Acid Control

Aug 16, 2008 - A revised one-step IMAC method with low sample loss and high specificity can be rationally designed by controlling salt, pH and the str...
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Immobilized Metal Affinity Chromatography Revisited: pH/Acid Control toward High Selectivity in Phosphoproteomics Chia-Feng Tsai,† Yi-Ting Wang,‡ Yet-Ran Chen,§,¶ Chen-Yu Lai,§,# Pei-Yi Lin,§ Kuan-Ting Pan,| Jeou-Yuan Chen,⊥ Kay-Hooi Khoo,|,O and Yu-Ju Chen*,§,| Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan, Department of Applied Chemistry, National Chia-Yi University, Chiayi, Taiwan, Institute of Chemistry and Genomic Research Center, Academia Sinica, Taipei, Taiwan, National Core Facilities for Proteomics Research, National Science Council, Taipei, Taiwan, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, and Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Received May 20, 2008

Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, a highly specific enrichment protocol is still a challenge in large-scale studies. Here, we report a simple pH/acid control method that addresses the poor specificity seriously criticized in IMAC. Detailed evaluation of the capture and release mechanism in IMAC revealed that pH, buffer and salt yield a complex interplay in enrichment of phosphopeptides, yet they play individual roles in recovery and specificity. A revised one-step IMAC method with low sample loss and high specificity can be rationally designed by controlling salt, pH and the structure and concentration of organic acid. Without methyl esterification, the one-step IMAC enrichment with single LC-MS/MS identified 386 phosphoproteins in 550 µg of non-small-cell lung cancer cell lysate with 96% specificity. Additional fractionation by SDS-PAGE from 4 mg of cell lysate revealed the comprehensive proteome map, identifying 2747 phosphorylation sites from 2360 nondegenerate phosphopeptides and 1219 phosphoproteins with a false discovery rate of 0.63%. To our knowledge, this pH/acid-controlled IMAC procedure provides higher specificity than any other one-step IMAC purification procedure. Furthermore, the simple and reproducible IMAC protocol can be adapted to other solid supports, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome. Keywords: IMAC • SDS-PAGE • phosphoproteomics • mass spectrometry • lung cancer

Introduction Protein phosphorylation is a reversible and dynamic process that is controlled by protein kinases and phosphatases. Phosphorylation regulates various cellular processes ranging from signal transduction, cell differentiation, cellular development, cell cycle control, metabolism and enzyme structure maintenance. An increasing number of human diseases have been discovered to involve mutations, overexpression, or malfunction of protein kinases and phosphatases as well as of their regulators and effectors.1 To elucidate the molecular basis of these processes, it is crucial to identify the specific phospho* To whom correspondence should be addressed. Yu-Ju Chen, Institute of Chemistry, Academia Sinica, Taipei, Taiwan. Phone, +886-2-2789-8660; fax, +886-2-2783-1237; e-mail, [email protected]. † National Taiwan Normal University. ‡ National Chia-Yi University. ¶ Current address: Agricultural Biotechnology Research Center, Academia Sinica. § Institute of Chemistry and Genomic Research Center, Academia Sinica. # Current address: Division of Molecular and Genomic Medicine, National Health. | National Core Facilities for Proteomics Research, National Science Council. ⊥ Institute of Biomedical Sciences, Academia Sinica. O Institute of Biological Chemistry, Academia Sinica.

4058 Journal of Proteome Research 2008, 7, 4058–4069 Published on Web 08/16/2008

rylation sites and quantitate their temporal and dynamic changes. Recently, mass spectrometry (MS) has emerged as a reliable and sensitive method to identify protein phosphorylation sites. Despite the biological significance of protein phosphorylation and the advances in MS, characterization of site-specific phosphorylation is still challenged by the technical difficulties2 associated with their dynamic modification patterns, substoichiometric concentrations, heterogeneous forms of phosphoproteins and low MS response. Methodologies that specifically enrich the transient phospho-subproteome in a robust, comprehensive manner are important for studying phosphorylation-dependent cellular signaling. On the basis of the interaction between metal ion and phosphopeptide, immobilized metal affinity chromatography (IMAC) was originally discovered by Andersson and Porath as an easy, economic and generic enrichment protocol for all types of phosphorylation3–5 and rapidly evolved for large-scale enrichment protocol of phosphopeptides in the proteome scale.6,7 The nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA) are the two most commonly used chelating compounds to conjugate to a solid support such as chromatographic resins or magnetic beads. The NTA/IDA moiety will chelate Zn(II), Fe(III), Ga(III) or other metal ions,4,8–10 leaving unoccupied 10.1021/pr800364d CCC: $40.75

