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Zirconium-Cation-Immobilized Core/Shell (Fe3O4@Polymer) Microspheres as an IMAC Material for the Selective Enrichment of Phosphopeptides Leyou Zheng,† Huaping Dong,*,‡ and Liujiang Hu*,‡ ‡

College of Chemistry and Chemical Engineering, Shaoxing University, 508 Huancheng West Road, Shaoxing, Zhejiang 312000, P. R. China † NHU Co. Ltd. of Zhejiang, 4 Jiangbei Road, Xinchang, Zhejiang 312500, P. R. China S Supporting Information *

ABSTRACT: Immobilized metal affinity chromatography (IMAC) is a widely employed method for the enrichment of phosphopeptides from complex proteolytic digests prior to mass spectrometric analysis. In this work, a novel IMAC material, zirconium-phosphate (Zr4+-PO3)-modified magnetic Fe3O4/GMA-co-EDMA (core/shell) (Zr4+-Fe3O4@polymer) microspheres, has been synthesized in a facile manner and used for the selective capture of phosphopeptides from protein tryptic digests for mass spectrometry analysis. The enrichment conditions were optimized using a protein digest solution of β-casein. By virtue of a thick and biocompatible poly(GMA-co-EDMA) shell, high tolerance of contaminants (salt, nonphosphopeptide) was demonstrated for the use of Zr4+-Fe3O4@polymer microspheres for the highly selective enrichment of phosphopeptides from a protein tryptic solution that contained a high concentration of NaCl (6.2 M) or urea (8 M) and a high ratio of nonphosphoprotein (BSA) to phosphoprotein (α-casein) (1:500). The performance of the Zr4+-Fe3O4@polymer microspheres was further successfully examined by the phosphoproteome analysis of mouse liver lysate.

1. INTRODUCTION Protein phosphorylation of serine, threonine, and tyrosine plays a crucial role in the regulation of cellular and molecular processes. The aberrant regulation of phosphorylation might be involved in several diseases including cancers.1 To elucidate this regulation change at the molecular level, the comprehensive identification of protein phosphorylation is a crucial process. To date, mass spectrometry has been confirmed as a powerful and reliable tool for proteome analysis. However, the presence of highly abundant nonphosphorylated peptides in complex proteolytic samples has been demonstrated to strongly suppress the ionization of phosphopeptides,2 which consequently causes much greater difficulty for phosphorylation detection in the mass spectrometric analysis of protein digests. Isolation of the phosphopeptides from complex peptide mixtures has been considered a promising approach to meet the analysis requirements for protein phosphorylation before protein sequence information can be obtained. Immobilized metal affinity chromatography (IMAC) is wellknown as the most widely employed and powerful method for the highly specific isolation of phosphopeptides, and many efforts have been made to develop this method based on the interactions between metal−ligand complexes and specific functional groups. Fe3+ and Ga3+ are the metal ions usually applied for affinity chromatographic adsorbents,3 which are immobilized through acidic chelating ligands of iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA). However, because the presence of some nonphosphopeptides containing glutamic and aspartic acidic residues and peptides containing histidine easily causes serious interference in the detection of target phosphopeptides by nonspecifically binding to the adsorbents, © XXXX American Chemical Society

any improvements in the selectivity or specificity of IMAC materials toward phosphopeptides are still of interest. Blocking the acidic groups of peptides through esterification has been found to be an efficient approach to enhance the specificity of phosphopeptide enrichment.4 Nevertheless, the incompleteness and side reactions of peptide esterification can make sample preparation, mass spectrometry analysis, and the subsequent data analysis complicated. Metal oxide affinity chromatography (MOAC) using titanium dioxide (TiO2),5 zirconium dioxide (ZrO 2),6 aluminum oxide (Al 2O3),7 or niobium oxide (Nb2O5)8 has also gained much attention and showed various degrees of specific binding characteristics to capture phosphopeptides from complex proteolytic digests. In addition, protocol modifications of phosphopeptide enrichment processes (such as chelating ligands, loading buffer composition, pH, ionic strength, and elution buffers) have also been reported to improve the analysis of phosphopeptides.5b,7a,9 However, the IMAC and MOAC techniques are still inconvenient and laborious, as some samples require centrifugation at high speed3b,6b,9c or need to be packed into a column or tips5b,8,10 or deposited on a plate.7b Thus, reduc ing sample loss and ncreas ing the specificity of phosphopeptides are still of importance to high-throughput phosphoproteome analysis. For the purpose of achieving convenient and rapid isolation of adsorbents from sample solutions, magnetic particles modified with metal oxides (titania,11 zirconia,12 alumina13 Received: January 28, 2013 Revised: May 16, 2013 Accepted: May 18, 2013

