CoreShell Microspheres for Selective Enric - American Chemical Society

May 13, 2008 - Immobilized metal affinity chromatography (IMAC) is a popular way to enrich phosphopeptides; however, con- ventional IMAC lacks enough ...
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Novel Fe3O4@TiO2 Core-Shell Microspheres for Selective Enrichment of Phosphopeptides in Phosphoproteome Analysis Yan Li,† Xiuqing Xu,† Dawei Qi, Chunhui Deng,* Pengyuan Yang, and Xiangmin Zhang* Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China Received September 7, 2007

Abstract: Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Immobilized metal affinity chromatography (IMAC) is a popular way to enrich phosphopeptides; however, conventional IMAC lacks enough specificity for efficient phosphoproteome analysis. In this study, novel Fe3O4@TiO2 microspheres with well-defined core-shell structure were prepared and developed for highly specific purification of phosphopeptides from complex peptide mixtures. The enrichment conditions were optimized using tryptic digests of β-casein, and the high specificity of the Fe3O4@TiO2 core-shell microspheres was demonstrated by effectively enriching phosphopeptides from the digest mixture of R-casein and β-casein, as well as a five-protein mixture containing nonphosphoproteins (bovine serum albumin (BSA), myoglobin, cytochrome c) and phosphoproteins (ovalbumin and β-casein). The Fe3O4@TiO2 core-shell microspheres were further successfully applied for the nano-LC-MS/MS analysis of rat liver phosphoproteome, which resulted in identification of 56 phosphopeptides (65 phosphorylation sites) in mouse liver lysate in a single run, indicating the excellent performance of the Fe3O4@TiO2 core-shell microspheres. Keywords: magnetic microspheres with core-shell structure • titania • selective enrichment • phosphopeptides • phosphoproteomics • proteomics

Introduction Phosphorylation is one of the most important post-translational protein modifications in living cells. Organisms use reversible phosphorylation of proteins to control many cellular processes including signal transduction, gene expression, cell cycle, cytoskeletal regulation, and apoptosis. Although phosphorylation is observed on a variety of amino acid residues, by far the most common and important sites of phosphorylation in eukaryotes occur on serine, threonine, and tyrosine residues.1,2 Owing to the importance of protein phosphorylation in cellular signaling, various methods for protein phos* To whom correspondence should be addressed. Prof. Dr. C. H. Deng and Prof. Dr. X. M. Zhang, E-mail: [email protected]. Tel: +86-21-65643983. Fax: +86-21-6564-1740. † These authors contributed equally.

2526 Journal of Proteome Research 2008, 7, 2526–2538 Published on Web 05/13/2008

phorylation site mapping have been developed through the years. However, this task remains a technical challenge, and there is an intense interest in development of technologies and methods for studying phosphorylation events. Mass spectrometry (MS) has been widely used as a powerful tool to characterize protein modifications including phosphorylation due to its high sensitivity and capability of rapid sequencing by tandem mass spectrometric technique.3–6 For the phosphoproteome analysis, satisfied results often can not be obtained by direct mass spectrometric analysis of protein digest. This is because phosphopeptides are present at low abundance in the digest and the mass spectrometric response of a phosphopeptide is seriously suppressed by unphosphorylated peptides. To reduce the suppression, it is crucial to purify the phosphorylated peptides from complex peptide mixtures. Therefore, selective enrichment of phosphopeptides from enzymatic digest product of proteins remains an interesting research topic in the filed of bioanalytical chemistry. A number of strategies have been applied to separate phosphorylated proteins and peptides from the nonphosphorylated. One such strategy involves the antibodies specific for phosphorylated amino acids,7–9 which is limited due to the availability, high costs, and specificity of the antibodies. Another commonly used strategy is immobilized metal ion affinity chromatography (IMAC), which relies on the affinity of the phosphorate group to metal ions (Fe3+ or Ga3+) immobilized on a matrix such as agarose via acidic compounds like iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA).10–15 However, the specificity of those IMAC adsorbents is still not high enough. Some unphosphorylated peptides (typically acidic peptides) are also strongly bound to the adsorbents, which results in serious interference for the analysis of target phosphopeptides. The poor specificity for phosphopeptides by IMAC may be partially overcome by esterification of the acidic side chains of glutamate and aspartate residues prior to IMAC purification;16 however, it may also increase sample complexity and interfere with subsequent mass spectrometry analysis because of incomplete reactions. During the past decades, metal oxide affinity chromatography (MOAC) has gained more and more attention. Metal oxides such as titania17–19 or alumina20–23 coated adsorbents have been demonstrated to be effective materials for the enrichment of phosphopeptides. MOAC seems to have higher selectivity for phosphopeptides because of their reduced nonspecific binding compared to IMAC, and the results are more reproducible. Guo and Xia et al.24,25 had successfully made use of porous anodic alumina membranes to selectively enrich phosphopeptides from a mixture of synthetic peptides and tryptic digest product of 10.1021/pr700582z CCC: $40.75

