Optimized IMAC−IMAC Protocol for Phosphopeptide Recovery from

May 10, 2010 - Immobilized metal ion affinity chromatography (IMAC) is widely used for ... was established using Fe(III)-NTA IMAC resin and it proved ...
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Optimized IMAC-IMAC Protocol for Phosphopeptide Recovery from Complex Biological Samples Juanying Ye, Xumin Zhang, Clifford Young, Xiaolu Zhao, Qin Hao, Lei Cheng, and Ole Nørregaard Jensen* Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Received January 26, 2010

Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)-nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile-water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)-NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 µg of mouse sample or 50 µg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC-IMAC and LC-MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC-IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects. Keywords: protein phosphorylation • phosphopeptide enrichment • phosphoproteomics • IMAC • MS analysis • LC-MS/MS

Introduction Protein phosphorylation is one of the most important posttranslational modifications in cellular processes. It plays a crucial role in governing signal transduction pathways, in modulating intrinsic biological activity of proteins, and in controlling protein-protein interactions.1 It is estimated that more than 30% of all cellular proteins are phosphorylated, often at multiple sites, so it is feasible that protein phosphorylation can affect cellular functions directly or indirectly.2 The detection of phosphorylated proteins and characterization of phosphorylation sites are therefore important for understanding protein regulation in biological processes. Mass spectrometry is a powerful technology for proteomic studies, allowing the detection and mapping of covalent post translational modifications, such as protein phosphorylation.3,4 However, the experimental analysis of phosphorylated proteins * To whom correspondence should be addressed. Ole Nørregaard Jensen, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark. E-mail: jenseno@ bmb.sdu.dk. Tel: +45 6550 2368. Fax: +45 6593 2661. 10.1021/pr100075x

 2010 American Chemical Society

is still challenging for a number of reasons. First, many phosphorylated proteins are generally present at low levels. As these proteins can also be phosphorylated on different sites, the lower abundance of each phosphorylated species makes it more difficult to detect them. Second, the mass spectrometer is normally used in positive ion mode, meaning the ionization of phosphopeptides is inherently affected by its nonphosphorylated counterparts. Third, in collision-induced dissociation (CID) fragmentation, the lability of the phosphate group on a phosphopeptide impairs identification of the phosphopeptide and the localization of the phosphorylation site. Normally, the fragmentation spectra of phosphopeptides in CID are dominated by one peak that is related to the neutral loss of the phosphate group and water, especially when the phosphate group is attached to a serine (S) or threonine (T).5–7 This hampers the level of backbone fragmentation, which is essential for determining the amino acid sequence. To circumvent these limitations, the selective isolation or enrichment of phosphopeptides prior to MS analysis becomes necessary.8 Numerous phosphopeptide enrichment approaches have been established and successfully employed to reveal phosJournal of Proteome Research 2010, 9, 3561–3573 3561 Published on Web 05/10/2010

research articles phoproteins and phosphorylation sites from complex biological samples.8 Examples include immunoprecipitation with specific antibodies,9 enrichment with strong cation exchange chromatography, 10–12 strong anion exchange chromatography13–15 strong cation and anion exchange chromatography,16,17 immobilized metal ion affinity chromatography (IMAC),12,18,19 metal oxides/hydroxides such as titanium dioxide (TiO2),20–23 zirconium dioxide24 and aluminum hydroxide,25 calcium phosphate precipitation,26 sequential elution from IMAC,27 and chemical derivatization methods.1,28–30 Currently, IMAC is one of the most widely used techniques for phosphopeptide enrichment prior to mass spectrometry.18,19,31,32 Phosphopeptide enrichment by IMAC is based on the strong affinity between positively charged metal ions (Fe(III), Ga(III), Al(III) or Zr(IV)) and the negatively charged phosphate groups. Both Ga(III)- and Fe(III)-based methods have been successfully applied in different large-scale phosphoproteomics studies.19,33,34 Although IMAC can predominantly enrich phosphopeptides, it may suffer from insufficient specificity, as acidic peptides can bind to the resin.18 Efforts have been made to improve the specificity of this method, including the use of 1,1,1,3,3,3hexafluoroisopropanol as loading and washing solution to enhance the efficiency of IMAC phosphopeptide enrichment.35 Imanishi et al. optimized the phosphopeptide elution conditions in Fe(III)-IMAC utilizing the combination of phosphoric acid (PA) and acetonitrile (ACN).36 The combination of 0.1% TFA in 50% ACN as both binding and washing solutions increased the selectivity of Phos-Select IMAC material.37 Ficarro and co-workers established a method to overcome the nonspecific binding of acidic peptides by performing methyl esterification. Since the carboxylate groups are neutralized and the only negatively charged groups remaining are the phosphoryl groups after esterification, the specificity of phosphopeptide enrichment can be improved considerably.38,39 However, the efficiency of esterification, hydrolyzation of the esters and other side reactions are the biggest limitations of this method.21,40 Therefore, there is still a need for IMAC development and optimization to produce higher selectivity and sensitivity with minimal sample loss. Here, we developed an optimized IMAC procedure by initially measuring the ionization efficiency difference between PA and the carboxyl group of acidic amino acids. This resulted in an improved IMAC method with high selectivity and minimal sample loss when tested on a phosphopeptide mixture. Phosphoproteomic studies on mouse and Drosophila cell lysates were conducted utilizing the optimized IMAC protocol in a tandem setup, that is, IMAC-IMAC, for separation of singly and multiply phosphorylated peptides.