A

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and gallium oxide14) have been successfully applied as affinity probes for the enrichment of phosphopeptides from tryptic digests by magnetic isolation. Some of these magnetic particles could be directly spotted on the matrix-assisted laser desorption/ionization (MALDI) plate so as to avoid elution steps and reduce sample loss. However, magnetic particles coated with metal oxides not only suffer from nonspecific binding with acidic peptides but also lack the biocompatibility and spacer arms that can reduce the steric hindrance for binding phosphopeptides efficiently. Zirconium-phosphate and zirconium arsenate titanium−NTA-modified Fe 3 O 4/SiO2 (core/shell) nanoparticles were developed for the selective enrichment of phosphopeptides. Yet, the nonspecific adsorption of nonphosphorylated peptides on the Fe3O4/SiO2 core/ shell nanoparticles is still a challenge owing to the presence of silanol groups in the SiO2 shell.15 Magnetic particles directly coated with polymer have been developed for phosphopeptide enrichment; however, they still suffer from the disadvantage of nonspecific adsorption.16 Although poly(ethylene glycol) methacrylate-brush-decorated Fe3O4@SiO2 exhibited great specificity for phosphopeptide enrichment, the procedures for the required atom-transfer radical polymerization were severe and tedious.17 Herein, zirconium-phosphate (Zr4+-PO3)-modified magnetic Fe3O4/poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) (core/shell) microspheres (designated as Zr4+-Fe3O4@polymer microspheres) were prepared in a facile manner for the specific capture of phosphopeptides, by procedures including coating the Fe3O4/SiO2 core with a poly(GMA-co-EDMA) shell, converting the epoxy groups into phosphonate groups, and loading zirconium ions. The prepared Zr4+-Fe3O4@polymer microspheres were then applied in the analysis of phosphopeptides of tryptic digests of α-casein, βcasein, and other nonphosphorylated proteins, demonstrating the high efficiency, good specificity, operational convenience, and high tolerance to salts and denaturants of this material. These novel Zr4+-Fe3O4@polymer microspheres were further examined by the phosphoproteome analysis of mouse liver lysate.

Fe3O4/GMA-co-EDMA (core/shell) microspheres was then carried out. Briefly, the obtained Fe3O4@polymer microspheres were first aminated with 0.5 M 1,6-diaminohexane solution at 70 °C for 6 h. After being dried in a vacuum, the aminated microspheres were incubated in an anhydrous acetonitrile solution (50 mL) containing 40 mM POCl3 and 40 mM 2,4,6collidine for 12 h at ambient temperature and then rinsed with ACN followed by water. The resulting phosphonate-modified Fe3O4@polymer microspheres were incubated in a 50 mM ZrOCl2 solution with gentle stirring overnight for the loading of Zr4+ cations. Finally, the prepared zirconium phosphonatemodified Fe3O4@polymer microspheres (designated as Zr4+Fe3O4@polymer microspheres) were rinsed with deionized water to remove the nonspecifically adsorbed Zr4+ cations and dried under a vacuum at 30 °C for 12 h. 2.3. Tryptic Digestion of Proteins. Separately, α- and βcasein (1 mg) dissolved in 1 mL of NH4HCO3 buffer solution (50 mM, pH 8.2) were digested by trypsin at an enzyme-tosubstrate ratio of 1:40 (w/w) at 37 °C for 16 h. BSA was first dissolved in 1 mL of 50 mM NH4HCO3 buffer solution containing 8 M urea. After the addition of 20 μL of 50 mM DTT, the solution was incubated at 60 °C for 40 min to reduce the disulfide bonds of BSA; then, 40 μL of IAA (50 mM) was added, and the mixture was incubated in the dark at room temperature for an additional 30 min. Finally, the solution was diluted 10-fold with 50 mM NH4HCO3 for tryptic digestion at 37 °C for 16 h at an enzyme-to-substrate ratio of 1:40 (w/w). Mouse liver proteins were obtained according to the procedure described in the literature.18 The concentration of extracted mouse liver proteins was determined by the Bradford protein assay. The tryptic digestion of the obtained mouse liver proteins was the same as that used for BSA. All of the prepared protein digests were stored at −20 °C before further mass spectrometry analysis. 2.4. Capture of Phosphopeptides by Zr4+-Fe3O4@ Polymer Microspheres. Protein digests were diluted with loading buffer (pH 3.47) containing 10.0% (v/v) HAc and 50% (v/v) ACN. For a typical phosphopeptide enrichment procedure, a protein digest solution of β-casein (250 fmol/ μL, 1 μL) was added to 2.5 μL of a suspension of Zr4+-Fe3O4@ polymer microspheres (15 mg/mL) in loading buffer, and the mixture was then incubated at room temperature for 30 min. After that, the magnetic nanoparticles were held by a magnet for removal of the supernatant by Eppendorf pipet and subsequently rinsed sequentially with 20 μL of loading buffer solution containing 200 mM NaCl, 20 μL of loading buffer solution, and 20 μL of an aqueous solution containing 0.1% (v/ v) HAc and 30% (v/v) ACN. After solvent evaporation, 1 μL of MALDI matrix (25 mg/mL of 2,5-DHB solution containing 70% ACN and 1% H 3 PO 4 ) was added and mixed homogeneously with the dried microspheres. Finally, a 0.5 μL aliquot of the matrix/microsphere suspension was deposited on a target for matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) analysis. For phosphoproteome analysis of mouse liver lysate, similar procedures were carried out. Specifically, the magnetic microspheres (300 μL, 15 mg/mL) were incubated with the tryptic digest of 200 μg of mouse liver lysate and then rinsed sequentially with 400 μL of solutions containing 0.5% (v/v) HAc and 50% (v/v) ACN with and without 200 mM NaCl, followed by loading buffer containing 10.0% (v/v) HAc and 50% (v/v) ACN and a solution of 0.1% HAc (v/v) and 30% (v/v) ACN. Afterward, the phosphopeptides trapped by the

