Optimization of Enrichment Conditions on TiO2 Chromatography

Nov 18, 2013 - Justine V. Arrington , Chuan-Chih Hsu , Sarah G. Elder , W. Andy Tao ... Muhammad Naeem Ashiq , Matthias Rainer , Christian W. Huck ...
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Optimization of Enrichment Conditions on TiO2 Chromatography Using Glycerol As an Additive Reagent for Effective 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 S Supporting Information *

ABSTRACT: Metal oxide affinity 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 efficiency by optimizing purification conditions. However, there are some differences in phosphopeptide selectivity and specificity enriched by various TiO2 materials and procedures. Here, we report that binding/ wash buffers 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 confirmed 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 identified. Additionally, some dephosphorylations of those were identified by treatment with dasatinib for a kinase inhibitor. These results indicate that our method is applicable to understanding the profiling of kinase inhibitors such as anticancer compounds, which will be useful for drug discovery and development. KEYWORDS: phosphoproteomics, metal oxide affinity chromatography, TiO2, phosphopeptide enrichment, mass spectrometry, dasatinib, prostate cancer

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INTRODUCTION Protein phosphorylation is one of the most important posttranslational modifications (PTM) with roles in regulating cell growth, adhesion, proliferation, and differentiation via a variety of signaling pathways.1−3 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 effects,7 and analyses of signaling pathways.8−10 In phosphoproteomic analysis, enrichment of phosphopeptides prior to MS is the key factor to success because it is difficult 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 scan11−13 and substitution of phosphate groups by chemical modification such as βelimination coupled with the Michael addition reaction14 have also been reported, the more effective and simple solution for the improvement of detection is considered to be improvement of the purification steps to increase the population of phosphopeptides as much as possible. Many enrichment techniques have been utilized to concentrate phosphopeptides prior to MS analysis.15−18 Although immobilized metal ion affinity chromatography (IMAC) is a well-known method for concentrating phosphopeptides, metal oxide affinity 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 nonspecific binding of Received: June 10, 2013 Published: November 18, 2013 5587

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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 nonspecific peptide contaminants as much as possible is a critical factor in purification for successful identification of phosphopeptides. In previous studies, these problems of TiO2 columns have been overcome by using acidic washing buffers, such as trifluoroacetic acid (TFA)20 or heptafluorobutyric acid (HFBA),21 elution buffers such as ammonium phosphate22 or pyrrolidine,23 and several additives such as 2,5-dihydroxybenzoic acid (DHB),24−26 glutamic acid,27,28 and hydroxy acid.23,29−32 In particular, these optimized additives used under conditions of low pH can markedly prevent nonspecific 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 clarified, 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,33−35 although it was first 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 confirm the practical applicability of our methodology to phosphoproteomic analysis. In the present study, we investigated the effects 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 kinase−drug interactions, providing useful information for future drug discovery and development.

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Preparation of the Phosphopeptide Standard Mixture

To confirm the selectivity of the additive effect 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 buffer (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 μL of 40 mg/ mL DTT, followed by further incubation at 37 °C for 1 h in the dark with 150 μL of 100 mg/mL IAA. Then, the 8-fold diluted BSA solution was digested by adding with 10 μg of trypsin (MS grade; Promega, Madison, WI), followed by incubation at 37 °C overnight. After digestion, the solution was acidified 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 buffer (0.1% TFA) and elution buffer (0.1% TFA, 80% ACN) and then dried on a savant SPD2010 SpeedVac system (Thermo Fisher Scientific, 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 μL tips (10 μm particles, 3 mg of TiO2) supplied with a Titansphere Phos-TiO kit (GL Sciences, Tokyo, Japan) were used in this study. To confirm the selectivity by the additive effect using a phosphopeptide standard mixture, we prepared wash buffer A (5% TFA, 80% ACN) and wash buffer 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 μg) was made up to 300 μL with wash buffer B and vortexed for 30 min. The tip was preequilibrated by adding 100 μL of wash buffer A, followed by 100 μL of wash buffer 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 flow-through fraction was passed through the system again. Subsequently, the tip was washed three times with 100 μL of wash buffer B with centrifugation at 3000g for 1 min, followed by three further washes with 300 μL of wash buffer A with centrifugation at 3000g for 1 min each time. Finally, the binding phosphopeptides were eluted with 100 μL of 5% NH4OH with centrifugation at 1000g for 5 min. Immediately, the eluate was acidified with 200 μL of 10% TFA prior to desalting with a Monospin C18 spin column (GL Sciences) using wash buffer (0.1% TFA) and elution buffer (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 5588

