Cerium Ion-Chelated Magnetic Silica Microspheres for Enrichment

Feb 29, 2008 - With the optimized enrichment conditions, the performance of the Ce4+-chelated magnetic microspheres was compared with the Fe3+-chelate...
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Cerium Ion-Chelated Magnetic Silica Microspheres for Enrichment and Direct Determination of Phosphopeptides by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Yan Li, Dawei Qi, Chunhui Deng,* Pengyuan Yang, and Xiangmin Zhang* Department of Chemistry & Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China Received June 21, 2007

Abstract: In this study, we employed, for the first time, the Ce4+-chelated magnetic silica microspheres to selectively concentrate phosphopeptides from protein digest products. Cerium ions were chelated onto magnetic silica microspheres using the strategy we established before. After enrichment, the phosphopeptide-conjugated magnetic microspheres were separated from the sample solution just by using a magnet. With the optimized enrichment conditions, the performance of the Ce4+chelated magnetic microspheres was compared with the Fe3+-chelated microspheres using tryptic digested peptides originating from ovalbumin, a five protein mixture containing phosphoproteins and nonphosphoproteins, as well as a mixture of β-casein and BSA with a molar ratio of 1:50. Compared to Fe3+, Ce4+-chelated magnetic microspheres exhibited more selective isolation ability for concentrating phosphopeptides from complex mixtures. Even when the amount of the tryptic digest product of BSA is 50 times higher than that of β-casein in the sample solution, the trace phosphopeptides derived from β-casein can still be concentrated effectively by the Ce4+-chelated magnetic microspheres in only 30 s. Furthermore, we initially utilized the Ce4+-chelated magnetic microspheres to directly enrich phosphopeptides from human serum without extra purification steps or tedious treatment, which opens up a possibility for their further application in phosphoproteomics. Keywords: cerium ion • magnetic microspheres • phosphorylation • blood serum • MALDI MS

Introduction In recent years, new mass spectrometric techniques and instrumentation have revolutionized our ability to analyze the cellular proteome on a large scale and to permit a global analysis of reversible protein phosphorylation which plays a key role in regulating most aspects of cellular processes including cell growth, division, and differentiation, cytoskeleton dynamics, signal transduction, gene expression, metabolism, and memory.1–4 Proteins are commonly phosphorylated at specific serine or threonine residues and, less commonly, at * Corresponding authors. E-mail: [email protected]; xmzhang@ fudan.edu.cn. Tel.: +86-21-6564-3983. Fax: +86-21-6564-1740. 10.1021/pr070385l CCC: $40.75

 2008 American Chemical Society

tyrosine as well as other amino acid residues.5 Although it has been estimated that approximately one-third of mammalian proteins contain covalently bound phosphate and many are subject to regulation by multisite phosphorylation,6,7 the abundance of the individual phosphorylated forms is frequently low. MALDI and electrospray MS have been established as powerful tools for investigation of protein phosphorylation.8,9 MS is a sensitive and specific analytical method for phosphoprotein characterization because covalent modification of polypeptides by phosphate is measured by an increase in protein or peptide molecular mass. Furthermore, phosphate release from phosphorylated molecules can be induced by tandem MS to produce highly specific and diagnostic reporter ion signals. Although mass spectrometry is an attractive technique, analysis of phosphopeptides still presents formidable challenges to the mass spectrometrist for a number of reasons. First, MS analysis of proteolytic digest of proteins rarely provides 100% coverage of the protein sequence, and regions of interest are easily missed. Second, negatively charged modifications can hinder proteolytic digestion by trypsin, the protease of choice in many applications. Third, biochemical analysis is complicated not only by low concentrations of phosphoproteins of interest in cells but also possibly by incomplete phosphorylation at individual sites in a protein. Thus, the mass spectrometric response of a phosphopeptide may be suppressed relative to its unphosphorylated counterpart, and this suppression tends to be enhanced in the presence of other peptides. Therefore, without specific enrichment, only the most abundant phosphoproteins would be identified. Several strategies have been developed to enrich the sample for phosphoproteins or phosphorylated peptides before analysis. At the level of phosphoproteins, mostly tyrosine-phosphorylated proteins have been successfully enriched by immunoprecipitation,10–12 although the approach is feasible in principle with serine and threonine-phosphorylated proteins.13 Enrichment strategies on the level of phosphopeptides have the advantage of identifying both the protein and the phosphorylated residues, which is otherwise a challenging task. Specific capture of phosphopeptides is possible by β-elimination of the phosphate group and subsequent introduction of an affinity tag,14 by covalent capture and release,15 by immobilized metal affinity chromatography (IMAC),16–18 or by metal oxides such as TiO2,19–21 ZrO2,22,23 and Al2O3.24–27 The former two methods have been designed for enhanced specificity but involve complex chemistry and have therefore not been widely used. Journal of Proteome Research 2008, 7, 1767–1777 1767 Published on Web 02/29/2008