2. EXPERIMENTAL SECTION 2.1. Materials and Reagents. α- and β-caseins, trypsin from porcine pancreas, bovine serum albumin (BSA), 2,5dihydroxybenzoic acid (2,5-DHB), and zirconium oxychloride (ZrOCl2·8H2O) were purchased from Sigma-Aldrich (St. Louis, MO). Urea, ammonium bicarbonate, dithiothreitol (DTT), and iodoacetamide (IAA) were purchased from BioRad (Hercules, CA). 2,4,6-Collidine, ammonium hydroxide, phosphorus oxychloride (POCl3), and 1,6-diaminohexane were obtained from Tianjin chemical plant (Tianjin, China). Phosphorous acid was analytically pure. Acetonitrile (ACN) and trifluoroacetic acid (TFA) were purchased from Merck (Darmstadt, Germany). Commercial Fe3+-IMAC beads (POROS 20 MC beads) were activated according to the instructions of the manufacturer, Applied Biosystems Co. Ltd. (Foster, CA). Water was purified by a Milli-Q system (Millipore, Milford, MA). Adult female C57 mice were purchased from Dalian Medical University (Dalian, China). 2.2. Preparation of Zr4+−PO3-Modified Magnetic Fe3O4/GMA-co-EDMA (Core/Shell) Microspheres. The details of the synthesis of magnetic Fe3O4/GMA-co-EDMA (core/shell) microspheres are described in the Supporting Information. The functionalization of the prepared magnetic B

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the criteria were set as follows: cross-correlation value (Xcorr) ≥ 2.0, 2.5, and 3.8 for single, double, and triple charges, respectively; ΔCn ≥ 0.1;21 and false discovery rate (FDR) < 5.0%. For the identification of phosphopeptides by matching the assigned sequences derived from MS2 and MS3 data, the inhouse-developed software called APIVASE (Automatic Phosphopeptide Identification Validation for SEQUEST)22 was used to validate the identification. With this software, the MS2/MS3 target-decoy database search approach ( also called the MS2/ MS3 approach) was applied to identify the most likely phosphorylation sites by following four steps: (i) extracting the valid MS2/MS3 pairs by remov ing the MS2/MS3 pairs with incorrect charge states, with MS3 not triggered, and with the intensity of the peak corresponding to neutral loss less than 50% that of the base peak in MS2; (ii) performing MS2 and MS3 target-decoy database searches with the valid MS2 and MS3 spectra, respectively; (iii) reassigning the peptide scores in the SEQUEST output to generate the peptide lists with the reassigned scores for MS2 and MS3; and (iv) filtering the candidate phosphopeptides with new defined parameters (Rank′m, ΔCn′m, and Xcorr′s) to achieve phosphopeptide identification with specific FDR. In this study, to achieve a false discovery rate (FDR) of 1%, the following filter criteria were used: Rank′m = 1, ΔCn′m ≥ 0.1, and Xcorr′s ≥ 0.60. The phosphoproteins identified by the same phosphopeptide(s) were grouped; if the group contained more than one phosphoprotein, then only one was kept according to the method described by He et al.,23 as all proteins in each group are highly homologous, generally belonging to the same superfamily or just different alternative splicing isoforms. Finally, to investigate the reliability of the peptide identification, PhosphoSitePlus (http://www.phosphosite.org) was used to distinguish known from novel phosphorylation sites.