Experimental Procedures Materials. Modified trypsin was from Promega (Madison, WI). Poros MC 20 and Oligo R3 reversed phase material were from PerSeptive Biosystems (Framingham, MA). Pure water was obtained from a Milli-Q system (Millipore, Bedford, MA). PhosSelect, FeCl3 (Cat. No: 10025771 purity >97%) and ammonia-water (NH3 · H2O) were from Sigma-Aldrich (St. Louis, MO). 2,5Dihydroxybenzoic acid (DHB) was from Fluka (St. Louis, MO). GELoader tips were from Eppendorf (Hamburg, Germany). 3 M Empore C8 disks were from 3 M Bioanalytical Technologies (St. Paul, MN). FeCl3 (Cat. No: 103943 purity >99%) was from Merck. Ni-NTA silica resin was from Qiagen (Mat. No. 1014059, 3562

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Ye et al. Hilden, Germany). All other reagents and solvents were of the highest commercial quality and were used without further purification. Digestion of Standard Bovine Phosphoproteins. R-Casein, β-casein, serum albumin (BSA), β-lactoglobulin and carbonic anhydrase were obtained from Sigma-Aldrich. Each protein was dissolved in 25 mM ammonium bicarbonate, reduced with dithiothreitol (DTT) and alkylated with iodoacetamide before digestion at 37 °C. Mouse Cell Lysate Protein Extraction and Digestion. 3T3L1 (Mouse embryonic fibroblast - adipose like cell line) cells (kindly provided by Dr. Karsten Kristiansen, Department of Biology, University of Copenhagen) were grown to confluence in Dulbeccos Modified Eagle Medium containing 10% fetal calf serum. The cells were rinsed twice in PBS, trypsinized and pelleted by centrifugation at 3300 rpm for 5 min at 4 °C. After washing twice with PBS, the cell pellet was resuspended in 200 µL solution containing 1% sodium dodecyl sulfate and 5 mM DTT. The solution was sonicated intermittently for 3 min (2 s sonication time followed by 5 s intervals) before centrifugation at 20 000× g for 20 min at room temperature. The supernatant was subjected to acetone precipitation. The solution was mixed with eight volumes of ice-cold acetone and kept at -20 °C for 2 h to allow protein precipitation. The acetone was removed after centrifugation at 20 000× g for 20 min at 4 °C. After two washes with ice-cold acetone, the pellet was completely dried in a speed-vacuum. The protein pellet was resuspended in 4 M guanidine monohydrochloride and the protein concentration was determined by Bradford assay. After adjusting the pH to 8, 20 µg of the protein solution was reduced by 5 mM DTT at 37 °C for 45 min and alkylated with 15 mM iodoacetamide at room temperature for 45 min. After an 8-fold dilution with 100 mM ammonium bicarbonate, the sample was digested overnight at 37 °C with 2.5% (w/w) trypsin. Note that the pH value should be around 8 and was checked before each digestion step. Desalting. Protein digests were acidified and individually loaded onto a homemade Poros R3 microcolumn.41 To avoid the potential loss of hydrophilic peptides, the flowthrough from each R3 column was further purified with a graphite powder microcolumn. To minimize sample loss, the length of the column was varied depending on the sample amount (approximately 1 µL bed volume of R3 material per 10 µg of sample). After washing the R3 or graphite resins twice with 5% formic acid (FA), the bound peptides were eluted with IMAC loading solution (60% ACN/100 mM acetic acid (HAc)). Both the R3 and graphite eluates were pooled for subsequent phosphopeptide enrichment analysis. Fe(III)-NTA Immobilized Metal Ion Affinity Chromatography. A slurry of Fe(III)-loaded NTA-silica resin was prepared as previously described, with minor modifications.18 The resin was treated in turn with 100 mM EDTA, 200 mM HAc, 50 mM FeCl3 in 100 mM HAc, and finally twice with 200 mM HAc. The activated IMAC resin was stored in 200 mM HAc. An aliquot of the IMAC material was added into the dried peptide and the pH was adjusted to around 3 with 100 mM HAc to a total volume of 30 µL. The peptide solution and IMAC material were incubated for 1 h with a slow end-over-end rotation at room temperature. After incubation, the slurry was packed into a GELoader tip. The beads were washed with washing solution containing 100 mM HAc and 30% ACN. Bound peptides were eluted with DHB matrix solution (20 mg/mL in 50% ACN, 1% PA) since it has been demonstrated to be efficient for MALDI