 2008 American Chemical Society

Fe3O4@TiO2 Core-Shell Microspheres β-casein by a direct MALDI-TOF MS analysis. Recently, Håkansson’s group26 has demonstrated the enrichment of phosphorylated peptides on the surface of zirconium dioxide loaded in a microtip. According to their statements, zirconium oxide microtips have similar performance to titanium oxide microtips but possess a unique selectivity for singly phosphorylated peptides. Magnetic materials, such as superparamagnetic iron oxide particles, have been extensively used for both in vitro and in vivo application, such as cell separation,27,28 magnetically assisted drug delivery,29,30 and enzyme immobilization.31–35 Besides, magnetic materials are the most commonly selected substrates as affinity probes because of the ease of isolation of the magnetic material-target conjugate from the sample solution based on their magnetic properties. Affinity probes with magnetic properties lead the isolation of the trapped species from sample solutions very conveniently and rapidly; therefore, the time required for separation can be dramatically reduced. Much effort has been devoted to synthesis of core-shell structured magnetic composite microspheres by encapsulation of magnetic particles with various matrix such as organic polymer (e.g., polystyrene) and inorganic species (e.g., silica) to endow magnetic particles with useful surface functionalities for practical applications. Chen et al. synthesized magnetic titania particles (composite of Fe3O4, SiO2, and TiO2 with illdefined structure) with an aim to combine the magnetic property of magnetite particles and affinity of TiO2 toward phosphopeptides for fast enrichment of phosphopeptides.17 In their work, they successfully applied the magnetic titania particles to the enrichment of phosphopeptides in standard phosphoprotein digestion. However, the TiO2 coated magnetic particles they synthesized had the disadvantage of nonspecific binding of nonphosphopeptides. This can be explained by the material with ill-defined structure. The application of the TiO2 coated magnetic particles for selective enrichment of phosphopeptides in complex biological samples is quite limited. Recently, we developed an innovative approach for the synthesis of TiO2-coated magnetite microspheres (donated as Fe3O4@TiO2 microspheres) with well-defined core-shell structure36 (Supporting Information). At first, magnetic microspheres were synthesized and then carbon was coated on them to form Fe3O4@C microspheres. Nanosized titanium oligomers were absorbed onto the Fe3O4@C microspheres and finally converted into titanium by calcination, leading to the formation of Fe3O4@TiO2 microspheres. In the present work, we demonstrate the high efficiency and convenience of the smart application of the Fe3O4@TiO2 microspheres as specific captures of phosphopeptides for MALDI-TOF MS analysis, and further applied to the phosphoproteome analysis of rat liver with nanoLC-MS/MS analysis.

Experiment Procedures Reagents and Materials. Bovine β-casein and casein (from bovine milk), chicken egg albumin (ovalbumin), myoglobin horse heart, cytochrome C, bovine serum albumin (BSA), trypsin (from bovine pancreas, TPCK treated), phosphoric acid (H3PO4), ammonium bicarbonate (NH4HCO3), and 2,5-dihydroxybenzoic acid (2,5-DHB) were purchased from Sigma Chemical (St. Louis, MO). Acetonitrile (ACN) was purchased from Merck (Darmstadt, Germany). All aqueous solutions were prepared using Milli-Q water by Milli-Q system (Millipore, Bedford, MA). All other chemicals and reagents were of the highest grade commercially available.

technical notes Synthesis of Fe3O4@C Magnetic Microspheres. Fe3O4 microspheres with diameter of 280 nm were first synthesized via a solvothermal reaction as previously described.36 In the next step, 0.05 g microspheres were ultrasonicated for 10 min in 0.1 M HNO3, followed by washing with deionized water. Then, the treated Fe3O4 microspheres were redispersed in 0.5 M aqueous glucose solution. After vigorous stirring for 10 min, the suspension was transferred to autoclaves and kept at 180 °C for 4 h. After reaction, the autoclaves were cooled naturally in air, and the suspensions was isolated with the help of a magnet and washed with deionized water and alcohol three times, respectively. The final sample was obtained after ovendrying at 80 °C for more than 4 h. Synthesis of Fe3O4@TiO2 Core-Shell Microspheres. Tetrabutyltitanate (5 mL) was dissolved in ethanol (35 mL) to form a clear solution. Fe3O4@C magnetic microspheres (∼100 mg) were then dispersed in the freshly prepared solution with the aid of ultrasonication for 5 min. A 1:5 (v/v) mixture of water and ethanol was added dropwise to the suspension of Fe3O4@C magnetic microspheres with vigorous magnetic stirring over a period of approximately 15 min. Thereafter, the suspension was stirred for a further 1 h before separation and washing with ethanol. After five cycles of separation/washing/redispersion with ethanol, the powder obtained was oven-dried and calcined according to the procedure described above. Preparation of Tryptic Digest of Standard Proteins. Bovine β-casein, R-casein (from bovine milk), chicken egg albumin (ovalbumin), myoglobin horse heart, cytochrome C, and bovine serum albumin (BSA) were each dissolved in 25 mM ammonium bicarbonate buffer at pH 8.0 and treated with trypsin (50:1, w/w) for 12 h at 37 °C respectively. All the Peptide mixtures were diluted with 50% acetonitrile and 0.1% TFA aqueous solution (v/v). Preparation of the Lysate of Rat Liver. Rats were sacrificed and the livers were promptly removed and placed in ice-cold homogenization buffer consisting of 8 M urea, 4% CHAPS (w/ v), 65 mM DTT, 1 mM EDTA, 0.5 mM EGTA, and a mixture of protease inhibitor (1 mM PMSF) and phosphatase inhibitors (0.2 mM Na3VO4, 1 mM NaF) and 40 mM Tris-HCl at pH 7.4. After mincing with scissors and washing to remove blood, the livers were homogenized in a Pottter-Elvejhem homogenizer with a Teflon piston, using 4 mL of the homogenization buffer per 1 g of tissue. The suspension was homogenized for approximately 2 min, vortexed at 0 °C for 30 min, and centrifuged at 22 000 g for 1.5 h. The supernatant contained the total liver proteins. Appropriate volumes of protein sample were precipitated as above, lyophilized to dryness, and redissolved in reducing solution (6 M guanidine hydrochloride, 100 mM ammonium bicarbonate, pH 8.3) with the protein concentration adjusted to 2 µg/µL. Then, 200 µg of this protein sample (100 µL volume) were mixed with 10 µl of 0.5 M DTT. The mixture was incubated at 37 °C for 1 h, and then 20 µL of 0.5 M IAA were added and incubated for an additional 30 min at 37 °C in the dark. The protein mixtures were exchanged into 50 mM ammonium bicarbonate buffer, pH 8.5, and incubated with trypsin (50:1) at 37 °C overnight. The digested peptide mixture was lyophilized and then dissolved in 0.1% TFA. Enrichment of Phosphopeptides by Fe3O4@TiO2 Core-Shell Microspheres. Suspension of Fe3O4@TiO2 core-shell microspheres (5 µL of 10 mg mL-1) was added into 200 µL of Peptide mixture originating from tryptic digestions respectively. Then the mixed solutions were vibrated at 37 °C for 30 s. After that, Journal of Proteome Research • Vol. 7, No. 6, 2008 2527