microspheres were eluted with NH4OH solution (pH 11.5, 200 μL) and then subjected to 15 min of sonication. By holding the magnetic microspheres with a magnet, we collected the eluted phosphopeptides and then lyophilized them and redissolved them in 4 μL of 0.1% formic acid for nano-liquid chromatography−electrospray ionization−tandem mass spectrometry (nano-LC-ESI-MS/MS) and MS/MS/MS (MS3) analysis. Enrichment of phosphopeptides using commercial Fe3+-IMAC beads was performed according to the optimized protocols recently reported by Lee et al.19 2.5. Mass Spectrometry. All MALDI-TOF mass spectra were acquired in linear positive-ion mode on a Bruker Autoflex time-of-flight mass spectrometer (Bruker, Bremen, Germany) equipped with a delayed ion-extraction device and a 337-nm pulsed nitrogen laser at an accumulation of 30 laser shots. The delay time for ion extraction and the extraction voltage were set at 90 ns and 20 kV, respectively. All nano-LC-ESI-MS/MS analyses were performed on a Thermo Finnigan LTQ linear ion-trap mass spectrometer with a nanospray ion source and a Surveyor high-performance liquid chromatography (HPLC) system (Thermo Finnigan, San Jose, CA). The flow rate of the HPLC pump was split to 200 nL/min by a cross for capillary separation. A bare fused silica capillary (75 μm i.d. × 120 mm length) was manually pulled to form a MS emitter tip (ca. 5 μm i.d.) at one end, which was then packed in house with C18 AQ particles (5 μm, 120 Å, Michrom BioResources, Auburn, CA) using a pneumatic packing device. The packed capillary column was directly coupled to the LTQ mass spectrometer. Water containing 0.1% formic acid was used as mobile phase A, and acetonitrile containing 0.1% formic acid was used as mobile phase B. The LTQ mass spectrometer was operated in positive-ion mode. A voltage of 1.8 kV was applied to the cross. About 1 μL (50 μg) of redissolved peptides was loaded onto a C18 capillary column as the sample loop and separated on the capillary column under a linear gradient with mobile phase B ramped from 5% to 35% in 120 min. For the detection of phosphopeptides, a full MS scan along with data-dependent MS/MS (MS2) acquisition for the three most intense ions was carried out, and an MS3 analysis was automatically triggered when the three most intense peaks from the MS2 spectrum corresponded to the neutral loss event of 98, 49, and 33 ± 1 Da for the precursor ion with 1+, 2+, and 3+ charge states, respectively. 2.6. Database Search and Data Analysis. The peak lists for MS2 and MS3 spectra were generated from raw data obtained from BioWorks (Thermo Electron Corporation, Madison, WI) using the following parameters: mass range, 600−3500 Da; intensity threshold, 1000; minimum ion count, 10. The MS2 and MS3 spectra were searched using SEQUEST20 (version 2.7) against a composite database including both original database and the reversed version of the forward data with the following parameters: enzyme, trypsin (KR/P); enzyme limits, fully enzymatic (cleaves at both ends); precursor-ion mass tolerance, 2 Da; fragment-ion mass tolerance, 1 Da; missed cleavages, 2; static modification, Cys (+57). For searches with MS2 data, the dynamic modifications were set for oxidized Met (+16) and phosphorylated Ser, Thr, and Tyr (+80). For searches with MS3 data, in addition to the parameters for MS2, dynamic modifications were also set for water loss on Ser and Thr (−18). The original database is the nonredundant mouse protein database of the International Protein Index (ipi.MOUSE.3.17.fasta), which includes 51446 entries. For the identification of nonphosphopeptides by MS2,

3. RESULTS AND DISCUSSION 3.1. Preparation and Evaluation of Zr4+-Fe3O4@ Polymer Microspheres. Magnetic particles coated with a polymeric shell of poly(GMA-co-EDMA) were synthesized according to the reported procedures.24 The synthesis details are presented in the Supporting Information. The epoxy groups on the poly(GMA-co-EDMA) shell were subsequently converted into amine groups and phosphate groups by reaction with 1,6-diaminohexane and POCl3, respectively. Afterward, zirconium cations (Zr4+) were immobilized on the resultant phosphate groups of the Fe3O4@polymer (core/shell) microspheres to form Zr4+-Fe3O4@polymer microspheres for the selective capture of phosphopeptides through the specific affinity between the immobilized Zr4+ ions and the phosphoryl groups of the phosphopeptides. The transmission electron microscopy (TEM) image in Figure S1a (Supporting Information) shows that the Fe3O4 magnetic particles were nearly spherical in shape and had a mean diameter of 240 nm. After further coating with a poly(GMA-co-EDMA) shell and modification with zirconium phosphonate groups, the obtained Zr4+-Fe3O4@polymer microspheres (2.5−17.5 μm) were much larger in diameter than the Fe3O4 magnetic particles, as characterized by scanning electron microscopy (SEM) (Figure S1b, Supporting Information), indicating that the magnetic particles had been successfully coated with a thick shell. For the enrichment of phosphopeptides, the main merit of Zr4+Fe3O4@polymer microspheres is that the thick polymer shell of the microspheres not only can protect the Fe3O4 magnetic particles from leaching in acidic media but also have a strong C

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decontaminating capability because of the weak interaction between the poly(GMA-co-EDMA) shell and contaminants, such as inorganic salts and acidic peptides. Therefore, phosphopeptides can be easily and selectively enriched from highly contaminated sample solutions by Zr4+-Fe3O4@polymer microspheres. The second merit is that the epoxy groups on the poly(GMA-co-EDMA) shell can be conveniently converted to amine groups by reaction with 1,6-diaminohexane, which endows the Zr4+-Fe3O4@polymer microspheres with a long spacer arm between the magnetic polymer support and the Zr4+ ions, providing the Zr4+ ions on the Fe3O4@polymer microspheres with a better chance to interact with the phosphate groups of phosphopeptides. The third merit is that the Zr4+-Fe3O4@polymer microspheres exhibit superparamagnetic properties as well as high magnetization, which enables a rapid response to an applied magnet, facilitating their manipulation in enrichment procedures without centrifugation. To evaluate the effectiveness of the prepared Zr4+-Fe3O4@ polymer microspheres in the selective capture of phosphopeptides, two commonly used standard phosphoproteins, namely, α- and β-casein, were employed as model proteins. The detailed procedure for phosphopeptide enrichment with Zr4+-Fe3O4@ polymer microspheres is shown in Scheme 1. Here, two aspects Scheme 1. Schematic Diagram of the Selective and Rapid Enrichment of Phosphopeptides Using Zr4+-Fe3O4@Polymer Microspheres