technical notes In contrast, the IMAC method is widely used for selective enrichment of phosphopeptides from the mixture since it is fast, easy to use, and economic. In our previous work, we have initially synthesized Fe3+immobilized magnetic silica microspheres with high magnetic responsivity and applied them for enrichment and identification of phosphopeptides from tryptic protein digest through a direct MALDI-TOF MS analysis.28 The magnetic property of the microspheres enables easy isolation by positioning an external magnetic field which made the whole process of enrichment very simple and quick. The resulting phosphopeptides absorbed Fe3+-immobilized magnetic microspheres can directly be analyzed by MALDI-TOF MS analysis without elution from the microspheres. These results opened up new possibilities for the enrichment of phosphopeptides. In IMAC, Fe3+ served as a generally useful chromatographic medium for isolation of phosphorylated peptides, 16–18 and it was reported that Ga3+, Al3+, Sc3+, Lu3+, Th3+, In3+, Ru3+, Y3+, and Zr4+ could retain phosphorylated species as well.29–31 However, to our knowledge, the use of Ce4+ for effective isolation of phosphopeptides has not previously been reported in the literature. Pilot studies were thus initiated to prepare Ce4+-chelated magnetic silica microspheres for selective enrichment of phosphopeptides. With the optimized enrichment conditions, the feasibility of using Ce4+-chelated magnetic microspheres for rapid purification and characterization of phosphopeptides was demonstrated using both tryptic digests of standard proteins and blood serum. Moreover, the binding specificity of Ce4+-chelated magnetic microspheres was compared with Fe3+-chelated magnetic microspheres.

Experimental Section Chemicals and Materials. 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. Preparation of Ce4+-Chelated Magnetic Silica Microspheres. According to the same method for the immobilization of Fe3+ on magnetic silica microspheres,28 Ce4+-chelated magnetic silica microspheres were prepared. At first, magnetic silica microspheres were synthesized and modified via reactions described in a previous work.28 Briefly, Fe3O4 magnetic microspheres were prepared via a solvothermal reaction and then were coated with silica generated from the hydrolysis and condensation of tetraethyl orthosilicate (TEOS). Subsequently, the magnetic silica microspheres obtained were grafted with a silane coupling agent (GLYMO-IDA) derived from reacting 3-glycidoxypropyltrimethoxysilane (GLYMO) with iminodiacetic acid (IDA), which provided chelate sites (carboxylate groups) on the surface of magnetic silica microspheres. For further immobilization of Ce4+ on these obtained microspheres, the product was redispersed in an aqueous solution of Ce(NO3)4 (20 mL, 0.2 M), and the dispersion was stirred for 2 h. Thereafter, the excessive Ce4+ was removed by repeated washing with deionized water and vaccuum-dried overnight at 60 °C, and Ce4+-chelated magnetic silica microspheres were prepared. Sample Preparation. All buffers for peptide loading and washing were aqueous solutions containing 50% ACN, except 1768