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Optimized IMAC-IMAC Protocol MS analysis of phosphopeptides or NH3 · H2O (pH 10.5) for LC-MS analysis. The eluate was immediately acidified with 1 µL FA, and desalted using Poros R3 microcolumns prior to LC-MS analysis. Poros MC 20. Poros MC 20 beads were activated according to Seo et al.43 A 1 µL bed volume of activated beads was equilibrated in 30 µL binding solution (ACN/MeOH/water (1: 1:1, v/v/v) in 0.01% HAc) before sample loading. The dried samples were dissolved in 30 µL of binding solution and mixed with MC 20 beads before incubated at room temperature with shaking for 1 h. After incubation, the slurry was loaded onto a GELoader tip. The resulting beads were washed twice with 20 µL of washing solution (ACN/MeOH/water (1:1:1, v/v/v) in 0.01% HAc).44 The bound phosphopeptides were eluted onto the MALDI target with DHB matrix solution. Phos-Select. Phos-Select material was rinsed twice with 30 µL of TFA/water/ACN (0.1/50/50) (TWA). The dried samples were dissolved in 30 µL of TWA and mixed with Phos-Select material. The peptide solution and Phos-Select material were incubated for 1 h with a constant slow end-over-end rotation at room temperature. After incubation, the slurry was loaded onto a GELoader tip and then washed with 20 µL of TWA.37 The bound phosphopeptides were eluted onto a MALDI target with DHB matrix solution. MALDI-TOF MS and MS/MS Analysis. Analyses by MALDITOF MS were used for screening the sample quality and composition. Prior to MALDI analysis, the peptide samples were desalted on Poros R3 microcolumns and directly eluted with matrix solution onto the sample supports. DHB (20 mg/ mL in 50% ACN, 5% PA) was used as the matrix solution. MALDI MS and MS/MS were performed using a Bruker Ultraflex Tof/Tof MS (Bremen, Germany). All spectra were obtained in positive reflector mode. Mass spectrometric data analysis was performed using the Bruker Daltonics FlexAnalysis Software v2.4. Sequence analyses and peptide assignments were accomplished using GPMAW software version 8.1 (http:// www.welcome.to/gpmaw). Nano LC-MS/MS Analysis. LC-MS/MS analysis was performed using a nanoliter flow EasyLC system (Thermo Fisher Scientific, Odense, Denmark) interfaced to a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The redissolved peptide samples were injected onto an in-house packed fused silica column (18 cm length, 100 µm inner diameter, 375 µm outer diameter, ReproSil, C18 AQ 3 µm (Dr. Maisch, Ammerbuch, Germany)). The peptides were eluted with a 50 min gradient of 0-34% B solvent (A solvent: 0.1% FA, B solvent 0.1% FA in 90% ACN). Peptide masses were measured in the Orbitrap at a resolution of 60 000 (m/z 400). Up to five of the most intense peptides (minimum intensity of 15 000 counts) were selected from each MS scan and fragmented using multistage activation in the linear ion trap. Database Searches. Raw data were processed using Proteome Discoverer software (version 1.1, Thermo Fisher Scientific). The resulting mgf files were searched against the IPI_mouse protein sequence database (Dec 09, 2008; 59 265 sequences) and the Drosophila melanogaster protein database (dmel-all-translation-r5.18.fasta from Flybase (www.flybase. org), May 16, 2009; 43 568 sequences) using an in-house Mascot server (version 2.2.04, Matrix Science, London, UK). The following parameters were specified in the protein database searches: only tryptic peptides with up to two missed cleavage sites were allowed; 5 ppm mass tolerances for MS and 0.6 Da for MS/MS fragment ions; carbamidomethylcysteine as fixed 42

modification; and protein N-acetylation, oxidized methionine and phospho_STY (serine, threonine and tyrosine) permitted as variable modifications. Phosphopeptides identified were considered to be potential candidates if the expectation value (p) was lower than 0.05 and the peptides were top ranked. Decoy database searches in Mascot revealed a false positive rate lower than 1% at the peptide level. Since the purpose of the study was to evaluate the performance of the different enrichment methods, but not to perform high confidence protein and phosphorylation site identification, it was determined that this procedure was adequate. The phosphorylation sites were assigned according to the Mascot results and counted manually.

Results and Discussion Comparison of Three IMAC Materials. To start the optimization work, three widely used IMAC materials, Ni-NTA silica from Qiagen, Poros MC 20 from Perseptive Biosystems and Phos-Select from Sigma-Aldrich were selected and tested. Prior to any application, the two former resins were activated to Fe(III)-IMAC using the methods by Stensballe et al.18 and Seo et al.,45 respectively. An examination of their phosphopeptide enrichment capabilities were accomplished using a 1 pmol trypsin digested test mixture (R-casein/β-casein/BSA/β-lactoglobulin/carbonic anhydrase ) 1:1:50:50:50). To investigate the performance of the IMAC resins for phosphopeptide enrichment, three optimized loading and washing conditions for three different IMAC resins were employed: loading with 100 mM HAc and washing with 100 mM HAc in 30% ACN for Qiagen Fe(III)-NTA resin, as reported by Stensballe et al.;18 loading and washing with 50% ACN in 0.1% TFA for Phos-Select material, reported by Kokubu et al.;37 and loading and washing with ACN/MeOH/water (1:1:1, v/v/v) in 0.01% HAc for Poros MC 20 resin as reported by Ndassa et al.44 Phosphopeptide enrichment performance was evaluated by counting the number of phosphopeptide peaks and comparing the phosphopeptide signal intensities obtained from MALDI MS spectra (Figure S1A-C, Supporting Information). Since the Qiagen Fe(III)-NTA resin showed the best performance in all three conditions, it was chosen for further optimization studies. Ionization Efficiency Test. Since phosphopeptide enrichment by IMAC is based on the affinity between positively charged metal ions and the negatively charged phosphoryl group of phosphopeptides, the selectivity and efficiency of phosphopeptide enrichment by IMAC are theoretically determined by the degree of ionization (DOI) of the phosphopeptide. In practice, the peptides containing multiple acidic residues such as aspartic acid (Asp) and glutamic acid (Glu) are difficult to eliminate from the enriched products since they are also negatively charged. Therefore, the selectivity and efficiency of the IMAC method depends on the difference of DOI between the phosphoryl group (phosphopeptide) and carboxyl group (acidic nonphosphopeptide). The DOI is also affected by the solvent, so it is possible to maximize the ionization ability difference between the phosphoryl group and carboxyl group with an appropriate binding solution. Since ACN-containing solutions are commonly used as loading solution for IMAC enrichment, the DOI in different ACN solutions was tested. PA was used to mimic the phosphopeptide, while Asp and Glu were used to mimic the acidic nonphosphopeptide in this binding test with Fe(III)-NTA. Journal of Proteome Research • Vol. 9, No. 7, 2010 3563