Figure 1. MALDI-TOF-MS analysis of tryptic digests of (a) 50 fmol of lyophilized β-casein (250 amol/μL, 200 μL), (b) 50 fmol of β-casein (250 amol, 200 μL) treated with Zr4+-Fe3O4@polymer microspheres and eluted with NH3·H2O, (c) 50 fmol of β-casein (250 amol/μL, 200 μL) treated with Zr4+-Fe3O4@polymer microspheres and directly spotted on a MALDI plate, and (d) 10 fmol of β-casein (100 amol/μL, 100 μL) treated with Zr4+-Fe3O4@polymer microspheres and directly spotted on a MALDI plate. The numbers in parentheses represent the S/N ratios of the peaks.

for a standard phosphoprotein with a minute phosphorylation site because of the interference of nonphosphopeptides generated from the tryptic digestion of the protein. Therefore, the molecular prefiltration of phosphopeptides from the tryptic digest is required prior to mass spectrometry analysis. Next, the same amount of tryptic digest of β-casein (50 fmol) was first treated with Zr4+-Fe3O4@polymer microspheres, eluted with ammonium hydroxide (NH3·H2O), lyophilized as part of the collected eluate, and finally subjected to MALDI-TOF-MS analysis (Figure 1b). Comparing panels a and b of Figure 1, one can see that the overwhelming nonphosphopeptides resulting from the tryptic digestion of β-casein in Figure 1a are absent from Figure 1b after treatment with Zr4+-Fe3O4@polymer microspheres and only the enhanced phosphopeptide peak of β1 remains. This indicates that the phosphopeptide was selectively captured by Zr4+-Fe3O4@polymer microspheres depending on the specific affinity interaction between the

should be mentioned: First, adjustment of the pH of the loading buffer is essential to the high selectivity and good recovery of the phosphopeptides.9b Second, the addition of H3PO4 to the matrix solution is for the enhancement of the phosphopeptide signals in the MALDI-TOF MS analysis.25 The list of identified phosphorylated peptides is provided in Table S1 (Supporting Information). Figure 1 presents a typical analysis of the tryptic digest of β-casein by MALDI-TOF-MS with and without the use of Zr4+-Fe3O4@polymer microspheres. Figure 1a is the direct MALDI-TOF-MS analysis of the tryptic digest of 50 fmol of β-casein (250 amol/μL, 200 μL), which was lyophilized and reconstituted with 1 μL of 2,5-DHB matrix. As can be seen from Figure 1a, only one phosphopeptide (β1) was detected, with a weak signal intensity at m/z 2061.94, that was almost overwhelmed by other peaks for nonphosphopeptides in this spectrum. This spectrum suggests that it is difficult to analyze phosphopeptides even D

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immobilized Zr 4+ and the phosphoryl groups of the phosphopeptide. To avoid the probable sample loss during ammonium hydroxide elution, the elution procedure was replaced by the on-plate MALDI-TOF-MS analysis of phosphopeptides bound to Zr4+-Fe3O4@polymer microspheres. Figure 1c presents the resulting MALDI-TOF-MS spectrum of a tryptic digest of β-casein (50 fmol) with the direct spotting of phosphopeptides captured by Zr4+-Fe3O4@polymer microspheres on the MALDI plate, where four phosphopeptide peaks labeled β1, β2, β3, and β32+ at m/z 2061.94, 2556.93, 3122.56, and 1561.24 Da can be clearly observed. The enhancement of the signal intensities and signal-to-noise (S/ N) ratios of these phosphopeptide peaks can most likely be attributed to the avoidance of sample loss during elution steps. Even upon decreasing the amount of β-casein to 10 fmol (100 amol/μL, 100 μL), the two phosphopeptides β1 and β3 were still detected with signal-to-noise ratios of 6.8 and 9.4, respectively (Figure 1d). 3.2. Incubation Conditions for Zr4+-Fe3O4@Polymer Microspheres. To evaluate the adsorption capacity of Zr4+Fe3O4@polymer microspheres for protein, 1 μL aliquots of tryptic digests of β-casein varying in concentration from 50 to 4000 fmol/μL were treated with the same amount of Zr4+Fe3O4@polymer microspheres (37.5 μg) and then analyzed by MALDI-TOF-MS. Figure 2 presents a plot of the average peak

Figure 3. Effect of incubation time on the peak intensity (averaged, n = 6) of phosphopeptides of β-casein (250 fmol, 1 μL) captured by Zr4+-Fe3O4@polymer microspheres.