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Li et al. that buffer 1 contained 0.1 M H3PO4; buffer 2 contained 0.1 M H3BO4; buffer 3 contained 0.1 M HAC; buffer 4 contained 0.1% TFA; and buffer 4 contained 0.1% TFA and 20 mg/mL of DHB. Bovine β-casein, chicken egg albumin (ovalbumin), myoglobin, cytochrome c, and bovine serum albumin (BSA) were purchased from Sigma Chemical (St. Louis, MO). Each protein was dissolved in 50 mM ammonium bicarbonate, pH 8.0, and treated with trypsin (2%, w/w) for 12 h at 37 °C, respectively. The resulted peptide mixtures were stored at -20 °C until further use. Peptide Mixture 1. Peptide mixture 1 contained peptides originating from tryptic digestion of 50 nM of β-casein and 2.5 µM of BSA which results in a molar ratio of 1:50. Peptide Mixture 2. Peptide mixture 2 contained peptides originating from a tryptic digestion of 20 nM of the phosphorylated proteins (β-casein and ovalbumin) and nonphosphorylated proteins (myoglobin, cytochrome c, and BSA), respectively. A nonfat milk sample was treated according to the procedure reported by Chen et al.23 Briefly, proteins in nonfat milk (0.25 mL) were denatured by incubating the milk with aqueous ammonium bicarbonate solution (0.25 mL, 50 mM) containing urea (8 M) for 30 min at 37 °C. The mixture was then modified by DTT and IAA and was finally incubated with trypsin in aqueous ammonium bicarbonate (50 mM) at 37 °C for 16 h. The digestion product (5 µL) was acidified by diluting in 50% acetonitrile (containing 0.15% TFA). Blood samples from Zhongshan Hospital were collected in 8.5 mL, allowed to clot at room temperature for up to 1 h, and centrifuged at 4 °C for 5 min at 1000 rpm. Sera (upper phase) were aliquoted and stored frozen at -80 °C. Before use, 10 µL of the serum sample was diluted with 20 µL of 50% acetonitrile (0.15% TFA) aqueous solution (v/v), and without any other purification and tedious treatment, the serum sample was ready for enrichment. Enrichment of Phosphopeptides with Metal Ion-Chelated Magnetic Silica Microspheres. A suspension of Ce4+-/Fe3+chelated magnetic silica microspheres (5 µL of 20 mg/mL) was added into 200 µL of peptide mixture originating from tryptic digestions or 30 µL of diluted serum sample, respectively. Then the mixed solutions were vibrated at 37 °C for 30 s. After that, with the help of a magnet, the phosphopeptide-loaded Ce4+-/ Fe3+-chelated magnetic silica microspheres were collected by removal of the supernatant and washed with 50% acetonitrile and 0.15% TFA aqueous solution (v/v) three times. Then the obtained peptide-loaded Ce4+-/Fe3+-chelated magnetic silica microspheres were redispersed in 10 µL of 50% acetonitrile aqueous solution (v/v). MALDI-TOF-MS/MS Process. The above peptide-loaded Ce4+-/Fe3+-chelated magnetic silica microsphere slurry was deposited on the MALDI target using the dried droplet method. An amount of 0.5 µL of the slurry was deposited on the plate, and then another 0.5 µL of a mixture of 20 mg/mL of 2,5-dihydroxybenzoic acid (in 50% acetonitrile aqueous solution, v/v) and 1% (v/v) H3PO4 aqueous solution, 1:1 (v/v), was introduced as a matrix. MALDI-TOF MS experiments were performed in the positive ion mode on a 4700 Proteomics Analyzer (Applied Biosystems, USA) with the Nd:YAG laser at 355 nm, a repetition rate of 200 Hz, and an acceleration voltage of 20 kV. For peptide mass fingerprinting (PMF) data, 800 laser shots were accumulated for each spectrum. The TOF/TOF tandem mass spectra were acquired by the data-dependent acquisition method. Data from the TOF/TOF were searched using Mascot (Matrix Sciences, London, U.K.) as the search

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Figure 1. Effect of various acids on the selective binding of phosphorylated peptides to Ce4+-chelated magnetic microspheres. The peptides used for enrichment were peptide mixture 1 (β-casein:BSA ) 1:50). Shown are MALDI mass spectra obtained without enrichment (a) and from Ce4+-chelated magnetic microsphere enrichment using a loading/washing buffer of 0.1 M H3PO4 (b), 0.1 M H3BO4 (c), 0.1 M HAC (d), 0.1% TFA (e), and 0.1% TFA + 20 mg/mL of DHB (f). All buffers contained 50% ACN. Phosphopeptide ions derived from β-casein and R-casein are marked with asterisks and sharp, respectively. The metastable losses of phosphoric acid are indicated with ∆. The data in parentheses are S/N of the corresponding peaks.

engine. The identifications of proteins from blood serum were preformed against the NCBI database. GPS Explorer (Applied Biosystems) was used for submitting data acquired from TOF/ TOF for database searching. The mass calibration was done externally on the target using a myoglobin digest peptide.

Results and Discussion Preparation and Characterization of Ce4+-Chelated Magnetic Microspheres. Recently, metal ion-chelated magnetic silica microspheres with high magnetic responsivity were prepared using a simple method for the first time in our group.28 The synthesized metal ion-chelated magnetic silica microspheres were then initially used for successful enrichment and identification of phosphopeptides from tryptic digest through a direct MALDI-TOF MS analysis. In that work, only Fe3+ was chelated onto the magnetic silica microspheres to demonstrate the feasibility of the strategy for easy and selective enrichment of phosphopeptides, but there is no doubt that it can be broadened to other metal ions which have an affinity for phosphorylated proteins or peptides. Accordingly, in this study, we attempted to prepare cerium ion-chelated magnetic silica microspheres and utilized them for rapid and effective enrichment of phosphopeptides.

