in molecular properties
of additive between carboxy acid and alcohol.
Although the reasons for this feature of glycerol additive are
still unknown, it may be due to the dierence in polarity
between lactic acid and glycerol. The acid dissociation constant
(pKa) values of lactic acid and glycerol are 3.9 and 14.2,
respectively. Therefore, the TiO2 surface bound with lactic acid
additive is potentially more acidic. On the other hand, glycerol
additive with a higher pKa is considered to provide a milder
environment on the TiO2 surface, which may confer advantages
such as binding elds onto TiO2 adsorption sites to
monophosphopeptides, which could not be competitively
entrapped by carboxy groups of lactic acid additive. On the
basis of these results, we applied the best match of glycerol
additive and two-step elution with NH4OH and bis-Tris
propane as an optimized TiO2 enrichment method.
Subsequently, we conrmed the eciency of phosphopeptide
identication between the conventional method (lactic acid
additive/NH4OH elution) and the optimized method (glycerol
additive/NH4OH + bis-Tris propane elution). Resulting from
this examination, 1286 phosphopeptides (83.5%) and 724
phosphoproteins (89.2%) were overlapped between two
methods, and an additional 788 phosphopeptides and 285
phosphoproteins were identied by the present optimized
method (Figure 3). Furthermore, we compared the identied
contents of phosphorylation residues between these two
methods. The results demonstrated that pSer/pThr/pTyr in
phosphopeptides enriched by the conventional and optimized
method were 1850 (88.9%):212 (10.2%):18 (0.9%) and 2374
(88.5%):281 (10.5%):27 (1.0%), respectively. The detected
frequencies on phosphorylated residues by the optimized
method showed the same values as those by the conventional
method, and these frequencies also indicated similar to the
reported values such as pSer (86.4%)/pThr (11.8%)/pTyr

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To evaluate the eects of combinations of additive and elution

conditions by TiO2 chromatography for phosphopeptide
enrichment, we compared the number of identied unique
phosphopeptides using PC3 cell lysate (Figure 2). The best

Figure 2. The distribution of the number of phosphopeptides

obtained from PC3 cell lysate digest by TiO2 chromatography with
each additive in combination with elution conditions. LC-MS/MS
measurement was performed in duplicate. Red bar, monophosphorylated peptide; blue bar, multiphosphorylated peptide; gray bar,
nonphosphorylated peptide.

optimized method indicating the highest phosphopeptide

recovery was the combination of glycerol additive and twostep elution (NH4OH and bis-Tris propane), which showed
that the distributions of mono- and multiphosphorylated
peptides were 986.0 25.9 and 320.7 41.5 (mean SD),
respectively. On the other hand, the distributions of mono- and
multiphosphorylated peptides obtained by the conventional
method (lactic acid additive and NH4OH elution) were 654.7
94.0 and 300.3 79.9, respectively. Therefore, total
phosphopeptides obtained by the optimized method were
increased by 1.4-fold compared to the conventional method. In
addition, bis-Tris propane elution combined with NH4OH
elution was eective for improving phosphopeptide recovery
when glycerol additive was used, but no improvement was
shown using lactic acid additive.

Figure 3. Comparison of the number of phosphopeptides and phosphoproteins obtained from PC3 cell lysate digest by the conventional method
(lactic acid additive/NH4OH elution) and the optimized method (glycerol additive/two-step elution with NH4OH and bis-Tris propane). (A)
Phosphopeptides and (B) phosphoproteins.
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Figure 4. Comparison of the number of phosphopeptides and phosphoproteins obtained from PC3 cell lysate digest in the presence or absence of
300 nM dasatinib. (A) Phosphopeptides and (B) phosphoproteins.

Figure 5. Ion chromatograms and Western blotting patterns of representative phosphorylation sites in PC3 cells suppressed by treatment with
dasatinib. (A) LIEDNEpY 419 TAR of SRC, (B) VLEDDPEATpY 772 TTSGGK of EPHA2, (C) YMEDSTYpY 577 K of FAK, (D)
pY827ATPQVIQAPGPR of ACK1. The red line and blue line on the ion chromatogram indicate the peak area intensity of treatment with 0 nM
dasatinib (DMSO) and that of treatment with 300 nM dasatinib, respectively. Western blotting analysis was performed using 20 g of PC3 cell lysate
per lane cultured in the presence of 0, 100, or 300 nM dasatinib for 1 h. Each band was detected by specic antibody against total or phosphorylated
protein.

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(1.8%) analyzed by strong cation exchange (SCX)-TiO250 and

pSer (89.6%)/pThr (9.9%)/pTyr (0.5%) analyzed by HILICTiO2.51 These results indicate that our optimized method is
possible to enrich the larger number of phosphopeptides
without unexpected biases.

kinase of dasatinib.60 In addition, ACK1 specically enhanced

androgen receptor phosphorylation in prostate cancer, which
induces promotion of tumor growth.61 Furthermore, inhibition
of SRC and/or ACK1 by dasatinib suppressed the progression
of prostate cancer.62 These ndings indicate that ACK1
overexpressed in prostate cancer may also participate in
tissue-specic promotion of cell growth via the SRC-related
signaling pathway. Thus, inclusive suppression of ACK1 and
SRC phosphorylation by dasatinib is considered to be a
potentially eective means of treatment for prostate cancer.
Finally, these results demonstrated that our methodology is
useful for exploring the dominant targets and signal transducers
of kinase inhibitors. In comprehensive phosphoproteomic
analysis, the combination of our optimized phosphopeptide
enrichment method using TiO2 column with MS analysis may
be applicable to rapidly investigate phosphorylation changes in
cells of interest to determine the eectiveness of various drugs.