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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 buffer (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 buffer A (5% TFA, 80% ACN) and wash buffer B (5% TFA, 60% ACN, 5% glycerol) were used. The procedures were almost the same as described above except the following elution buffers 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 buffer A (5% TFA, 80% ACN) and wash buffer B (5% TFA, 60% ACN) containing 25% DL-lactic acid or 5% glycerol. In addition, elution buffers 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 first elution was performed by adding 100 μL of 5% NH 4 OH with centrifugation at 1000g for 5 min. The second elution was then performed by further adding 100 μL of 1 M bis-Tris propane with centrifugation at 1000g for 5 min. These eluates were mixed and acidified immediately by adding 400 μL 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 buffer A (5% TFA, 80% ACN), wash buffer B (5% TFA, 60% ACN, 5% glycerol), and the elution buffer (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 buffer (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 μL of 40 mg/mL DTT, followed by further incubation at 37 °C for 1 h in the dark with 150 μL of 100 mg/mL IAA. Then, the lysate solution was diluted 2-fold with 100 mM TEAB and digested by adding 20 μg 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 μg of trypsin (MS grade; Promega). Subsequently, the peptide solutions were acidified 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 Ä 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 buffer 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 μm particles, 100 Å pore size; Merck, Darmstadt, Germany) at a flow rate of 0.5 mL/min with buffer A and eluted with buffer B (0.013% TFA, 5% ACN) according to the following time courses and linear gradient B%: 0−40 min, 0−40%; 40−43 min, 40−100%; 43−50 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 Ultraflex II MALDITOF/TOF mass spectrometer (Bruker Daltonics). After the TiO2-enriched samples were dissolved in 10 μL of matrix solution (10 mg/mL DHB, 0.1% TFA, 30% ACN, 1% H3PO4), 1 μL 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-reflectron; laser power, 15% constant; accumulated sample positions, 20 positions; total laser shot number, 1000 shots; mass range, m/z 800−5000. To evaluate the selectivity of the TiO2 column with each additive, the obtained MS spectra were cut off 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 Scientific) and a Qexactive hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific). Phosphopeptide samples were dissolved in 5 μL of 0.1% TFA containing 2.5% ACN and loaded onto a reverse-phase C18 analytical column (50 μm i.d. × 150 mm, 2 μm particles, 100 Å pore size; Thermo Fisher Scientific) at a flow rate of 300 nL/min with buffer A (0.1% FA) and buffer B (0.1% FA, 99.9% ACN) with an Easy-nLC system with the following linear gradient B%: 0−60 min, 0− 35%; 60−62 min, 35−90%; 62−70 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% penicillin−streptomycin solution (Life Technologies) and cultured at 37 °C in a humidified atmosphere with 5% CO2. For stimulation, 80−90% confluent 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 5589

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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 300−1650) 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 identification of the target kinases of dasatinib.

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MS Data Analysis

RESULTS AND DISCUSSION

Phosphopeptide Selectivity on TiO2 Column by Additive Effect

To determine the optimal elution conditions and the combination of additives and elution conditions for enrichment using TiO2 chromatography, peptide identification of obtained product ion spectra was conducted by Proteome Discoverer software, version 1.3 (Thermo Fisher Scientific) 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; fixed modifications, carbamidomethylation of Cys residues; variable modifications, oxidation of Met residues and phosphorylation of Tyr, Ser, and Thr residues; MS tolerance, 3 ppm; MS/MS tolerance, 0.02 Da. Phosphopeptides with confidence interval (CI) > 95.0% were extracted with a MASCOT peptide ion score cutoff >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 identified 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 identified as described above except the application of the SEQUEST search engine (Thermo Fisher Scientific). For the quantification, representative MS peak area intensities of identified phosphopeptide were calculated using Xcalibur software (Thermo Fisher Scientific).