technical notes with the help of magnet, the peptides-Fe3O4@TiO2 core-shell microspheres were collected by removal of the supernatant and washed with 50% acetonitrile and 0.1% TFA aqueous solution (v/v) for three times. Then the obtained peptide-loaded Fe3O4@TiO2 microspheres were redispersed in 10 µL of 50% acetonitrile aqueous solution (v/v). For phosphoproteome analysis of the mouse liver lysate, 1 mg of Fe3O4@TiO2 core-shell microspheres were mixed with digest of 100 µg moule liver lysate, and then the total volume was adjusted to 1 mL by adding 50% acetonitrile and 0.1% TFA aqueous solution (v/v). Except the volume was 1 mL, the washing and elution steps were the same as for standard protein digests. Sample Preparation for MALDI-TOF MS. The above peptides-Fe3O4@TiO2 core-shell microspheres slurry was deposited on the MALDI target using dried droplet method. The slurry (0.5 µL) was deposited on the plate, and then another 0.5 µL of a mixture of 20 mg mL-1 2,5-DHB (in 50% acetonitrile aqueous solution, v/v) and 1% (v/v) H3PO4 aqueous solution, 1:1(v/v), was introduced as a matrix. Sample Preparation for Nano-LC-LTQ-MS/MS/MS Analysis. After the extraction of phosphopeptides from tryptic digest by Fe3O4@TiO2 core-shell microspheres, 100 µL of 12.5% NH3•H2O were added to elute the phosphopeptides from Fe3O4@TiO2 core-shell microspheres. After incubation for 5 min, the supernatant containing phosphopeptides was collected and lyophilized to dryness, and finally redissolved in 10 µL of 0.1% formic acid and 5% acetonitrile for nano-LC-MS/ MS/MS analysis with the help of a magnet. Apparatus. MALDI-TOF MS experiments were performed on positive ion mode on a 4700 Proteomics Analyzer (Applied Biosystems) with the Nd:YAG laser at 355 nm, a repetition rate of 200 Hz and an acceleration voltage of 20 kV. Nano-LC-MS/MS and MS/MS/MS were performed on the µRPLC-MS/MS system. A LTQ linear ion trap mass spectrometer (Thermo-electron) with a nanospray source was used with a LC-Packings chromatography system. The pump flow rate was split by a cross to achieve a flow rate at 500 nL/min. The column was Michrom, Magic C18, 5 µm, 100 Å. The separation of phosphopeptides enriched from the tryptic digest of mouse liver lysate was performed using 75 min linear gradient elution. The mobile phase consisted of mobile phase A, 0.1% formic acid and 5% acetonitrile in H2O, and mobile phase B, 0.1% formic acid in 95% acetonitrile. The LTQ instrument was operated at positive ion mode. A voltage of 1.8 kV was applied to the cross. About 1 µL (20 µg) redissolved peptides was loaded onto the C18 capillary column using a 75 µm i.d. × 220 mm length capillary column as sample loop. The detection of phosphopeptides was performed in which the mass spectrometer was set as a full scan MS followed by three data-dependent MS/MS (MS2). Subsequently MS/MS/MS (MS3) spectrum was automatically triggered when the most three intense peaks from the MS/MS spectrum corresponded to a neutral loss event of 98, 49, and 32.7 Da for the precursor ion with 1+, 2+, 3+ charge states, respectively. Database Searching and Manual Validation. The obtained MS2 and MS3 spectra were searched with the SEQUEST algorithm against the rat protein database from IPI (ipi. Rat. V.3.27,), respectively. In MS2 searching, differential modifications of 80 Da to Ser, Thr, and Tyr residues were selected, and for MS3 database searching, besides above modifications, -18 Da were added to Ser, Thr, and Tyr residues. The obtained result of database searching was first filtrated by setting Xcorr as 2.5, 2.63, and 2528

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Li et al. 3.11 corresponding to 1+, 2+, and 3+ charge states, respectively. Furthermore, phosphorylated peptides identified by SEQUEST were manually validated using the following criteria. First, the MS/MS spectrum must be of good quality, with fragment ion peaks clearly above baseline noise with at least three sequential members of the b- or y-ion series observed. Second, the phosphoric acid neutral loss peak must be detected in the MS/MS spectrum and be the dominant signal for peptides that are singly phosphorylated. Third, the MS/MS/ MS spectrum should be consistent with the determined peptide sequence. Peptides with only MS/MS data but no MS/MS/MS data were confirmed as phosphorylated peptides only if their phosphoric acid neutral loss peak was clearly defined.

Results and Discussion Preparation and Evaluation of Fe3O4@TiO2 Core-Shell Microspheres. Herein, we present an innovative approach for synthesis of TiO2-coated magnetite microspheres (donated as Fe3O4@TiO2 microspheres) with well-defined core-shell structure, and by using the Fe3O4@TiO2 microspheres with the assistance of applied magnetic field, we demonstrate the high efficiency of the smart application of these microspheres as specific captures of phosphopeptides for MALDI-TOF MS analysis. The synthesis route for the preparation of Fe3O4@TiO2 microspheres have been reported in our previous work involving three steps.36 Briefly, 280-nm sized magnetite microspheres were first synthesized via a solvothermal reaction using FeCl3 as source of magnetite and ethylene glycol as both solvent and reductant. Then, the magnetite microspheres were coated with a thin layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction, resulting in Fe3O4@C microspheres. Finally, tetrabutyltitanate was prehydrolyzed and absorbed onto the microspheres and eventually converted into titania by calcination in nitrogen. The detailed procedures for the preparation of Fe3O4@TiO2 microspheres are demonstrated in Scheme S1a (Supporting Information). Transmission electron microscopy images and Fourier transform infrared spectra of the as-made Fe3O4, Fe3O4@C, and Fe3O4@TiO2 microspheres are shown in Figure S1 and Figure S2 (Supporting Information), which demonstrated that Fe3O4@TiO2 microspheres were successfully synthesized. Notably, as can be seen in the TEM image of high magnification (Figure S1d, Supporting Information), the outer shell of the obtained Fe3O4@TiO2 core-shell microspheres consists of larger amount of titania nanoparticles, which may provide high specific area and is beneficial for specific trapping of phosphopeptides when they are used for enrichment application. The magnetic properties of the microspheres enable the Fe3O4@TiO2 microspheres to make rapid response to applied magnetic field, thus allowing a fast enrichment procedure. The wide-angle X-ray diffraction measurement result also indicates that TiO2 nanoparticles in the outer layer of the Fe3O4@TiO2 microspheres are of anatase phase (JCPDS No. 211272) (Figure S3, Supporting Information). To investigate the selectivity and efficiency of the obtained Fe3O4@TiO2 microspheres for enrichment of phosphopeptides, bovine β-casein was first chosen as a model compound due to its well characterized phosphorylation sites. Scheme 1b displays the detailed procedures for phosphopeptide enrichment with the Fe3O4@TiO2 core-shell microspheres. The tryptic β-casein digest was mixed with the Fe3O4@TiO2 core-shell microspheres and was incubated for 30 s, then simply with the help of magnet, the phosphopeptides were isolated, washed, and then