the four phosphopeptides increased as the incubation time increased to 30 min and then remained constant after 30 min. This indicates that the chelating interaction between the zirconium cations of the Zr4+-Fe3O4@polymer microspheres and the phosphoryl groups of the phosphopeptides achieved equilibrium. In other words, an incubation time of 30 min was found to be necessary to obtain reliable and better results in the analysis of phosphopeptides by MALDI-TOF-MS. It is well-known that salts and other contaminants can severely interfere with the detection of phosphopeptides by MALDI-TOF-MS because of the disruption of sample cocrystallization on the MALDI plate,26 resulting in mass spectra of poor quality. Therefore, desalting steps are usually required for sample preparation before MALDI-TOF-MS analysis. In this work, tryptic digests of β-casein (2.50 fmol/ μL, 100 μL) separately containing the highest (saturated) salt concentrations of 6.2 M for NaCl and 8 M for urea were utilized as sample solutions for the investigation of whether the presence of contaminants in the sample would severely disturb the results of the MALDI-TOF-MS analysis using Zr4+-Fe3O4@ polymer microspheres as the matrix assistance material for the enrichment of phosphopeptides. In addition, the commercial Fe3+-IMAC method was used for comparison. Figure S2 (Supporting Information) displays the MALDI-TOF mass spectra obtained in this comparative study, with the peaks of the phosphopeptides labeled with the corresponding S/N ratios. It is notable that all of the phosphopetides were detected with much higher S/N ratios and intensities after enrichment with Zr4+-Fe3O4@polymer microspheres compared to commercial Fe3+-IMAC beads in the presence of 6.2 M NaCl or 8 M urea. In addition, in comparison with the sample solution without salt treated with Zr4+-Fe3O4@polymer microspheres, the values of S/N ratio for the phosphopeptide decreased by only about a factor of 2.5 or 3 for the highest concentrations of 6.2 M for NaCl or 8 M for urea, respectively. In contrast, the enrichment of phosphopeptides with the commercial Fe3+IMAC beads was more strongly affected by salt: Specifically, compared with the sample solution without salt treated with commercial Fe3+-IMAC beads, the values of the S/N ratio for the phosphopeptide decreased by about a factor of 7 in the presence of 6.2 M NaCl and 8 in the presence of 8 M urea. This expected decrease of the peak intensity is due to the strong chelation interaction between the zirconium cations and the phosphoryl groups of the phosphopeptides, as well as the weak interaction between the biocompatible polymer shell and the contaminants. These results show that Zr4+-Fe3O4@polymer microspheres have a high salt tolerance and can be used to efficiently enrich phosphopeptides at trace levels without a

Figure 2. Effect of the ratio of protein to microspheres [fmol (μg/ μL)−1] on the signal intensity (averaged, n = 6) of phosphopeptides from the tryptic digest of β-casein treated with Zr4+-Fe3O4@polymer microspheres.

intensities of the four isolated phosphopertides (β1, β2, β3, β32+) as a function of the protein/microsphere ratio (femtomoles of protein per microgram of microspheres). As can be seen in Figure 2, the MS intensities of the four phosphopeptides all increased as the protein/microsphere ratio was increased from 1.33 to 53.33; however, after the protein/microsphere ratio was increased above 53.33, the MS intensities of the four phosphopeptides remained approximately constant, indicating that 37.5 μg of Zr4+-Fe3O4@polymer microspheres showed saturated adsorption toward 2000 fmol of tryptic digests of βcasein, so the optimum ratio of protein to microspheres was determined to be 53.3. To obtain better MALDI-TOF-MS results, the effects of the incubation time of the Zr4+-Fe3O4@polymer microspheres with tryptic digest of protein, the ratio of the amount of the protein to the amount of Zr4+-Fe3O4@polymer microspheres, and the salt concentration of the protein sample were all investigated. Figure 3 displays the relationship between the peak intensities of phosphopeptides β1, β2, β3, and β32+ and the incubation time of the Zr4+-Fe3O4@polymer microspheres with the tryptic digest of β-casein. It can be seen that the MS peak intensities of E