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 specifically 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 efficiency 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 efficiency of glycerol additive was 80.6 ± 4.7% (mean ± SD, Table 1). Propylene Table 1. The Enrichment Efficiency of Phosphopeptides Using Phosphopeptide Standard Mixture by TiO2 Chromatography with Various Additivesa

Western Blotting Analysis

Aliquots of 20 μg of dasatinib-treated (0, 100, and 300 nM at 37 °C for 1 h) PC3 whole-cell lysate in SDS sample buffer were denatured at 70 °C for 10 min, followed by 4−12% SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred onto PVDF membranes, which were blocked with blocking buffer (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 μg/mL; Thermo Fisher Scientific), anti-pY827ACK1 rabbit polyclonal antibody (1 μg/mL; Biorbyt Ltd., Cambridge, UK), antitotal EPHA2 rabbit monoclonal antibody (1:1000; Cell Signaling Technology), and antitotal ACK1 mouse monoclonal antibody (1 μg/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 purification) 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

efficiencyb (%)

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

a

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).

b

glycol and trimethylene glycol additives also observed high efficiency 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 5590

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lysates. The phosphopeptides bound to the TiO2 column were eluted by each alkaline solution and identified 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 sol−gel method40−42 or as a cross-linking agent in the condensation of TiO2 particles.43,44 However, this is the first 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 titanium−glycerol complexes coordinated via three C−O bonds of the trihydroxy groups, i.e., “tridentate chelation or bridging.” This capping effect 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 (−Ti−OH) or titanium-oxo bridges (−Ti−O−Ti−). 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 efficiency 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 nonspecific 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 α-hydroxy group of lactic acid is more important than the α-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 first key structure identified for high phosphopeptide selectivity. However, simple 1,2-diols such as propylene glycol were not sufficient to improve selectivity. These results suggest that the further oxygen atom neighboring 1,2-diol structure, derived from the hydroxy group (−CH2−OH) at the 3-carbon position of glycerol and the carbonyl group (>CO) 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 findings 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 specific selectivity to phosphopeptides.

Table 2. Numbers of Phosphopeptides Obtained from PC3 Cell Lysate Digests by TiO2 Chromatography with Various Alkaline Elution Buffers

a

elution buffer

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

identified 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 buffer had the highest pH, but the number of phosphopeptides obtained was not significant. Furthermore, NH4HCO3 elution buffer, 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 first-choice elution buffer. 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 findings suggest that effective 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 5.0−6.0 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 buffer to determine which parameters made major contributions to the effective 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 5591

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Figure 1. The property distributions of phosphopeptides obtained from PC3 cell lysate digest by TiO2 chromatography with each elution buffer. (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 buffers are represented as follows: (■) bis-Tris propane, (●) NH4OH, (□) TEA, (△) Tris, (⧫) NaOH, (○) NH4HCO3, and (×) bis-Tris.

particular 0−40% 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 buffer 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 C−O 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 buffer after NH4OH elution to improve the recovery and identification of longer and/or hydrophilic phosphopeptides.

Figure 1A shows the distribution of molecular weight of phosphopeptides obtained from TiO2 chromatography with each elution buffer using PC3 cell lysate digest. Although NH4OH has generally been used as elution solution, it tended to elute shorter phosphopeptides (1000−1500 Da). On the other hand, bis-Tris propane eluted from shorter to longer phosphopeptides (1000−4000 Da). These results suggest that bis-Tris propane elution in combination with NH4OH elution would make it possible to increase the phosphopeptide identification 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 (0−0.5) tended to be strongly eluted by TEA in comparison to NH4OH and bis-Tris propane. Moreover, we confirmed 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 5592

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Effects 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 specific 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 differences 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 difference 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 fields 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 confirmed the efficiency of phosphopeptide identification 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 identified by the present optimized method (Figure 3). Furthermore, we compared the identified 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

To evaluate the effects of combinations of additive and elution conditions by TiO2 chromatography for phosphopeptide enrichment, we compared the number of identified 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 effective 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. 5593

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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 μg 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 specific 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 specifically 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 findings indicate that ACK1 overexpressed in prostate cancer may also participate in tissue-specific 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 effective 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 effectiveness of various drugs.

Exploring Target Kinases of Dasatinib Using Optimized Enrichment Method

To confirm 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 effective 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 identified 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 identified under these conditions, respectively (Figure 4B). These identified 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 confirmed 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 effects 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 effect 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

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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 effective 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 differences 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 identified, 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.

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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 identified 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 quantification data corresponding to peak area intensities. These materials are available free of charge via the Internet at http:// pubs.acs.org. 5595

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-6-6331-5967. Fax: +81-6-6332-6385. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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