technical notes

Fe3O4@TiO2 Core-Shell Microspheres

Scheme 1. (a) Synthesis of Fe3O4@TiO2 Core-Shell Microspheres and (b) Procedures of Using Fe3O4@TiO2 Core-Shell Microspheres to Enrich Phosphopeptides from a Sample Solution

Table 1. Phosphopeptides Identified in β-Casein no.

1 2 3

AA

phosphopeptide sequences

[M + H]+

33-48 FQ[pS]EEQQQTEDELQDK 2061.70 33-52 FQ[pS]EEQQQTEDELQDKIHPF 2555.82 1-25 RELEELNVPGEIVE[pS]L[pS][pS][pS]EESITR 3121.98

ready for MS analysis using 1% (v/v) phosphoric acid and 2,5DHB (in 50% acetonitrile aqueous solution, v/v), 1:1, (v/v), as a matrix. A list of the theoretical tryptic phosphorylated peptides derived from β-caseins and their molecular masses is shown in Table 1. A direct analysis of a tryptic digestion of 10 fmol of commercial β-casein by MALDI MS using a dried droplet sample preparation in which the peptide mixture is mixed with DHB matrix solution (including 1% phosphoric acid) resulted in detection of only one theoretical phosphorylated peptides with weak intensity (Figure 1a). After enrichment using the Fe3O4@TiO2 core-shell microspheres for only 30 s, all three

phosphopeptides derived from β-casein appeared in the mass spectrum, marked with 2061.70, 2555.82, and 3121.98 (see Figure 1b). Compared to the tryptic β-casein digest prior to isolation (Figure 1a), the signal-to-noise ratio (S/N) of the MS signal of the phosphorylated peptides enriched by Fe3O4@TiO2 core-shell microspheres was significantly improved. These results indicate that the Fe3O4@TiO2 core-shell microspheres can be used for efficient enrichment of phosphopeptides. Optimization of the Conditions for Phosphopeptides Enrichment using Fe3O4@TiO2 Core-Shell Microspheres. Initial optimization of the loading buffer was performed using tryptic peptides originating from β-casein. Almost all IMAC protocols employ acetic acid in the buffer system to decrease the interaction of nonspecific binders such as acidic peptides to the IMAC beads. It is believed that strong acids such as TFA prevent the interaction of phosphopeptides with IMAC by the protonation of phosphate groups. Nevertheless, Oda and his co-workers compared four different acids with different acid Journal of Proteome Research • Vol. 7, No. 6, 2008 2529

technical notes

Li et al. should be achievable with TiO2 upon careful selection of the pH. In this study, TFA was added to the loading buffer to adjust to a certain pH value (2.0, 4.0, 6.0), and the selectivity of Fe3O4@TiO2 core-shell microspheres was assessed for capture of the phosphopeptides at the given pH. The data in Figure 3 indicate that high selectivity can be obtained when the pH value of the loading buffer at both 2.0 and 4.0 (Figure 3a and b). When pH value further increased to 6.0, phosphopeptides still dominate the mass spectrum; however, nonphosphorylated peptide at m/z 2163 was observed in the spectrum, indicating the compromising selectivity at higher pH (Figure 3c). Compared with IMAC,16,42 Fe3O4@TiO2 core-shell microspheres could selectively enrichment phosphopeptides in wider pH. On the basis of the results obtained above, the use of 50% ACN with 0.1% TFA (pH∼2.0) not only enhanced the specific enrichment on Fe3O4@TiO2 core-shell microspheres but also washed away nonphosphorylated peptides from the microspheres due to the existence of ACN, and was thus chosen as the loading/washing buffer.

Figure 1. MALDI mass spectra of (a) the tryptic digest product of β-casein without any pretreatment, 2 × 10-8 M, 0.5 µL and (b) on bead analysis of phosphopeptides obtained when using Fe3O4@TiO2 core-shell microspheres to selectively trap target peptides from the tryptic digest product of β-casein at the following concentrations and extraction volumes: 2 × 10-8 M, 200 µL. The data in parentheses are S/N of the corresponding peptides.

strengths as additives for enriching phosphopeptides of ovalbumin using IMAC tips,37 and their results revealed that the best selectivity was obtained when using TFA solution as loading buffer. In this work, we used acetic acid and TFA in the loading buffer to investigate their effect on the selective binding of phosphorylated peptides to Fe3O4@TiO2 core-shell microspheres. Another major factor in nonspecific binding on affinity columns is hydrophobic interaction. Oda et al.38 have proved that acetonitrile (ACN) was very effective in eliminating nonphosphorylated peptides and thus improved the specificity on IMAC. In this study, we also investigated the impact of ACN on improving specificity of Fe3O4@TiO2 core-shell microspheres. Compare Figure 2a and b, c and d, when the loading buffer contained acetonitrile, almost no nonphosphopeptides were enriched, because acetonitrile could break up interactions between hydrophobic peptides and the metal oxides. Both acetic acid- acetonitrile and TFA- acetonitrile buffers show good selectivity in the enrichment of phosphopeptides (Figure 2a and c). Prior studies indicated that phosphopeptides are not well retained by IMAC resins and metal oxides at alkaline pH values.38–40 Titania is known to have amphoteric properties, that is, it can react either as a Lewis acid or base depending on the pH of the reaction solution.41 In acidic solution, TiO2 behaves as a Lewis acid with positively charged titanium atoms, thereby displaying anion-exchange properties. The binding constant of phosphate ions is markedly higher than for other Lewis bases, suggesting that high binding selectivity for phosphorylated peptides over nonphosphorylated acidic peptides 2530