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Figure 1. (A) pH effects in solvents containing varying concentrations of ACN. (, pH value of 2.5 mM PA; ), pH value of 100 mM HAc; 4, pH value of 2.5 mM Asp; 2, pH value of 2.5 mM Glu. (B) pH effects of different acids and HAc in the solvent containing various concentrations of ACN. O, pH values of 100 mM HAc, which were set as baseline. (, indicates the pH difference between 2.5 mM PA and 100 mM HAc; 4, pH difference between 2.5 mM Asp and 100 mM HAc; 2, pH difference between 2.5 mM Glu and 100 mM HAc.

The dissociation and ionization of an acid in solution is given by: HX + H2O h H3O+ + X-

(1)

According to the electric charge equilibration in a solution, [H3O+] ) [OH-] + [X-]

(2)

Under acidic conditions, [H3O+] . [OH-], thus we can use the approximation: [H3O+] ≈ [X-]

(3)

Equation 3 indicates that the concentration of H3O+ is almost equal to the concentration of the ionized product X-. The observed change of pH can therefore be used as an indicator of the change of DOI when the sample concentration is constant. The pH changes of four different acids as a function of ACN concentration are shown in Figure 1A. Because the solubility of Asp and Glu is quite low in the solvent with high ACN content (around 5 mM in 80% ACN) and the concentration of phosphopeptides is often substoichiometric in real biological samples, the concentrations of Asp, Glu and PA were set at 2.5 mM. HAc at 100 mM was also measured in this test because it is the recommended IMAC loading solution to inhibit the ionization of Asp and Glu residues.18 As shown in Figure 1A, it is evident that the pH (-log[H3O+]) of the PA solution is almost constant in ACN. However, the pH values of the carboxylic group containing solutes (Asp, Glu and HAc) were higher when the ACN concentration was increased. Thus, increased ACN content reduces the ionization of Asp, Glu and HAc, whereas it does not have any obvious effects on the ionization of PA. We note that the pH difference (∆pH ) pH(HX) - pH(HAc)) is more important in practice, because HAc is often used in IMAC loading solutions to maintain the pH. This ∆pH parameter is 3564

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negatively correlated to the DOI of the solute in the HAc containing solution. Figure 1B shows ∆pH as a function of ACN concentration relative to 100 mM HAc. It is clear that the value of pH(PA) - pH(HAc) decreases as the ACN percentage increases, meaning that a higher concentration of ACN enhances the ionization of PA in 100 mM HAc. A series analysis of the pH changes of PA and HAc in various acetonitrile-water solutions was previously reported.46,47 These studies found that the pH of PA showed a very slight increase when the content of ACN increased from 0 to 60%, whereas the pH of HAc increased greatly. This indicates the pH difference between PA and HAc is enlarged with higher ACN content, which is in agreement with our results. Regarding Asp and Glu, the ∆pH were almost constant when the content of ACN was varied from 0 to 70%, demonstrating that the increased ACN content does not have a significant effect on the dissociation of Asp and Glu in HAc containing solutions. In summary, these results suggest that the higher ACN concentration in 100 mM HAc increases the ∆pH between PA and the acidic amino acids Asp and Glu. Therefore, 100 mM HAc is an appropriate solution to assist phosphopeptide enrichment as its pH value is between PA and the acidic amino acids. IMAC of Phosphoprotein Digests. Next, we evaluated the effects of ACN concentration for enriching phosphopeptides by IMAC. Experiments were carried out using 100 mM HAc and varying ACN concentrations as the sample loading solution for IMAC. For each experiment, 1 pmol tryptic R- and β-casein digests were used. If the loading solution had a low ACN content (e30%), 100 mM HAc in 30% ACN was used as washing solution to avoid unspecific hydrophobic binding, otherwise loading solution was also used as the washing solution. Figure S2A-D (Supporting Information) show MALDI mass spectra of the IMAC enriched samples under different binding conditions, with the corresponding flowthrough and wash fractions. Similar phosphopeptide recovery was achieved using different loading conditions (Figure S2A, Supporting Information). However, a notable difference among the spectra was the increase in relative intensity of the m/z 1951.9 peak as ACN

Figure 2. (A) Phosphopeptide enrichment of 1:50 peptide mixture (R-casein/β-casein/BSA/β-lactoglobulin/carbonic anhydrase ) 1:1:50:50:50) at different ACN concentrations in the loading solution (labeled in each spectrum). HAc at a concentration of 100 mM was constant in the loading solution and a 1 µL bed volume of Fe(III)-IMAC resin was used. The sample was incubated with loading solution for 1 h. (B) Magnified area from m/z 1925 to 1960. All labeled signals indicate phosphorylated peptides from Table S2 (Supporting Information).