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Exploring Target Kinases of Dasatinib Using Optimized

Enrichment Method

To conrm the practical applicability of the optimized method,

we performed comprehensive detection of phosphopeptides by
exploring phosphorylation sites of kinases in PC3 prostate
cancer cells suppressed by dasatinib. For comprehensive
phosphoproteomic analysis, HILIC has been shown to be
eective for the separation of phosphopeptides.52,53 Therefore,
we combined HILIC fractionation of the digest of PC3 wholecell lysate prior to phosphopeptide enrichment. LC-MS/MS
analysis identied 6841 and 5844 phosphopeptides in the
absence (DMSO control) or presence of 300 nM dasatinib,
respectively (Figure 4A). As shown at the protein level, 2387
and 2128 phosphoproteins were identied under these
conditions, respectively (Figure 4B). These identied phosphopeptides were listed as Table S1 in the Supporting
Information.
Next, in order to validate the feasibility of our TiO2
enrichment methods for analyzing the large phosphoproteomic
data, we conrmed the correlativity between ion chromatograms and Western blotting patterns about well-known
dasatinib-related phosphorylation sites such as SRC and
EPHA2 (Figure 5). In this study, the ion chromatograms of
these phosphopeptides containing their active sites, such as
LIEDNEpY419TAR of SRC (Figure 5A) and VLEDDPEATpY772TTSGGK of EPHA2 (Figure 5B), were decreased by 27.5
and 10.4 fold with 300 nM dasatinib treatment, respectively.
Their Western blotting patterns also revealed dephosphorylation of these phosphorylation sites with 300 nM dasatinib. In
particular, the strong suppression of the Y419-SRC phosphorylation site was observed. In some cases, it is indicated that
dasatinib may simultaneously act through the multiple signaling
pathways regulated by SRC-family kinases in cancer cells, which
will make it possible to suppress the cell growth strongly. With
regard to biological eects of dasatinib on PC3 cells, previous
studies indicated that Y419-SRC dephosphorylation caused by
dasatinib was strongly involved in inhibition of cell growth,54
antimetastasis eect via the induction of E-cadherin overexpression,55 and interruption of tumor-cell induced angiogenesis in vivo.56 Therefore, our results are also considered to
suggest that SRC dephosphorylation is an important event in
the action of dasatinib for the reduction of PC3 cells.
Next, we investigated the changes in SRC-related downstream phosphorylation sites, such as focal adhesion kinase
(FAK) and activated cell division cycle (CDC) 42-associated
kinase 1 (ACK1). The ion chromatograms of YMEDSTYpY577K of FAK (Figure 5C) and pY827ATPQVIQAPGPR of
ACK1 (Figure 5D) were suppressed 15.4 and 8.2 fold,
respectively, and Western blotting patterns also correlated
with their peak area intensities from those of the ion
chromatograms. Both FAK and ACK1 have been known as
direct substrates of SRC via the receptor tyrosine kinases
(RTK)/integrin signaling pathway,57,58 indicating that their
activations are regulated under SRC. Regarding ACK1, this
molecule is thought to be overexpressed in prostate cancers and
related to the integrin signaling pathway.59 Recently, computational analysis has indicated that ACK1 is a potential target

CONCLUSIONS
We investigated the conditions of TiO2 chromatography for the
phosphopeptide enrichment and established a method for
comprehensive phosphoproteomic analysis. In this study,
glycerol, one of the most eective additives, showed marked
improvement of phosphopeptide selectivity. In particular,
glycerol as an additive played a characteristic role in the
enrichment of singly phosphorylated peptides compared to the
conventional conditions using lactic acid as an additive, but
there were no dierences in the numbers of multiply
phosphorylated peptides. In addition, the elution conditions,
such as a combination of NH4OH and bis-Tris propane, made
it possible to recover phosphopeptides of a wide range of
peptide lengths including more hydrophilic and/or more acidic
residues. These optimized additive and elution conditions are
expected to achieve the maximal phosphopeptide recovery
compared to conventional methods.
For the feasibility study of our optimized method, we tried to
search for target kinases of dasatinib in PC3 prostate cancer
cells. As a result, a total 8296 phosphopeptides were identied,
and Western blotting analysis revealed that the phosphorylation
changes of phosphopeptides, such as SRC and EPHA2 tyrosine
kinases, were correlated to their MS peak area intensities. These
results indicate that our optimized method could be applicable
for a large phosphoproteomic analysis. In summary, the
preparation of highly pure phosphopeptides obtained by our
TiO2 enrichment technique could facilitate subsequent MS
analysis. Our method is a powerful tool for phosphoproteomics
and will be useful for observations of a variety of complicated
cellular activations via intersecting signaling pathways in the
future.

ASSOCIATED CONTENT

S Supporting Information
*

Figure S1 (PDF): Representative MALDI-TOF MS spectra of

phosphopeptides obtained from phosphopeptide standard
mixture by the TiO2 chromatography with each additive.
Table S1 (XLSX): A list of identied phosphopeptides
obtained from digests from PC3 cells in the absence
(DMSO) or presence of 300 nM dasatinib treatments by the
optimized TiO2 enrichment method, including their quantication data corresponding to peak area intensities. These
materials are available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-6-6331-5967. Fax: +81-6-6332-6385. E-mail:

atsushi.morita@shionogi.co.jp.
Notes

The authors declare no competing nancial interest.

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dx.doi.org/10.1021/pr400546u | J. Proteome Res. 2013, 12, 55875597