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We further investigated whether the enrichment efficiency of Fe3O4@TiO2 core-shell microspheres were improved by extending the enrichment time. Figure 4a-d display the MALDI mass spectra obtained after incubating the tryptic digest product of β-casein with Fe3O4@TiO2 core-shell microspheres under vortex mixing for 0.5, 5, 15, and 60 min. The same three peaks derived from phosphopeptides of β-casein appeared in the mass spectra but when incubation time increases, signal abundance of peak 2556.09 and 3122.27 become higher. Nevertheless, the results indicated that phosphopeptides sufficient for MALDI MS analysis could be enriched within only 0.5 min. The effect of elution time on enrichment of phosphopeptides was also investigated. When the phosphopeptides conjugated microspheres eluted with 12.5% NH3•H2O for 1 min, despite the fact that all three phosphopeptides derived from β-casein were detected in the mass spectrum (Figure 5a), they were with low intensities. When the elution time increased to 5 min, the intensities of these three ion signals remarkably improved, while more peaks which could be assigned to phosphopeptides derived from R-casein (due to impurity of the β-casein we used) appeared in the mass spectrum (Figure 5b). No apparent improvement of the peak intensities was observed when we further increased the elution time to 15 and 60 min (Figure 5c and d). The results indicate that 5 min is long enough for eluting the conjugated phosphopeptides from the Fe3O4@TiO2 core-shell microspheres. With the optimized enrichment condition, even when the concentration of the sample is lowered to 2 × 10-9 M, as shown in Figure 6, all three phosphopeptide derived from β-casein can still be observed in the mass spectrum after enrichment, suggesting the ability of the Fe3O4@TiO2 core-shell microspheres for enrichment of phosphopeptides from samples with low concentration. The binding and recovery of phosphopeptides to the Fe3O4@TiO2 core-shell microspheres were also evaluated in our study. Peptides originating from tryptic digest of β-casein (2 × 10-8 M, 200 µL) were enriched by Fe3O4@TiO2 core-shell microspheres. After enrichment, the phosphopeptide-conjugated microspheres were redispersed in 10 µL of loading buffer, and 0.5 µL of the dispersion were directly deposited onto the MALDI plate for MS analysis (Figure S4a, Supporting Information). In a parallel experiment, the phosphopeptides conjugated to the microspheres were then eluted with 10 µL of 12.5% NH3•H2O for 5 min. The elution was then neutralized with formic acid, and 0.5 µL of them was used

Fe3O4@TiO2 Core-Shell Microspheres

technical notes

Figure 2. Effect of loading buffer on Fe3O4@TiO2 enrichment of phosphopeptides. Shown are MALDI mass spectra obtained from 2 × 10-8 M β-casein trypsin digest (a) enriched using 50% ACN-TFA as loading buffer, (b) enriched using H2O-TFA as loading buffer, (c) enriched using 50% ACN-ACOH as loading buffer, and (d) enriched using H2O-ACOH as loading buffer.

Figure 3. Effect of sample pH on Fe3O4@TiO2 enrichment of phosphopeptides. Shown are MALDI mass spectra obtained from 2 × 10-8 M β-casein trypsin digest at (a) pH ) 2, (b) pH ) 4, and (c) pH ) 6.

for MALDI MS analysis (Figure S4b, Supporting Information). As shown in Figure S4a and S4b, (Supporting Information), similar results were obtained with both direct on-bead

analysis and analysis of elution. The results indicate that if the phosphopeptides were eluted from the Fe3O4@TiO2 core-shell microspheres, they can be effectively detected. Journal of Proteome Research • Vol. 7, No. 6, 2008 2531

technical notes

Li et al.

Figure 4. Effect of incubation time on Fe3O4@TiO2 enrichment of phosphopeptides. Shown are MALDI mass spectra obtained from 2 × 10-8 M β-casein trypsin digest after (a) 30 s, (b) 5 min, (c) 15 min, and (d) 60 min.

Figure 5. Effect of elution time on Fe3O4@TiO2 enrichment of phosphopeptides. Shown are MALDI mass spectra obtained from 2 × 10-8 M β-casein trypsin digest after (a) 1 min, (b) 5 min, (c) 15 min, and (d) 60 min.

We further compared the performance of Fe3O4@TiO2 microspheres with commercial Fe3+-IMAC resin for selective enrichment from a semicomplex sample (tryptic digested peptides of β-casein and bovine serum albumin (BSA) with a molar ratio of 1:50). A direct analysis of tryptic digestion of 0.5 2532

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µL of Peptide mixture 1 by MALDI MS was carried out, and the obtained mass spectrum is shown in Figure S5a (Supporting Information). Except for the peak at m/z 2061.76 with low S/N, which can be assigned to phosphopeptide of β-casein, other phosphopeptides from β-casein were difficult to distinguish

Fe3O4@TiO2 Core-Shell Microspheres

technical notes

Figure 6. MALDI mass spectra of (a) the tryptic digest product of β-casein without any pretreatment, 2 × 10-9 M, 0.5 µL and (b) on bead analysis of phosphopeptides obtained when using Fe3O4@TiO2 microspheres to selectively trap target peptides from the tryptic digest product of β-casein, 2 × 10-9 M, 200 µL.