Optimized IMAC-IMAC Protocol

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Figure 3. Fe(III)-IMAC phosphopeptide enrichment of trypsin digested protein mixtures at various dilutions (1:50, 1:200, 1:500, 1:1000). A 1 µL bed volume of Fe(III)-IMAC resin was used and 100 mM HAc in 60% ACN employed as loading and washing solutions. The sample was incubated with loading solution for 1 h. All labeled signals indicate phosphorylated peptides from Table S2 (Supporting Information).

content increased in the loading solution (Figure S2B, Supporting Information). This signal represents the peptide of YKVPQLEIVPNpSAEER (R-casein-S1, 119-134) with one missed tryptic cleavage site. Because it possesses only one phosphoryl group and two basic amino acids, its affinity to the Fe(III)-IMAC resin is weaker when compared to other phosphopeptides. Considerably fewer and lower intense phosphopeptide signals were obtained from the flowthrough and wash fractions when a higher ACN concentration was used (Figure S2C, Supporting Information). This is also reflected in the intensity of the m/z 1951.9 peak (Figure S2D, Supporting Information). Therefore, it is clear that increased ACN concentration is able to reduce the phosphopeptide loss during enrichment. It should be noted that the IMAC resin possesses a stronger affinity toward multiply phosphorylated peptides, since the corresponding ion signals were not observed from the flowthrough and wash fractions. These observations suggest that the increased ACN concentration enhanced the affinity between the IMAC resin and phosphopeptides. The sample loss was also reduced when we increased the ACN concentration, which is consistent with results obtained from the pH test. Evaluation with Digest Mixtures (1:50). Further tests were performed by using the optimized sample preparation method and the 1:50 tryptic digest mixture (R-casein/β-casein/BSA/β3566

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lactoglobulin/carbonic anhydrase ) 1:1:50:50:50). Figure 2A shows the highly specific enrichment obtained especially when the ACN content increases from 30 to 60%, which was also reported by Kokobu et al.37 As described in the previous test, the signal at m/z 1951.9 was more intense when the ACN content is higher than 30%. However, the results acquired from the two highest ACN concentrations are unsatisfactory (Figure 2B). Although all the phosphopeptides signals could be observed, specificity is poor due to numerous and intense peaks from nonphosphopeptides. This could be due to these peptides containing multiple acidic amino acid residues, or the binding properties of the IMAC resin was altered in high percentages of ACN solution. The ACN content was therefore set at 60% since it provided satisfactory selectivity and efficiency. Different concentrations of HAc (25, 50, 100, 200, and 400 mM) were also tested to obtain optimal IMAC phosphopeptide enrichment (Figure S3, Supporting Information). More nonphosphopeptides were present when the lower concentrations of HAc (25 and 50 mM) were used, whereas loss of singly phosphorylated peptides occurred when higher concentrations of HAc (200 and 400 mM) were utilized. Only the spectrum from the 100 mM HAc loading solution shows high selectivity and binding efficiency.

Optimized IMAC-IMAC Protocol

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Figure 4. Effects of Fe3+(FeCl3) on phosphopeptide enrichment of 1:50 peptide mixture with activated IMAC resins. A 1 µL bed volume of Fe(III)-IMAC resin was used and 100 mM HAc in 60% ACN was employed as loading and washing solutions. The sample was incubated with loading solution for 1 h. All labeled signals indicate phosphorylated peptides from Table S2 (Supporting Information).

In summary, a solvent consisting of 60% ACN and 100 mM HAc was found to be the optimal sample loading solution for Fe(III)-NTA-silica IMAC resin. Next, we optimized the IMAC washing solution, which is supposed to maintain the binding of the phosphopeptides and remove nonphosphopeptides from the resin. Various ACN washing solutions were tested (data not shown). A solvent consisting of 60% ACN and 100 mM HAc was selected due to its superior performance in sample loading containing low (Figure S4A, Supporting Information) or high (Figure S4B, Supporting Information) ACN concentrations. Several types of elution solvents were investigated (Figure S5, Supporting Information). We found that either 5% PA or 5% NH3.H2O were efficient elution solvents, which is in contrast to a previous report.36 The ratios of protein amount to IMAC resin volume (Figure S6, Supporting Information) as well as incubation time (Figure S7A-B, Supporting Information) were also investigated. The data suggests that the optimal incubation time is peptide sequence-dependent: a short incubation time is needed for phosphopeptides with acidic sequences and/or multiple phosphoryl groups, while longer incubation times are required for singly phosphorylated peptides with nonacidic sequences. Furthermore, selectivity does not seem to vary when excess IMAC resin is used. In summary, the best and most practical loading and washing solvents were found to be 100 mM HAc in 60% ACN for binding of phosphopeptides to Fe(III)-NTA IMAC resin. The incubation time for loading was set to 1 h. The preferred elution solvent was 20 mg/mL DHB in 50% ACN/5% PA for subsequent MALDI MS analysis, whereas 5% NH3 · H2O was preferred for subsequent LC-MS analysis.