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nonphosphprotein (BSA) at a molar ratio of 1:500.9c Similar results were also observed in other previous works.5,27 For Zr4+PO3-modified Fe3O4/SiO2, it was reported that phosphopeptides could be enriched when the ratio of phosphoprotein (αcasein) to nonphosphoprotein (BSA) was no lower than 1:100.15a Moreover, Li et al. reported that Fe3O4@ZrO2, Fe3O4@Ga2O3, and Fe3O4@Al2O3 microspheres could be used to enrich phosphopeptides only when the ratio of phosphoprotein (β-casein) to nonphosphoprotein (BSA) was a minimum of 1:50.12a,13c,14 These results indicate that the ability of Zr4+-Fe3O 4@polymer microspheres to enrich phosphopeptides is much greater than those of the other materials (i.e., Fe3O4@ZrO2, Fe3O4@Ga2O3, Fe3O4@Al2O3, IMAC-Fe3+, and Zr4+-PO3-modified Fe3O4/SiO2). The different performances among the mentioned materials might be due to the fact that the polymer shell used in this work (GMA-coEDMA) does not have strong Lewis acid sites as do metal oxides (ZrO2, Ga2O3, Al2O3) and is more biocompatible than s silanol shell for less nonspecific adsorption. Considering that the molar level of α-casein was 2 orders of magnitude lower than that of BSA in the sample tested in this work, the specificity of Zr4+-Fe3O4@polymer microspheres can be good enough for the selective capture of phosphopeptides from very complex samples. 3.4. Application of Zr4+-Fe3O4@Polymer Microspheres for Phosphoproteome Analysis of Mouse Liver. To further evaluate their performance in the selective capture of phosphopeptides in real complex biological samples, Zr4+Fe3O4@polymer microspheres were also applied to analyze the phosphoproteome of mouse liver lysate. After the phosphopeptides from 200 μg of mouse liver lysate digest had been enriched with Zr4+-Fe3O4@polymer microspheres, only onequarter of the sample was loaded onto a capillary C18 column and analyzed by LTQ mass spectrometer. To objectively demonstrate the performance of phosphopeptide enrichment by Zr4+-Fe3O4@polymer microspheres, an automatic phosphopeptide validation approach (MS2/MS3 target-decoy approach) was applied to strictly confirm the identification of the phosphopeptides and phosphopeptide sites without any manual validation stage. As a result, a total of 314 unique peptides including 106 nonphosphorylated peptides and 208 phosphorylated peptides were successfully identified by this novel approach. The specificity (ratio of the number of identified phosphopeptides to the number of all identified peptides) was 66.0%. The majority of these phosphopeptides had very high Xcorr scores. Among the enriched phosphopeptides, 37.5% contained a singly phosphorylated site, whereas 45.2% and 17.3% had two and three phophorylated sites, respectively. Finally, to investigate the reliability of the results, PhosphoSitePlus (http://www.phosphosite.org) was further used to distinguish the known from novel phosphorylation sites for the mouse liver lysate. Of the 208 identified phosphorylated peptides with 337 phosphorylated sites, it was found that 94.06% (317 sites) were identified as known phosphorylation sites (Table S2, Supporting Information). These results indicate that the phosphopeptides identified by both MS2 and MS3 spectra were of high confidence. They also confirm that the Zr4+-Fe3O4@polymer microspheres do have a high specificity for the capture of phosphopeptides in real complex biological samples and that they are very promising for use in the purification of phosphopeptides for phosphoproteome analysis.

desalting step, even when the samples contain abundant contaminants. 3.3. Purification of Phosphopeptides from Semicomplex Tryptic Digests by Zr4+-Fe3O4@Polymer Microspheres. To evaluate the performance of Zr4+-Fe3O4@polymer microspheres in the selective capture of phosphopeptides from an even more complex protein sample, mixtures of the standard proteins α-casein and BSA were used. Figure 4a displays the

Figure 4. MALDI-TOF mass spectra of tryptic digests of protein mixtures of α-casein (1 pmol, 2 μL) and BSA with molar ratio of 1:1: (a) by direct analysis and (b,c) by enrichment treatment of Zr4+Fe3O4@polymer microspheres with molar ratios of (b) 1:1 and (c) 1:500.

obtained MALDI mass spectra of the tryptic digest of the mixture with an α-casein/BSA molar ratio of 1:1. It can be seen that only seven weak phosphopeptide peaks could be detected without treatment using Zr4+-Fe3O4@polymer microspheres, whereas the remaining strong peaks were replaced by nonphosphopeptides in this mass spectrum. That is, nonphosphorylated peptides derived from the protein mixture interfered badly with the detection of phosphopeptides from the tryptic digest of the mixture of α-casein and BSA. However, after enrichment using Zr4+-Fe3O4@polymer microspheres, as shown in Figure 4b, 16 phosphopeptides including singly and multiply phosphorylated peptides were clearly detected. When the molar ratio of α-casein to BSA was further decreased to 1:500, 15 phosphopeptides could still be detected with a similar peak pattern of phosphopeptides, and the peak of only one phosphopeptide located at m/z 1238.35 is absent from Figure 4c. The identified phosphopeptides from α-casein, including amino acid sequences and phosphorylation sites, are listed in Table S1 (Supporting Information). In a previous report, it was found that the conventional Fe3+-IMAC method lacked sufficient specificity and cocaptured many nonphosphopeptides in the digest mixture of a phosphoprotein (α-casein) to a F