Figure 7. MALDI mass spectra of (a) 5 ng/µL tryptic casein digest without any pretreatment and (b) on bead analysis of phosphopeptides obtained when using Fe3O4@TiO2 microspheres to selectively trap target peptides from the 5 ng/µL tryptic digest product of casein, 200 µL. The phosphopeptides are marked with asterisks.

from the mass spectrum because of the presence of numerous abundant nonphosphopeptides peaks from BSA. When using Fe3O4@TiO2 microspheres for phosphopeptides enrichment, although the acidic nonphosphopeptides of BSA (at m/z 1955.85, S319-336, DAIPENLPPLTADFAEDK) can still be observed in the mass spectrum, all three phosphopeptides of β-casein (marked with asterisks) and peaks derived from their metastable losses of phosphoric acid (marked with ∆) can be detected in the spectrum with greatly improved S/N (Figure S5b). However, after enrichment with IMAC resins (Figure S5c), only two phosphopeptides (at m/z 2061.76 and 3122.95) were detected with low S/N, while many nonphosphopeptides can still be observed in the mass spectrum. Similar results were also obtained by other researchers. Larsen et al. 42 demonstrated that by using Fe3+-IMAC to enrich phosphopeptides from the digest mixture of phosphoproteins (R-casein, β-casein, ovalbumin) with nonphosphoproteins (serum albumin, β-lactoglobulin, carbonic anhydrase), when the ratio decreased to 1:50, the peaks of phosphopeptides could hardly be observed. Zou et al.43 also investigated the performance of Fe3+-IMAC to enrich phosphopeptides from the digest mixture of β-casein and BSA at different molar ratios, and it was found that phosphopeptides could not be specifically captured when the molar ratio decreased to 1:10. These results indicated that Fe3+IMAC lacks enough specificity to selectively capture phosphopeptides from complex peptide mixture. The results above

indicate a more selective binding of the phosphorylated peptides on the Fe3O4@TiO2 microspheres than on the Fe3+-IMAC resin. Purification of Phosphopeptides from Peptide Mixture Using Fe3O4@TiO2 Core-Shell Microspheres. Selectivity of these magnetic particles for phosphopeptides was further demonstrated with a tryptic digest of the mixture of casein (composed of R-S1, R-S2 units, and β-casein) at a low concentration (5 ng/µL). Figure 7a presents the direct MALDI mass spectrum of the tryptic digest of casein (5 ng/µL) prior to enrichment. Among the peaks, only four phosphorylated peptide-ions with low intensity were observed, marked with asterisks at m/z 2#1466.58 (S2/138-149), 4#1660.67 (S1/ 106-119), 8#2061.63 (β/33-48), and 13#3122.65 (β/1-25). After using the affinity probes to enrich phosphopeptides, phosphopeptide residue ions start to appear in the MALDI mass spectrum (Figure 7b). The peaks marked with the numbers 4, 5, 6, 7, and 11 are derived from R-S1-casein, whereas the peaks marked with the numbers 1, 2, 3, 10, and 12 are derived from R-S2-casein. The remaining peaks, marked with the numbers 8, 9, and 13 are derived from β-casein. The corresponding peptide sequences of these ions are listed in Table 2. All of the ions revealed in the mass spectrum are phosphopeptide residues. The results indicate that the Fe3O4@TiO2 core-shell microspheres can be used to selectively enrich phosphopeptides from a complex sample. Journal of Proteome Research • Vol. 7, No. 6, 2008 2533

technical notes

Li et al.

Table 2. Phosphopeptides Ion Peaks Observed in the MALDI Mass Spectrum of Tryptic Digest of Casein

a

no.

AA

peptide sequences

monoisotopic mass

1 2 3 4 5 6 7 8 9 10 11 12 13

R-S2/138-147 R-S2/138-149 R-S2/126-137 R-S1/106-119 R-S1/104-119 R-S1/43-58 R-S1/104-119 β/33-48 β/33-52 R-S2/2-21 R-S1/99-120 R-S2/2-22 β/1-25

TVDME[pS]TEVF TVDME[pS]TEVFTK EQL[pS]T[pS]EENSKK VPQLEIVPN[pS]AEER YKVPQLEIVPN[pS]AEER DIG[pS]E[pS]TEDQAMETIK YKVPQLEIVPN[pS]AEER FQ[pS]EEQQQTEDELQDK FQ[pS]EEQQQTEDELQDKIHPF NTMEHV[pS] [pS] [pS]EESII[pS]QETYK LRLKKYKVPQLEIVPN[pS]AEERL NTMEHV[pS] [pS] [pS]EESII[pS]QETYKQ RELEELNVPGEIVE[pS]L[pS][pS][pS]EESITR

1237.42 1466.51 1562.04a 1660.661 1832.66 1927.49 1951.76 2061.64 2555.86 2618.84 2703.60 2746.59 3121.70

Phosphopeptides ion peaks is [M + Na]+.

Figure 8. MALDI mass spectra of peptide mixture contained peptides originating from tryptic digestions of 2 × 10-8 M of β-casein, ovalbumin, myoglobin, cytochrome C, and bovine serum albumin (BSA). (a) Without any pretreatment, 0.5 µL; (b) on bead analysis of phosphopeptides obtained when using Fe3O4/TiO2 microspheres to selectively trap target peptides from the above tryptic digest mixture, 200 µL.