Selectivity and Sensitivity of IMAC. To test the sensitivity of the optimized IMAC method, we made a dilution series of the trypsin digested R- and β-caseins into a 50 pmol mixture of BSA, β-lactoglobulin and carbonic anhydrase (1:50, 1:200, 1:500, 1:1000). High phosphopeptide specificity was still achieved with the highly diluted (1:1000) phosphopeptide sample, where numerous phosphopeptide signals were detected (Figure 3). Ion signals at m/z 1816, 1896, 2939, 3190, and 3270 were only found in the highly diluted phosphopeptide samples (1:500 and 1:1000). Although the corresponding peptide sequences are unknown, all produced a 98 Da neutral loss (representing the diagnostic loss of H3PO4) in MS/MS analysis (Figure S8, Supporting Information). We found β-lactoglobulin was slightly contaminated with unknown phosphoproteins (data not shown). Regardless, the selectivity and sensitivity demonstrated here is superior to other recent reports on IMAC performance. Effects of Fe3+ Quality. We discovered IMAC performance varied greatly between different Fe3+ suppliers. As shown in Figure 4, using Fe3+ purchased from Merck, we observed much better selectivity under our test conditions compared to the Sigma-Aldrich product. A possible reason could be the difference in the purity of the FeCl3 batches (97% for Sigma-Aldrich and 99% for Merck), since the nonspecific binding was more pronounced in IMAC enrichment conducted with the SigmaAldrich reagent. Application of Optimized IMAC-IMAC Protocol to Phosphoproteome Analysis of Mouse Cells. We devised a strategy to apply the optimized IMAC protocol for analysis of complex phosphopeptide samples using a tandem setup of consecutive IMAC enrichment steps (Figure 5). A mouse cell lysate was used to evaluate the efficiency of the optimized IMAC method. Since Journal of Proteome Research • Vol. 9, No. 7, 2010 3567

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Figure 5. Strategy for enrichment of phosphopeptides from complex biological samples.

the sensitivity and selectivity of the IMAC approach can be severely affected by various buffers and detergents used in biochemical procedures,48 desalting becomes necessary prior to IMAC enrichment. It was reported that the use of graphite microcolumns provided a more complete coverage of dynamic phosphoproteomes, since the graphite powder binds small or hydrophilic peptides.49 Therefore, 20 µg of trypsin digested mouse cell lysate was first desalted with a R3 microcolumn to obtain large and hydrophobic peptides, with the flowthrough further purified with a graphite powder microcolumn to bind

small and hydrophilic peptides. The desalted and concentrated peptides were eluted from these resins with the optimized IMAC loading solution and directly used for phosphopeptide enrichment by IMAC. In a previous study, we demonstrated that sequential IMAC enrichment using a limited amount of IMAC material in each step leads to more phosphopeptide identifications by the separation of multiply phosphorylated peptides from singly phosphorylated peptides.26 This is also the case for IMAC and TiO2.27 Therefore, we first used a 5 µL bed volume of IMAC

Table 1. Overview of Identified Unique Phosphorylation Sites in IMAC-IMAC Enrichment Experiments of Mouse Cell Lysate (DI Indicates the First IMAC Enrichment, DII Indicates the Second IMAC Enrichment) first experiment

second experiment

phosphorylation site

DI

DII

DI + DII

DI

DII

DI + DII

total

S T Y total

909 108 5 1022

570 53 1 624

1269 141 6 1416

878 109 2 989

525 66 1 591

1307 163 3 1473

1818 219 7 2044

Table 2. Overview of Identified Unique Phosphopeptides in IMAC-IMAC Experiments Using 20 µg of Mouse Cell Lysate

Total peptides Total phosphopeptides Percentage of peptides that are phosphopeptides (%) Separation % of multiply phosphorylated peptides % of singly phosphorylated peptides 3568

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first IMAC

second IMAC

total

829/821 783/771 94.5/93.8 65.9/62.7 34.1/37.3

1163/1254 673/658 57.9/52.5 8.3/1.7 91.7/98.3

1697/1749 1246/1341 73.4/76.7 42.7/35.6 57.3/64.4

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Optimized IMAC-IMAC Protocol

Figure 6. Phosphopeptide enrichment from 20 µg of mouse cell lysate using the IMAC-IMAC method. Flowthrough and wash fractions from the first IMAC were enriched by the second IMAC. (A) Number of phosphorylated peptides identified in each IMAC step and the overlap between the two data sets. (B) Distribution of phosphoryl groups per phosphopeptide from different enrichment steps. (C) Amino acid distribution of the singly phosphorylated peptides from the first and second IMAC fractions. Table 3. Overview of Identified Unique Phosphopeptides and Phosphorylation Sites in IMAC-IMAC Experiments Using 50 µg of Drosophila Cell Lysate

Total peptides Phosphopeptides Percentage of peptides that are phosphopeptides (%) Multiply phosphorylated peptides Singly phosphorylated peptides Phosphorylation sites Phosphoserines Phosphothreonines Phosphotyrosines

resin in the enrichment experiment, with the flowthrough (void volume) and wash fractions from the IMAC column combined and directly applied to the second enrichment with 8 µL (bed volume) of IMAC material to capture unbound phosphopeptides, mostly singly phosphorylated peptides. The enrichment experiments were performed in duplicate to assess the reproducibility of the optimized IMAC-IMAC method. The phosphopeptide samples from the first and second IMAC enrichment steps were then individually analyzed by LC-MS/MS on a LTQ-Orbitrap XL hybrid tandem mass spectrometer. Using the IMAC-IMAC approach, we identified 1416 phosphorylation sites in 590 proteins in the first experiment and 1473 phosphorylation sites in 632 proteins in the second experiment. In combination, a total of 2044 phosphorylation sites were identified in 823 proteins. The annotated phosphorylation sites are listed in Table S3 (Supporting Information). Among 2044 identified phosphorylation sites, 1818 phosphorylation sites were assigned to serine, 219 were assigned to threonine and 7 were assigned to tyrosine (Table 1). The ratio