 2008 American Chemical Society

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Immobilized Metal Affinity Chromatography Revisited coordination sites to interact with negatively charged peptides, such as phosphopeptides. The metal ion has an empty orbital as the electron acceptor, which can interact with spare electron pairs in the negatively charged peptides through electrostatic interactions and coordination bonding. Neville and co-workers5 reported that Fe3+-NTA IMAC has better selectivity than Fe3+IDA IMAC to isolate phosphopeptides. However, nonspecific capture of peptides containing acidic amino acids, such as glutamic acid, aspartic acid, histidine and cysteine, presents a serious drawback in IMAC. To reduce nonspecific binding, optimized protocols to change buffer pH4,10 or washing conditions11,12 have been reported with various degrees of specificity for enrichment and identification of phosphopeptides. Despite the reported successes of standard proteins,4,8 the performance of IMAC at the proteome-wide level has low specificity (60%∼70%) and thus yields low number of identified phosphoproteins. Alternatively, methyl esterification before IMAC enrichment can eliminate nonspecific binding, and enable determination of a large number of phosphorylation sites from cell lysates.11,13–15 However, the additional chemical reaction may cause serious loss of sample.16,17 Until recently, tandem purification by either cation exchange chromatography (SCX)18 or anion exchange chromatography (SAX)9 in combination with IMAC has been interfaced to increase the enrichment specificity up to 75%. More recently, hydrophilic interaction chromatography (HILIC) combined with IMAC was reported as an effective strategy for large-scale identification of phosphoproteins. On the basis of separation of the strong hydrophilic phosphate group in the first HILIC fraction, subsequent enrichment by IMAC have near 99% purification specificity without additional derivatization or chemical modification.19 Because of the criticism of nonspecific enrichment in IMAC method, metal oxide chromatography using titanium dioxide (TiO2) was recently introduced by Pinkse et al.20 as a more successful alternative. Using an online TiO2 precolumn coupled with a reverse-phase capillary column, they demonstrated enrichment of phosphopeptides from β-casein and R-casein in bovine milk. However, the selectivity of this method was also compromised by the detection of several acidic nonphosphorylated peptides retained by the TiO2 column. In a later study,21 2,5-dihydroxybenzoic acid (DHB) was used to remove the nonspecific binding peptides, thereby enhancing the binding selectivity and detection sensitivity of phosphopeptides. Kweon et al.22 first showed that zirconium dioxide (ZrO2) chromatography provides comparable selectivity for phosphopeptides to TiO2 chromatography. Although they observed that the TiO2 chromatography selectively enriches multiply phosphorylated peptides whereas ZrO2 column possesses unique selectivity for monophosphorylated peptides, the tendency for enriching monophosphorylated peptides by ZrO2 was not observed by later reports.23,24 These findings may suggest complementary use of the two materials have potential for comprehensive phosphoproteomic profiling,24 presumably through different surface chemistry, coordination number and enrichment protocol.22 On the other hand, the comparison between IMAC and metal ion chromatography gains increasing attention. By detailed evaluation on the purification performance of standard protein mixtures, Larsen et al. discovered that TiO2 is more robust and tolerant toward the interference of salts, detergents, and small molecule composites than IMAC.23 Inclusion of detergent in loading buffer can enhance IMAC performance by avoiding adhesion of peptides to Eppendof, yet interestingly, favors enrichment of multiply phosphorylated peptides com-