Tempst has tried to activate metal ion affinity microtips with Ce(SO4)2 solution; however, due to the solubility constraint of Ce(SO4)2, the Ce4+ column could not be readily prepared.31 Herein, we chose Ce(NO3)4 for immobilization of Ce4+ on the magnetic silica microspheres because it has high solubility in water. To confirm that we had successfully chelated Ce4+ on the MS microspheres, energy-dispersive X-ray analysis (EDXA) was conducted by illuminating electron beams on the obtained Ce4+-chelated microspheres. We found that the atomic content of Ce is about 2.02%. Although the result obtained by EDXA is just semiquantitative, it can still prove the successful chelation of Ce4+ on the magnetic silica microspheres. Optimization of Enrichment Conditions for Ce4+-Chelated Magnetic Microspheres. IMAC suffers from many nonspecific interactions, which increase the chance of false-positive results. One of the major nonspecific binders is acidic peptides. Methyl esterification of carboxyl groups of peptides improved the selectivity of IMAC for phosphopeptides by eliminating the acidic bias.32,33 However, the chemical reaction is sometimes incomplete. Especially in multiple Asp/Glu-containing peptides, the solubility of peptides is decreased after esterification, and the attachment of chemical tags and removal of excess reagents can lead to sample loss. Almost all IMAC protocols employ Journal of Proteome Research • Vol. 7, No. 4, 2008 1769

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Li et al. acid (DHB) was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. Because the binding of phosphorylated peptides to TiO2 is attributed to its ion exchange properties20 and is similar to the binding observed in IMAC experiments, we thus also investigated the possibility of adding DHB to loading buffer to improve the enrichment selectivity.

Figure 2. Effect of TFA concentration on the selective binding of phosphorylated peptides to Ce4+-chelated magnetic microspheres. Peptide mixture 1 (β-casein:BSA ) 1:50) was enriched using different concentrations of TFA in the loading and washing buffer. The bar graph shows peak intensities in MALDI spectra of phosphopeptides, and the line graph (•) shows total peak intensities of nonphosphopeptides. Three spot replicates were taken in the experiments and were used to calculate the average ion count.

Figure 3. Effect of acetonitrile concentration on the selective binding of phosphorylated peptides to Ce4+-chelated magnetic microspheres. Peptide mixture 1 (β-casein:BSA ) 1:50) was enriched using different concentrations of TFA in the loading and washing buffer. The bar graph shows peak intensities in MALDI spectra of phosphopeptides, and the line graph (•) shows total peak intensities of nonphosphopeptides. Three spot replicates were taken in the experiments and were used to calculate the average ion count.

acetic acid in the buffer system because 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 different acids with different acid strengths as additives for enriching phosphopeptides of ovalbumin using IMAC tips,17 and their results revealed that the best selectivity was obtained when using TFA solution as loading buffer. In this work, we used four different acids in the loading buffer (boric acid, phosphoric acid, acetic acid, and TFA) to investigate the effect of various acids on the selective binding of phosphorylated peptides to Ce4+-chelated magnetic silica microspheres. On the other hand, Jørgensen et al.34 have proved that the effect of 2,5-dihydroxy benzoic 1770