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etry. Mol. Cell Proteomics 2003, 2 (11), 1234−1243. (b) Pan, C.; Ye, M.; Liu, Y.; Feng, S.; Jiang, X.; Han, G.; Zhu, J.; Zou, H. Enrichment of phosphopeptides by Fe3+-immobilized mesoporous nanoparticles of MCM-41 for MALDI and nano-LC-MS/MS analysis. J. Proteome Res. 2006, 5 (11), 3114−3124. (c) Posewitz, M. C.; Tempst, P. Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal. Chem. 1999, 71 (14), 2883−2892. (d) Raska, C. S.; Parker, C. E.; Dominski, Z.; Marzluff, W. F.; Glish, G. L.; Pope, R. M.; Borchers, C. H. Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads. Anal. Chem. 2002, 74 (14), 3429−3433. (e) Ueda, E. K.; Gout, P. W.; Morganti, L. Current and prospective applications of metal ion−protein binding. J. Chromatogr. A 2003, 988 (1), 1−23. (4) (a) Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E.; Bai, D. L.; Shabanowitz, J.; Burke, D. J.; Troyanskaya, O. G.; Hunt, D. F. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (7), 2193−2198. (b) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 2002, 20 (3), 301−305. (5) (a) Bodenmiller, B.; Mueller, L. N.; Mueller, M.; Domon, B.; Aebersold, R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat. Methods 2007, 4 (3), 231−237. (b) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell Proteomics 2005, 4 (7), 873−886. (6) (a) Kweon, H. K.; Hakansson, K. Selective zirconium dioxidebased enrichment of phosphorylated peptides for mass spectrometric analysis. Anal. Chem. 2006, 78 (6), 1743−1749. (b) Zhou, H.; Tian, R.; Ye, M.; Xu, S.; Feng, S.; Pan, C.; Jiang, X.; Li, X.; Zou, H. Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis. Electrophoresis 2007, 28 (13), 2201−2215. (7) (a) Sugiyama, N.; Masuda, T.; Shinoda, K.; Nakamura, A.; Tomita, M.; Ishihama, Y. Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/ MS in proteomics applications. Mol. Cell Proteomics 2007, 6 (6), 1103−1109. (b) Lu, J.; Liu, S.; Deng, C. Facile synthesis of alumina hollow spheres for on-plate-selective enrichment of phosphopeptides. Chem. Commun. 2011, 47 (18), 5334−5336. (8) Ficarro, S. B.; Parikh, J. R.; Blank, N. C.; Marto, J. A. Niobium(V) oxide (Nb2O5): Application to phosphoproteomics. Anal. Chem. 2008, 80 (12), 4606−4613. (9) (a) Ndassa, Y. M.; Orsi, C.; Marto, J. A.; Chen, S.; Ross, M. M. Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications. J. Proteome Res. 2006, 5 (10), 2789− 2799. (b) Tsai, C. F.; Wang, Y. T.; Chen, Y. R.; Lai, C. Y.; Lin, P. Y.; Pan, K. T.; Chen, J. Y.; Khoo, K. H.; Chen, Y. J. Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics. J. Proteome Res. 2008, 7 (9), 4058−4069. (c) Zhou, H.; Ye, M.; Dong, J.; Han, G.; Jiang, X.; Wu, R.; Zou, H. Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis. J. Proteome Res. 2008, 7 (9), 3957−3967. (10) (a) Kuroda, I.; Shintani, Y.; Motokawa, M.; Abe, S.; Furuno, M. Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn. Anal. Sci. 2004, 20 (9), 1313−1319. (b) Mazanek, M.; Roitinger, E.; Hudecz, O.; Hutchins, J. R. A.; Hegemann, B.; Mitulovic, G.; Taus, T.; Stingl, C.; Peters, J.-M.; Mechtler, K. A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes. J. Chromatogr. B 2010, 878 (5−6), 515−524. (11) (a) Chen, C. T.; Chen, Y. C. Fe3O4/TiO2 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using TiO2 surface-assisted laser desorption/ionization mass spectrometry. Anal. Chem. 2005, 77 (18), 5912−5919. (b) Li, Y.; Xu, X.;

4. CONCLUSIONS A novel material involving zirconium phosphate- (Zr4+-PO3-) modified magnetic Fe3O4/GMA-co-EDMA (core/shell) (Zr4+Fe3O4@polymer) microspheres has been shown to selectively bind and enrich phosphopeptides from complex protein tryptic digest mixtures. The present work demonstrates the convenience, simplicity, and specificity of this material for the enrichment of phosphopeptides. The microspheres have a high tolerance for salts (urea, NaCl) and nonphosphopeptides (tryptic solution from BSA). The feasibility of Zr4+-Fe3O4@ polymer microspheres was further illustrated by analysis of mouse liver lysate. All of the obtained results indicate that Zr4+Fe3O4@polymer microspheres have a quite high selectivity and sensitivity for the enrichment of phosphopeptides and the potential to be developed for many applications in the rapidly growing field of phosphoproteomics.

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ASSOCIATED CONTENT

S Supporting Information *

Procedure for the synthesis of magnetic Fe3O4/GMA-coEDMA (core/shell) microspheres; TEM image of magnetic particles (Fe3O4); SEM image of Zr4+-Fe3O4@polymer microspheres; MALDI-TOF-MS analysis of tryptic digests of β-casein (2.5 fmol/μL, 100 μL) in the presence of no salt, saturated NaCl (6.2 M), and saturated urea (8 M) treated with Zr4+Fe3O4@polymer microspheres or commercial Fe3+−IMAC; identification of phosphopeptides from α-casein and β-casein tryptic digests using Zr4+-Fe3O4@polymer microspheres as affinity probes in MALDI-TOF MS analysis; and data on the identified unique phosphopeptides from mouse liver lysate. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-575-88342592. Fax: +86-575-88341521. E-mail: huliujiang @163.com (L.H.), [email protected] (H.D.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Zhejiang Province, China (LY12B06003), and the Science and Technology Program of Shaoxing (2011A21056).

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REFERENCES

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