first IMAC

second IMAC

total

904 885 97.9 688 197 1320 1155 148 17

1063 793 74.6 44 749 747 671 69 7

1766 1496 84.7 708 788 1798 1580 197 23

of phosphoserine to phosphothreonine (1818:219) is consistent with the frequency ratio of phosphoserine: phosphothreonine: phosphotyrosine (1800:200:1) observed in previous studies,50,51 whereas the identified phosphotyrosine level seems to be higher than expected. This observation might be explained by the lability of the phosphate group on the different phosphorylated amino acids. For serine/threonine phosphorylated peptides, the loss of H3PO4 is dominant in the MS/MS spectra,5 while for tyrosine phosphorylated peptides, there is only a very low abundance of neutral loss (typically the loss of HPO3).6 This makes the identification and localization of the phosphoryl group on tyrosine phosphorylated peptides easier when compared to serine/threonine phosphorylated peptides. Next, we investigated the binding/elution behavior of the phosphopeptides from the first and second IMAC fractions in each of the two experiments. An overview of the duplicate enrichment experiments is shown in Table 2. It is noticeable that all the first IMAC fractions achieved high specificity, with more than 94% of the total identified peptides assigned as Journal of Proteome Research • Vol. 9, No. 7, 2010 3569

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Figure 7. Phosphopeptide enrichment from 50 µg of Drosophila melanogaster cell lysate using the IMAC-IMAC method. Flowthrough and wash fractions from the first IMAC were enriched by the second IMAC. (A) Number of phosphorylated peptides identified in each IMAC step and the overlap between the two data sets. (B) Distribution of phosphoryl groups per phosphopeptide from different enrichment steps. (C) Amino acid distribution of singly phosphorylated peptides in first and second IMAC fractions.

phosphopeptides. However, fewer than 60% of the total identified peptides in the second IMAC fractions were phosphopeptides. Because most of the multiply phosphorylated peptides and a subset of singly phosphorylated peptides were captured by the first IMAC stage, the second IMAC stage has a greater capacity to bind singly phosphorylated peptides and some acidic regular peptides. The separation of multiply and singly phosphorylated peptides by sequential IMAC enrichment is also apparent. In the first IMAC fractions, more than 60% of the identified phosphopeptides were multiply phosphorylated peptides. Conversely, more than 90% of the identified phosphopeptides were singly phosphorylated peptides from the second IMAC fractions. Figure 6A shows the number of phosphopeptides identified in each of the sequential IMAC eluates and the overlap between the two IMAC steps. It is clear that the phosphopeptide overlaps between the two sequential IMAC fractions are negligible. This finding supports our idea of using an optimized sequential IMAC for adequate recovery of phosphopeptides. To further understand the characteristics of the IMAC-IMAC enrichment, we considered the distribution of the number of phosphoryl groups per peptide (Figure 6B). In both of the first IMAC fractions, more than 60% of all the identified phosphopeptides were multiply phosphorylated peptides and approximately 30% were singly phosphorylated peptides, while only less than 10% of all the identified phosphopeptides were multiply phosphorylated peptides and more than 90% were singly phosphorylated peptides in all of the second IMAC enrichment fractions. The different nature of the phosphopeptides obtained with two consecutive IMAC enrichment steps suggests that sample complexity can be reduced by controlling the bed volume of IMAC resin. Finally, an examination of the amino acid composition on the total number of identified 3570

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singly phosphorylated peptides was performed using GPMAW software (Figure 6C). In all the first IMAC enrichment fractions, the frequencies of aspartic acid (D) and glutamic acid (E) make up almost half of the entire sequences. However, in the second IMAC enrichment fractions, the frequencies of the acidic amino acids are considerably decreased, while the frequencies of the basic amino acid residues (histidine (H), lysine (K) and arginine (R)) are increased slightly. These observations were also validated by the motif present in identified singly phosphorylated peptides from the IMAC fractions. The phosphopeptides identified from all the first IMAC fractions possess four phosphoserine motifs: xxxSDxE/xxxSDxD and xxxSDxx/xxxSExx, and two phosphothreonine motifs: xxxTPxx and xxxTxxE. However, the phosphopeptides identified from the second IMAC fractions possess phosphoserine motifs of RxxSxxx and xxxSPxx, and a phosphothreonine motif of xxxTPxx. This demonstrates that the IMAC material is not limited to enriching only acidic phosphopeptides under these conditions. Application to Phosphoproteome Analysis of Drosophila melanogaster Kc167 Cell Line. To investigate the applicability of the optimized IMAC method in different biological samples, we applied the protocol to a phosphoproteomic study of Drosophila Kc167 cells. Fifty micrograms of the trypsin digested cell lysate was desalted and subjected to the two step IMAC protocol. As described in Table 3, from the first IMAC fraction that contained 904 identified peptides, 885 were phosphopeptides (representative of 456 phosphoproteins with 1320 phosphorylation sites). In the second IMAC fraction, where 1063 peptides were identified, 793 were phosphopeptides (482 phosphoproteins containing 747 phosphorylation sites). In summary, we obtained 1496 phosphopeptides that possessed 1798 phosphorylation sites, altogether representing 693 phosphoproteins (Table S4, Supporting Information). As shown in