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Optimization of the procedure was performed using 200 µL of tryptic peptides originating from β-casein and BSA with a molar ratio of 1:50 (peptide mixture 1, see Experimental Section). For phosphopeptide enrichment, tryptic digests of proteins were mixed with the Ce4+-chelated magnetic silica microspheres and were incubated for 30 s under gentle vibration. Then, simply with the help of a magnet, the phosphopeptide-loaded magnetic microspheres were isolated, washed, and then ready for MALDI MS analysis using 1% (v/v) phosphoric acid and 2,5-dihydroxybenzoic acid (in 50% acetonitrile aqueous solution, v/v), 1:1, (v/v), as a matrix. A direct analysis of tryptic digestion of 0.5 µL of peptide mixture 1 by MALDI MS was carried out, and the obtained mass spectrum was shown in Figure 1a. The phosphopeptides from β-casein were difficult to be distinguished from the mass spectrum because of the presence of numerous abundant nonphosphopeptide peaks from BSA. The MALDI MS spectrum obtained from enrichment of phosphopeptides using the Ce4+-chelated magnetic microspheres with buffer 1 (0.1 M H3PO4, 50% ACN) is shown in Figure 1b. Compared with Figure 1a, nonphosphopeptides still dominate the spectrum, while only two peaks belonging to phosphopeptides of β-casein, marked with asterisks at m/z 2061.84 and 3122.27, can be observed with weak intensities. When using H3BO4 as loading and washing buffer (buffer 2), as shown in Figure 1c, although three phosphopeptides of β-casein (marked with asterisks) and peaks derive from their metastable losses of phosphoric acid (marked with ∆) can be observed in the spectrum, a significant number of nonphosphorylated peptides were observed together. When 0.1 M acetic acid (buffer 3) was employed, detection of phosphopeptides was significantly enhanced especially for the larger β-casein phosphopeptide (S16–40, obsd m/z ) 3122.23), which is a tetraphosphorylated peptide known for being hardly detected with MS, and dominated the spectrum (Figure 1d). However, several nonphosphorylated peptides still appear in the mass spectrum with strong intensities. Take the peak at m/z 1567.80 as an example. It can be assigned to a peptide derived from BSA (S347–359, DAFLGSFLYEYSR), which contains two acidic amino acid residues, including one aspartic acid (D) and one glutamic acid (E). The best result was obtained when using 0.1% TFA (buffer 4) as the loading and washing buffer. As depicted in Figure 1e, phosphopeptides from β-casein (marked with asterisks) dominate the mass spectrum with a high signal-tonoise ratio (S/N), and the phosphopeptide identification was supported by the observation of peaks at ∼ 93 m/z units below, indicative of metastable losses of phosphoric acid from the precursor ions (marked with ∆). The peaks from nonphosphorylated peptides are relatively very small. Since the β-casein we purchased was only 90% pure with contamination of traces of R-casein, the peak at m/z 1927.67 can be assigned to R-casein (R-S1, S43–58). The above results indicate that the enrichment selectively of Ce4+-chelated magnetic microspheres for phos-

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Figure 4. MALDI mass spectra of the tryptic digest products of ovalbumin (2.2 × 10-7 M) without enrichment (a) and after enrichment with Ce4+- (b) and Fe3+- (c) chelated magnetic microspheres. Phosphopeptides were bound in 0.15% TFA/50% ACN. Phosphopeptides are marked with asterisks. The metastable losses of phosphoric acid are indicated with ∆. The data in parentheses are S/N of the corresponding peaks.

phopeptides increased in the following order: TFA > HAC >H3BO4 >H3PO4. This is similar to the order of acid strength and in accordance with the results obtained by Oda et al.17 except for H3PO4. We suppose this is because strong acids protonated carboxyl groups but still dissociated phosphates, which allowed the discrimination of phosphoamino acids from acidic residues on Ce4+-chelated magnetic microspheres. As for phosphoric acid, although it has stronger acid strength than acetic acid and boric acid, the phosphate groups have effective competition with phosphorylated peptides, leading to a bad selectivity. After adding DHB in the buffer solution (buffer 5),

nearly no nonphosphorylated peptides were detected in the mass spectrum (Figure 1f). However, only the tetraphosphorylated peptide (m/z 3122.29) and peaks from its sequential metastable losses dominate the spectrum. The other two monophosphorylated peptides are both with low S/N, which suggests that most of these two phosphopeptides were not bound to the Ce4+-chelated magnetic microspheres. We suspect that the DHB molecules have competition for biding sites on Ce4+-chelated magnetic microspheres not only with nonphosphorylated peptides but also with phosphorylated peptides. The large molar excess of DHB thus effectively competes Journal of Proteome Research • Vol. 7, No. 4, 2008 1771

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Figure 5. MALDI mass spectra of the tryptic digest products originating from tryptic digestions of peptide mixture 2 (20 nM β-casein, ovalbumin, myoglobin, cytochrome C, and bovine serum albumin (BSA)) without enrichment (a) and after enrichment with Ce4+- (b) and Fe3+- (c) chelated magnetic microspheres. Phosphopeptides were bound in 0.15% TFA/50% ACN. Phosphopeptides are marked with asterisks. The metastable losses of phosphoric acid are indicated with ∆. The data in parentheses are S/N of the corresponding peaks.