To evaluate the ability of the Fe3O4@TiO2 core-shell microspheres to capture the phosphopeptides from an even more complicated sample, we mixed the tryptic digest products of five proteins, including nonphosphorylated proteins (cytochrome C, myoglobin and bovine serum albumin (BSA)) and phosphoproteins (β-casein and chicken egg albumin (ovalbumin)). Figure 8a displays the direct MALDI mass spectrum of the tryptic digest product (0.5 µL) of cytochrome C, myoglobin, BSA, β-casein, and ovalbumin, which are all of 2 × 10-8 M. At such a low concentration, only two weak phosphopeptide residue ions are observed in the MALDI mass spectrum prior to enrichment. The remaining peaks are all nonphosphorylated peptide residues derived from these proteins. However, after 2534

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enrichment, four phosphopeptide residue ion peaks and their corresponding double charge ion peaks appear in the mass spectrum (as shown in Figure 9a and 9b). The results demonstrate again that the magnetic microspheres coated with TiO2 are very effective in selectively trapping phosphopeptides from a very complex sample. Application of Fe3O4@TiO2 Core-Shell Microspheres for Phosphoproteome Analysis of Rat Liver. To further evaluate the performance of the Fe3O4@TiO2 core-shell microspheres for the capture of phosphopeptides, they were applied to analyze the phosphoproteome of mouse liver. Phosphopeptides from 100 µg rat liver lysate digest were enriched and loaded onto capillary C18 column and analyzed by LTQ mass spectrometer. Three replicate LC-MS runs were conducted for each sample. The acquired MS/MS spectra we searched by Sequest program and the searching results were filtered with Xcorr (>2.5, 2.63, and 3.11 for 1+, 2+, 3+ charged peptides) criteria. Prior to further confirmation, for the three replicate analysis, totally 1347 unique peptides were identified, with an average of 104 nonphosphopeptides and 332 phosphopeptides including 91 singly, 118 doubly, and 123 triply phosphopeptides each run. Among 1347 unique peptides, 76.3% were phosphopeptides and 23.7% were nonphosphopeptides. The low percentage of identified nonphosphopeptides indicated that the nonspecific adsorption of nonphosphopeptides on Fe3O4@TiO2 core-shell microspheres. The acquired MS/MS spectra were searched by Sequest program and the searching results were filtered with Xcorr values obtained by statistical calculation of reverse database searching results. For p < 0.05, the Xcorr values were >2.5, 2.63, and 3.11 for 1+, 2+, 3+ charged peptides respectively. However, because of the poor quality of the spectra for phosphopeptides, the scores of a spectrum passing the criteria does not necessarily mean true identification.3 The fragment ion generated by phosphate-loss in MS/MS stage can be further fragmented to generate MS/MS/MS (MS3) spectrum. It was observed previously that MS3 spectra were useful for the validation of phosphopeptides identified from MS2 spectra.3,44,45 In this study, the acquired MS3 spectra were also searched by Sequest program. As the peptide identifications derived from MS3 were used to confirm the identifications derived from MS2 relative poor spectra should be allowed.44 Comparing the phosphopeptides identified from MS2 and MS3 spectra, it was found that the sequences of 41 unique phosphopeptides were the same in both cases (33 singly phosphorylated, 7 doubly phosphorylated, and 1 triply phosphorylated). The sequences

Fe3O4@TiO2 Core-Shell Microspheres

technical notes

Figure 9. Example of MS/MS spectra of phosphopeptide TPEELDDS*DFETEDFDVR enriched by Fe3O4@TiO2 microspheres. (a) Neutral loss spectra, (b) MS/MS/MS spectra. Journal of Proteome Research • Vol. 7, No. 6, 2008 2535

technical notes

Li et al.

Table 3. Identified Phosphorylated Peptides after Enrichment with Fe3O4@TiO2 Microspheres from Mouse Liver protein accession

peptide sequence

no. of phosphopeptide sites

charge

XC

MS2

MS3

IPI00656420 IPI00767685 IPI00766722 IPI00734740 IPI00564566 IPI00558327 IPI00551815 IPI00480820 IPI00476899 IPI00476698 IPI00476178 IPI00471911 IPI00471584 IPI00393259 IPI00382376 IPI00382244 IPI00373197 IPI00370652 IPI00370209 IPI00369227 IPI00366370 IPI00365935 IPI00365929 IPI00365864 IPI00365663 IPI00365663 IPI00365149 IPI00359917 IPI00359172 IPI00358406 IPI00324618 IPI00210566 IPI00209277 IPI00208304 IPI00208277 IPI00200898 IPI00197900 IPI00194102 IPI00193648 IPI00191707 IPI00189138 IPI00366533 IPI00364925 IPI00363941 IPI00214258 IPI00210280 IPI00209618 IPI00208266 IPI00207601 IPI00202703 IPI00201103 IPI00200145 IPI00192480 IPI00191707 IPI00190024 IPI00188053

K.DADEEDS*DEETSHLER.S K.FNLFS*QELIDKK.S R.WLDES*DAEMELR.A K.KEES*EES*DEDMGFGLFD.R.T*LS*EIELIKVTR.A R.TGDLGIPPNPEDRS*PS*PEPIYNSEGK.R R.NYQQNYQNSESGEKNEGS*ES*APEGQAQQR.R K.EGEEPTVYS*DDEEPK.D K.YGPVSVADTTGSGAADAKDDDDIDLFGS*DDEEESEDAKR.L R.MLPHAPGVQMQAIPEDAVHEDS*GDEDGEDPDKR.I K.ESLKEEDES*DDDNM.K.GILAADES*VGTMGNR.L K.IEDVGS*DEEDDSGKDK.K R.PT*RAS*ISPGSPTSSAAT.R.SSGS*PYGGGYGSGGGSGGYGSR.R K.FHDS*EGDDTEETEDYR.Q R.LLKPGEEPSEYT*DEEDTK.D R.LGAS*PGGDAGTCPPVGRT*GLK.T K.VFDDS*DEKEDEEDTDVR.K R.ESPRPPAAAEAPAGS*DGEDGGRR.D K.TSFDENDS*EELEDKDSK.S K.DWEDDS*DEDMSNFDR.F K.DGELPVEDDIDLS*DVELDDLEKDEL.K.VLHGAQTS*DEEKDF.R.S*VDEVNYWDK.Q R.IGHHS*TSDDSSAYR.S R.YTDQS*GEEEEDYESEEQIQHR.I K.FIDKDQQPSGS*EGEDDDAEAALKK.E R.RES*GEGEEEVADSAR.L R.TPEELDDS*DFETEDFDVR.S K.LKDLGHPVEEEDES*GDQEDDDDELDDGDRDQDI.K.ESDDKPEIEDVGS*DEEEEEKK.D K.LSSQLS*AGEEK.W K.IYHLPDAES*DEDEDFKEQTR.L K.SLDS*DES*EDEDDDYQQK.R R.SAS*SDTSEELNAQDSPK.R K.KGATPAEDDEDNDIDLFGS*DEEEEDKEAAR.L R.FIIGSVSEDNS*EDEISNLVK.L K.SAS*PAPADVAPAQEDLR.T R.YHGHS*MS*DPGVS*YR.T K.VVDYSQFQES*DDADEDYGR.D K.NGIPYSFAFELRDTGY*FGFLLPEMLIK.P R.Y*PVAVSTLEEMAPGTAFK.P R.HSS*WGSVGLGGSLEASR.L K.EVEDKES*EGEEEDEDEDLSK.Y K.AIYQGPSS*PDKS.R.APTAAPS*PEPR.D K.AALGLQDS*DDEDAAVDIDEQIESMFNSK.K K.NFETNDLAFS*PK.G R.TLSNAEDYLDDEDS*D.R.PLS*PTAFSLESLR.K K.KEES*EESEDDMGFGLFD.R.VHGHS*DEEEEEEQPR.H R.YGMGTS*VER.A R.MAMPINVS*DPDLLR.H R.GDS*ETDLEALFNAVMNPK.T