Optimized IMAC-IMAC Protocol

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Figure 8. MS/MS spectra of multiply phosphorylated peptides identified from the first IMAC fraction of Drosophila cell lysate. (A) MS/MS spectrum of triply phosphorylated peptide AAAPAAVApSPAAAATSADApSPpSPAK. (B) MS/MS spectrum of doubly phosphorylated peptide RGGGGGDpSpSDEDVTTR. (C) MS/MS spectrum of doubly phosphorylated peptide QVpSIEpSPGPGAK. (D) MS/MS spectrum of doubly phosphorylated peptide LREEpSPDpSETDNQVNR. The observed y-ions and b-ions are indicated in the peptide sequences.

from the mouse cell lysate enrichments. Figure 8 shows the MS/MS spectra of four multiply phosphorylated peptides (one triply phosphorylated peptide and three doubly phosphorylated peptides) identified from the first IMAC fraction. We identified 1798 phosphorylation sites in total, while these phosphorylation sites were assigned to only 693 unique phoshoproteins. This indicates that there is a wide range of multiphosphorylated proteins in our data set. As shown in Figure 9, a total of 429 identified phosphoproteins possessed two or more phosphorylation sites. In particular, more than 90 phosphoproteins contained five or more phosphorylation sites. Figure 9. Distribution of identified phosphorylation sites per phosphoprotein identified from 50 µg Drosophila melanogaster sample.

Figure 7A, the overlap between the two IMAC fractions only accounts for 12.2% (183/1496) of the identified phosphopeptides. Most of the phosphopeptides from the first IMAC fraction were multiply phosphorylated peptides (77.7%), while very few of the phosphopeptides from the second IMAC fraction were multiply phosphorylated peptides (5.5%) (Figure 7B). The distribution of phosphorylation sites is illustrated in Figure 7C. The ratio of phosphoserine: phosphothreonine: phosphotyrosine was 1475:167:24, which is consistent with the results

Recent Drosophila melanogaster phosphoproteome analyses by the Aebersold group52–54 and the Gygi group55 identified many phosphoproteins (4583 and 2702, respectively) and phosphorylation sites (10 118 and 13 720). However, the overlap between the data sets was low at the phosphoprotein (48%) and phosphopeptide level (27%). This suggests further optimization of experimental and computational issues is required, or that the Drosophila melanogaster phosphoproteome is very large. As our study used considerably less starting material, direct comparison to our results is biased unfairly. Nonetheless, from the 661 identified phosphoproteins, 96 were not present in the Phosphopep database, while 103 were not found in the list reported by Zhai et al.55 A total of 52 phosphoproteins from our study were not found in either data set, which include two Journal of Proteome Research • Vol. 9, No. 7, 2010 3571

research articles proteins identified in highly phosphorylated forms; La-related protein (FBgn0260724) with 17 phosphorylation sites and the Hook-like protein, isoform A (FBgn0086441), which possesses 10 phosphorylation sites. Thus, using significantly less protein starting material than previously reported studies, the IMACIMAC method presented here adds novel phosphorylation sites to the knowledgebase, thereby demonstrating the sensitivity and specificity of the protocol.

Conclusion An optimized phosphopeptide enrichment method was established using Fe(III)-NTA IMAC resin, which focused on the binding, washing and elution conditions. The loading and washing conditions were optimized by considering the ∆pH between phosphoryl and carboxyl groups. The sensitivity of this method, the effect of incubation time on the binding efficiency, the ratio of sample amount to resin volume and the different Fe3+ sources were also addressed. The phosphoproteome studies of 20 µg protein obtained from a mouse cell lysate and 50 µg protein from a Drosophila cell lysate demonstrated that a highly specific phosphopeptide enrichment was achieved using the optimized IMAC-IMAC protocol. In addition, we showed that it is possible to reduce the complexity of the sample by efficiently separating the multiply phosphorylated peptides from singly phosphorylated peptides using successive IMAC enrichments. We foresee that more comprehensive phosphoprotein and phosphoproteome analysis can be performed with this IMAC-IMAC methodology due to its simplicity, sensitivity and specificity and its compatibility with both MALDI-MS/MS and LC-ESI-MS/MS based workflows. Abbreviations: DOI, degree of ionization; IMAC, immobilized metal ion affinity chromatography; LC, liquid chromatography; LTQ, linear ion trap; MALDI, matrix-assisted laser desorption/ ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; TOF, time-of-flight.

Acknowledgment. We thank Thomas Aarup Hansen for assistance in database searches and data analysis. We are grateful to Lene Jakobsen and Melanie Schulz for their help in LC-MS/MS measurements. This work was supported by The Lundbeck Foundation, The Danish Research Agency and the Danish National Research Foundation. Supporting Information Available: Supplemental data, figures, and tables. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Oda, Y.; Nagasu, T.; Chait, B. T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 2001, 19 (4), 379–82. (2) Hubbard, M. J.; Cohen, P. On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem. Sci. 1993, 18 (5), 172–7. (3) Mann, M.; Jensen, O. N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 2003, 21 (3), 255–61. (4) Jensen, O. N. Interpreting the protein language using proteomics. Nat. Rev. Mol. Cell. Biol. 2006, 7 (6), 391–403. (5) DeGnore, J. P.; Qin, J. Fragmentation of phosphopeptides in an ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 1998, 9 (11), 1175–88. (6) Tholey, A.; Reed, J.; Lehmann, W. D. Electrospray tandem mass spectrometric studies of phosphopeptides and phosphopeptide analogues. J. Mass Spectrom. 1999, 34 (2), 117–23. (7) Hoffert, J. D.; Knepper, M. A. Taking aim at shotgun phosphoproteomics. Anal. Biochem. 2008, 375 (1), 1–10.

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