with both nonphosphorylated peptides and monophosphorylated peptides for adsorption to the surface of Ce4+-chelated magnetic microspheres, leading to weak signals of these peptides in the mass spectrum. As for the tetraphosphorylated peptide, since it has much stronger affinity for immobilized cerium ions, it can still absorb on the Ce4+-chelated magnetic microspheres despite the existence of the DHB molecules. We also optimized the concentration of TFA in the buffer system for Ce4+-chelated magnetic microspheres as shown in Figure 2. Low concentration of TFA was insufficient for the protonation of carboxyl groups, and a higher concentration resulted in the loss of negative charge on phosphates, which are both ineffective for phosphopeptide enrichment using Ce4+-chelated magnetic microspheres. The results revealed that 0.1–0.5% TFA in buffer was suitable for selective enrichment 1772

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of phosphopeptides from a complex sample. Another major factor in nonspecific binding on affinity columns is hydrophobic interaction. Oda et al.17 have proved that ACN was very effective in eliminating nonphosphorylated peptides and thus improved the specificity on IMAC. In this study, we investigated the impact of ACN at different concentrations on improving specificity of Ce4+-chelated magnetic microspheres. From the obtained results (Figure 3), we find that 50% ACN in buffer has the best efficiency for improving the specificity. We therefore chose 50% ACN with 0.15% TFA as the optimized buffer for the following experiment because it not only enhanced the specific enrichment with Ce4+-chelated magnetic microspheres but also washed away nonphosphopeptides due to high concentration of ACN. We further investigated whether the

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Figure 6. MALDI mass spectra of the tryptic digest products originating from tryptic digestions of peptide mixture 1 (β-casein:BSA ) 1:50) and after enrichment with Ce4+- (a) and Fe3+- (b) chelated magnetic microspheres. Phosphopeptides were bound in 0.15% TFA/ 50% ACN. Phosphopeptides are marked with asterisks. The metastable losses of phosphoric acid are indicated with ∆. The data in parentheses are S/N of the corresponding peaks.

results would be improved by extending the enrichment time to 5, 15, 60, and 90 min, and no obvious improvements were observed. Comparison of Ce4+- and Fe3+-Chelated Magnetic Microspheres for Enrichment of Phosphopeptides. To compare the enrichment selectivity and efficiency of Ce4+-chelated magnetic microspheres with Fe3+-chelated microspheres, we applied the protocol established above to both of them for enrichment of phosphopeptides from tryptic digests of phosphoproteins. Ovalbumin was first chosen as a model phosphoprotein. More than thirty peaks were observed before enrichment in the mass spectrum (Figure 4a), while only the peak at m/z 2089.05 with low intensity belongs to phosphopeptide derived from ovalbumin. Figure 4b and 4c display the MALDI mass spectra obtained after using Ce4+- and Fe3+-chelated magnetic microspheres to selectively enrich phosphopeptides from the tryptic digest of ovalbumin (2.2 × 10-7 M), respectively. In both mass spectra, detection of the phosphopeptide at m/z 2089.0 (S340–359, EVVG[pS]AEAGVDAASVSEEFR) was significantly enhanced. The signal-to-noise ratio (S/N) for this ion peak improved from approximately 32 to 693 and 683 after enrichment using Ce4+- and Fe3+-chelated magnetic microspheres, respectively. And the phosphopeptide identification was supported by the observation of a peak derived from metastable loss of phosphoric acid from the precursor ion (marked with ∆). Further, the signal of the larger phosphorylated tryptic peptide at m/z 2902.3 (S59–84, FDKLPGFGD[pS]IEAQCGTSVNVHSSLR) appeared in both spectra after enrichment. A few

other nonphosphopeptide signals were detected in both spectra; however, their intensities were low. The results above indicate that both Ce4+- and Fe3+-chelated magnetic microspheres can be used for effective enrichment of phosphopeptides from tryptic digests of ovalbumin. To further evaluate the ability of the Ce4+- and Fe3+-chelated magnetic microspheres to capture the phosphopeptides from an even more complicated sample, we mixed the tryptic digest products of five proteins (peptide mixture 2), including nonphosphorylated proteins (cytochrome C, myoglobin, and bovine serum albumin (BSA)) and phosphoproteins (β-casein and chicken egg albumin (ovalbumin)). Figure 5a 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 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 enrichment with Ce4+-chelated magnetic microspheres, nine phosphopeptide residue ion peaks appear in the mass spectrum (as shown in Figure 5b). Among these peaks, m/z at 2061.95, 2556.23, and 3122.31 are derived from β-casein. The phosphopeptide marked with 2089.08 is derived from ovalbumin, whereas the peak at m/z 1466.69 is derived from R-casein due to the impurity of the β-casein we used. The peaks labeled with ∆ could be assigned to dephosphorylated fragments of phosphopeptides through the metastable loss of H3PO4 from the parent ions. No Journal of Proteome Research • Vol. 7, No. 4, 2008 1773