1 1 1 2 2 2 2 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 2 2 2 2 2 2 2 2 3 2 2 3 2 2 2 2 3 2 3 2 2 2 2 2 3 3 3 2 2 3 3 2 3 2 2 3 2 2 3 2 3 2 2 2 2 2 3 2 2 2 2 3 2 2 2

8.74 2.93 3.73 4.88 2.9 4.59 4.84 3.44 8.74 4.7 4.6 4.59 4.34 2.76 4.04 5.86 2.89 5.32 5.04 3.63 4.18 5.83 3.58 4.04 3.8 4.97 5.32 4.43 2.76 5.42 7.69 5.78 3.44 5.28 6.14 5.63 7.96 4.44 4.93 4.06 4.56 3.25 2.66 3.47 3.17 3.51 3.21 3.94 3.96 3.61 2.78 4.11 3.44 3.12 3.88 4.74

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

of theses peptides and their Xcorr scores are listed in Table 3. After manually checking of neutral loss of phosphoric acid for each phosphopeptide, it was found that their spectra were of high quality and so their identifications were considered as positive. These results indicated that confirmation of phosphopeptides identifications by MS3 data is very effective and are in accordance with the results obtained in Zou’s work.45 Figure 9 is an example for identification of doubly charged phosphopeptide TPEELDDS*DFETEDFDVR. From the spectra it can be seen that b- and y-ions series are consistent with the theoretically predicted peaks in both MS2 and MS3 spectra, and in MS2 spectrum that peak at m/z 1071.43 represents the doubly charged form of the selected precursor ion at m/z 1120.68 by losing H3PO4 group. The high quality of MS/MS and MS/MS/MS spectra showed the high performance of 2536

Journal of Proteome Research • Vol. 7, No. 6, 2008

Fe3O4@TiO2 core-shell microspheres for selective enrichment of phosphopeptides. Besides the above 41 phosphopeptides, there are more phosphopeptides identified from MS/MS spectra. Without the confirmation with MS/MS/MS data, these identifications should be manually validated carefully. Many multiply phosphorylated peptide identifications were observed after filtering with Xcorr criteria. However, until now, strictly universal validation criteria have not been established and defined; it has too many subjective factors for interpretation of the spectra of multiply phosphorylated peptides. Therefore, only singly phosphorylated peptide identifications were validate manually in this work. After manual validation, an additional 15 singly phosphorylated peptides were finally identified from MS/MS spectra. The sequences of the peptides finally identified from MS/MS and

technical notes

Fe3O4@TiO2 Core-Shell Microspheres

Abbreviations: MALDI, matrix-assisted laser desorption/ ionization; TOF, time-of-flight; LC-MS/MS, liquid chromatography tandem mass spectrometry; DHB, dihydroxybenzoic acid; IMAC, immobilized metal affinity chromatography.

Acknowledgment. The work was supported by the National Basic Research Priorities Program (Project: 2007CB914100/3), The National High Technology Research and Development Program of China 863 Project (No. 2006AA02Z4C5), the National Key Natural Science Foundation of China (Project: 20735005), and Shanghai Leading Academic Discipline Project (B109). Supporting Information Available: Patent details, Scheme S1, and Figures S1-5. This material is available free of charge via the Internet at http://pubs.acs.org. References

Figure 10. Types of phosphopeptides. (A) Ratio of singly (green), doubly (blue), and triply (brown) phosphorylated peptides. (B) Ratio of serine (green), threonine (blue), and tyrosine (brown) phosphorylation sites identified from rat liver tissue.

their Xcorr scores are listed in Table 3. Combing with the phosphopeptides identified by MS2 and MS3, a total of 56 phosphopeptides (65 phosphorylated sites) were identified from rat liver. Among them, 85.7% contained a single phosphorylation site, 12.5% had two phosphorylation sites, and 1.8% with three phosphorylation sites (see Figure 10a). In addition, 90.8% of the identified phosphorylation sites were phosphorylated on serine residues, 6.1% of the identified sites were phosphorylated on threonine residues, and tyrosine phosphorylation was 3.1% (see Figure 10b).

Conclusions In summary, we present an innovational approach to utilize Fe3O4@TiO2 microspheres with well-defined core-shell structure for the highly selective enrichment and identification of phosphopeptides from protein tryptic digest mixture via a direct MALDI-TOF mass spectrometry analysis and nanoLC-MS/MS analysis. The process of enrichment is very simple, quick, efficient, and specific. The resulting Fe3O4@TiO2 core-shell microspheres-absorbed phosphopeptides can be either directly analyzed by MALDI-TOF MS analysis without elution from the microspheres or eluted with NH3•H2O and further analyzed by nano-LC-MS/MS. In only 0.5 min, phosphopeptides sufficient for characterization can be enriched from very low concentrations of sample solutions. The Fe3O4@TiO2 core-shell microspheres were demonstrated to have high specificity to phosphopeptides by using standard phosphoproteins as well as real biological sample, which opens up a possibility for their application in phosphoproteome analysis.

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