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Figure 7. MALDI mass spectra of the tryptic digest products originating from tryptic digestions of the nonfat milk sample (a) and after enrichment with Ce4+- (b) and Fe3+- (c) chelated magnetic microspheres. Phosphopeptides were bound in 0.15% TFA/50% ACN. Phosphopeptides are marked with asterisks. The metastable losses of phosphoric acid are indicated with ∆.

nonphosphorylated peptides were observed in the spectrum. When Fe3+-chelated magnetic microspheres were used, the tetraphosphorylated peptide of β-casein could not be observed in the mass spectrum (Figure 5c), and three nonphosphopeptides were detected. Furthermore, we applied Ce4+- and Fe3+-chelated magnetic microspheres to selective concentrate phosphopeptides from tryptic peptides of β-casein and BSA with a molar ratio of 1:50 (peptide mixture 1). The obtained result (Figure 6a) indicates that the trace phosphopeptides derived from β-casein can be concentrated effectively by the Ce4+-chelated magnetic microspheres, even when the amount of the tryptic digest product of BSA is 50 times higher than that of β-casein in the sample solution. However, when using Fe3+-chelated magnetic microspheres, a peak derived from a nonphosphorylated peptide of BSA (at m/z 1955.97, S319–336, DAIPENLPPLTADFAEDK, con1774

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taining three aspartic acids and two glutamic acids) dominates the mass spectrum (Figure 6b). Only two ion signals of monophosphorylated peptide derived from β-casein can be observed in the spectrum with low intensities, while the tetraphosphorylated peptide cannot be detected. These results indicate that Ce4+-chelated magnetic microspheres are more powerful than Fe3+-chelated microspheres for selective and effective enrichment of phosphopeptides, especially when dealing with complex samples. To further compare the performance of Ce4+-chelated magnetic microspheres with Fe3+-chelated magnetic microspheres on the proteome-wide scale, we chose to use both of these materials to selectively trap phosphopeptides from tryptic digested nonfat milk, since that nonfat milk commonly contains abundant proteins including phosphoproteins, i.e., R- and β-caseins, and Chen et al.23,27 had successfully used it as a real

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Figure 8. (a) MALDI-TOF mass spectrum of phosphopeptides enriched by Ce4+-chelated magnetic microspheres from diluted normal human serum (30 µL). Phosphopeptide ions are marked with asterisks. MALDI-TOF/TOF mass spectra of the parent ion at m/z 1465.66 (b), the parent ion at m/z 1545.73 (c), and the parent ion at m/z 1616.59 (d) enriched by Ce4+-chelated magnetic microspheres. The amino acid sequence coverage is shown by yn ions. Journal of Proteome Research • Vol. 7, No. 4, 2008 1775

technical notes sample to demonstrate the feasibility of their nanoparticles as affinity probes to enrich phosphopeptides. Figure 7a presents the direct MALDI mass spectrum of the tryptic digest of the milk sample (100-fold diluted). Among the peaks, only two, with low intensities, are phosphopeptides generated from R-casein (S1/104–119) and β-casein, marked with asterisks at m/z 1951.97 and 3122.21, respectively. Figure 7b displays the MALDI mass spectrum of the sample enriched from the tryptic digests of the milk sample (100-fold diluted, 50 µL) using Ce4+-chelated magnetic microspheres. Eight peaks at m/z 1411.46 (R-S2/ 126–136), 1466.57 (R-S2/138–149), 1660.73 (R-S1/106–119), 1927.58 (R-S1/43–58), 1951.86 (R-S1/104–119), 2618.74 (R-S2/ 2–21), 2703.66 (R-S1/99–120), and 3007.80 (R-S2/46–70) marked with asterisks are derived from R-casein, while two peaks at m/z 2061.72 (33–48) and 3122.04 (1–25) are derived from β-casein. The peaks marked with ∆ are fragmentations of the phosphopeptides by metastable losses of phosphate groups. Details of the identified phosphopeptides are listed in Table S1. However, when the diluted tryptic digests of the milk sample were enriched by Fe3+-chelated magnetic microspheres (Figure 7c), many nonphosphopeptides were observed, and only four phosphopeptides derived from R-casein and one phosphopeptide derived from β-casein were detected in the mass spectrum, which indicted relatively poorer selective enrichment efficiency. The results again prove that Ce4+chelated magnetic microspheres have better selectivity and efficiency for enrichment of phosphopeptides over Fe3+chelated magnetic microspheres. Enrichment of Phosphopeptides from Human Blood Serum Using Ce4+-Chelated Magnetic Microspheres. Human serum contains thousands of peptides, most of which are thought to be fragments of larger proteins that have been partially degraded by endogenous, proteolytic enzymes, but the precise identities remain undetermined.35–38 The bulk of polypeptide mass, at total concentrations in excess of 60 mg/ mL, is made up of a handful of highly abundant species, such as albumin, immunoglobulins, transferrin, and others.39 Phosphorylated peptides are present in low abundance in serum but play a vital role in regulatory mechanisms and may serve as a casual factor in diseases. To enrich and analyze phosphorylated peptides directly from human serum is a challenge for the researchers involved in phosphoproteomics. Hence, we also initially investigated the feasibility of employing our Ce4+chelated magnetic microspheres for selective enrichment of phosphopeptides directly from dilution of human serum without any other pretreatment. Figure 8a presents the MALDI mass spectrum obtained using Ce4+-chelated magnetic microspheres to selectively enrich target species from dilution of human serum. Three remarkable peaks at m/z 1465.66, 1545.73, and 1616.59 appear in the mass spectrum. To confirm the identities of these peaks, MALDI-TOF-TOF mass spectrometry was employed for characterization. The tandem mass spectra obtained by selecting the peptide ion at m/z 1465.66, 1545.73, and 1616.59 for low-energy CID are shown in Figure 8b-d, respectively. All fragment ion spectra displayed a near complete series of y-ion fragments. In Figure 8c and d, the mass difference of 167 Da between the peptide fragment ions y13 (1263.60) and y14 (1430.77) corresponds to a phosphoserine residue and located the phosphorylation site at Ser2 and Ser3, respectively. Loss of phosphoric acid (H3PO4, 98 Da) from the precursor ion confirmed this assignment. These MS/MS ions were subjected into the NCBI protein database for matching the possible proteins. The MS/MS results of the three 1776

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Li et al. peaks are all matched to residues derived from fibrinopeptide A (gi|229185, ADSGEGDFLAEGGGVR) whose increased level can be found in hepatocellular, ovarian, urothelial, and gastric cancers.40–43 The peaks at m/z 1545.73 (D[pS]GEGDFLAEGGGVR) and 1616.59 (AD[pS]GEGDFLAEGGGVR) are phosphopeptides confirmed by both tandem MS spectra and database searching, while the peak at m/z 1465.66 has the same sequence with the peak at m/z 1545.73 only without phosphorylation. The results support that the Ce4+-chelated magnetic microspheres can be directly employed to enrich phosphopeptides from human serum without requiring any purification steps.

Conclusions We have successfully developed, for the first time, a simple and highly phosphopeptide-specific enrichment procedure using Ce4+-chelated magnetic microspheres. On the basis of their magnetic properties, microspheres that are conjugated with target phosphopeptides can be isolated easily from sample solution just by a magnet. The enrichment conditions were optimized, and the loading/washing buffer containing 50% ACN and 0.15% TFA was finally selected for phosphopeptide enrichment because it not only enhanced the specificity of Ce4+chelated magnetic microspheres but also washed away nonphosphopeptides due to high concentration of ACN. Furthermore, by comparing the enrichment efficiency with Fe3+-chelated magnetic microspheres, we have confirmed that these Ce4+chelated magnetic microspheres have better selectivity toward phosphopeptides in mixture solutions. A complete molecular understanding of these differences in affinity and selectivity, however, will require further investigation as the current study only provides comparative data from several model proteins. We also initially demonstrated that the Ce4+-chelated magnetic microsphere-based enrichment method combined with MALDIMS/MS analysis can be used to quickly enrich and characterize the identities of phosphopeptides directly from human serum without extra purification steps or tedious treatment.

Acknowledgment. The work was supported by the National Basic Research Priorities Program (Project: 2007CB914100/3), 863 Project (No. 2006AA02Z4C5), the National Key Natural Science Foundation of China (Project: 20735005), and the Shanghai Leading Academic Discipline Project (B109). Supporting Information Available: Figure S1 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

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