pared with TiO2. In a more recent report, they developed a sequential IMAC elution strategy for separation of monophosphorylated and multiply phosphorylation peptides under different acid and base elution.25 Compared to an optimized TiO2 chromatographic method, the batch-elution strategy demonstrated superior recovery of both monophosphorylated and particularly multiply phosphorylated peptides. The above results demonstrated that the experimental protocols significantly affect the performance of IMAC and the nature of purified phosphopeptides. A thorough comparison between IMAC and TiO2 chromatography of the global phosphoproteomic profiling of Drosophila melanogaster Kc167 cells was reported by Aebersold’s group.26 In terms of selectivity, combining methyl esterification with IMAC increased the sensitivity from 66% to 80%, which is comparable to TiO2-based enrichment. Surprisingly, little overlap (35%) in the identified phosphorylation sites was observed, implying that the two methods may be complementary to enrich different nature of phosphopeptides. The results suggest that IMAC is still essential to isolate phosphopeptides toward comprehensive coverage of phosphoproteome, which warrants further efforts to improve the selectivity and recovery without tedious methyl esterification or prior enrichment with SCX, SAX or HILIC. In the present study, we report a pH/acid-controlled IMAC protocol for comprehensive profiling of the phosphoproteome with low sample loss and high enrichment selectivity. From the chemical standpoint, enrichment versus nonspecific binding is determined by the binding affinity between metal ion and phosphopeptides and the competition by nonphosphopeptides or components in the buffer solution. To derive a rational IMAC protocol rather than trial-and-error optimization, we performed a series of experiments to gain insight into the chemical nature of the system components and their contribution to specific or competitive binding. Thus, we re-examined the specific roles of pH, structure and concentration of digestion buffer components, and sample loading and elution strategies toward obtaining high sample recovery and contamination-free purification. Finally, the optimized single-step IMAC protocol, combined with either high-resolution LC or SDS-PAGE fractionation, was further applied to large-scale profiling of the phosphoproteome of non-small-cell lung cancer cells. Lung cancer is one of the most common cancer and leading cause of cancer-related death worldwide.27 Among the types of lung cancer, ∼80% of patients were diagnosed with non-small-cell lung cancer.28 Large-scale profiling of phosphoproteome may enhance our understanding on the role of signaling pathways in lung cancer pathogenesis. Our results contradict the prevailing view that IMAC has low selectivity for phosphopeptide enrichment. A total of 926 defined phosphorylation sites with 96% selectivity were identified in a single LC-MS/MS analysis and incorporation of SDS-PAGE fractionation established the most comprehensive phosphoproteome map of non-small-cell lung cancer cells.

Experimental Procedures Materials. Triethylammonium bicarbonate (TEABC), triethylammonium formate (TEAF), ammonium bicarbonate (ABC), ammonium formate (AF) and iron chloride (FeCl3) were purchased from Sigma Aldrich (St. Louis, MO). The BCA protein assay reagent kit was obtained from Pierce (Rockford, IL). Ammonium persulfate and N,N,N′,N′-tetramethylenediamine were purchased from Amersham Pharmacia (Piscataway, NJ). Acetic acid (AA) was purchased from J. T. Baker (Phillipsburg, Journal of Proteome Research • Vol. 7, No. 9, 2008 4059

research articles NJ). Hydrochloric acid (HCl), trifluoroacetic acid (TFA), formic acid (FA) and HPLC-grade acetonitrile (ACN) were purchased from Sigma Aldrich. Modified, sequencing-grade trypsin was purchased from Promega (Madison, WI). Standard proteins, R-casein and β-casein, were purchased from Sigma Aldrich. Cell Culture and Lysate. The human non-small lung carcinoma cell line (H1299) was cultured in RPMI 1640 medium (HyClone Logan, UT) supplemented with 10% fetal bovine serum and 1% penicillin G (GibooBRL, Gaithersburg, MD) at 37 °C in a 5% CO2 atmosphere. Cells were stimulated with or without 500 µM pervanadate (pH 10 with 0.14% H2O2) from Sigma Aldrich for 1 h before harvesting cells. After stimulation, cells were washed with PBS 3 times and lysed in modified RIPA buffer (10 mM Tris-HCl, pH 7.4, 158 mM NaCl, 1 mM EGTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM DTT, 100 mM sodium vanadate, 100 mM sodium fluoride and 100 mM protease inhibitor). Preparative SDS-PAGE Separation and Proteolysis. The protein concentrations in cell lysate were determined by BCA assay (Pierce, Rockford, IL) before tryptic digestion. For largescale identification of phosphoprotein in H1299 cell, 4 mg of cell lysate was separated by 10% SDS-PAGE29 (8.6 cm × 6.8 cm × 1.5 mm). To visualize SDS-PAGE, a gel slice of roughly 2-cm-wide was cut and stained with Coomassie blue. The remaining gel was then cut into 10 gel slices based on molecular weight. Each band was cut into pieces of approximately 0.9 mm,3 washed with MilliQ water and destained twice with 25 mM TEABC (pH 8) in 50% (v/v) ACN for 15 min. Gel slices were dehydrated with 100% ACN and dried for 10 min under vacuum. For trypsin digestion, dry gel pieces were rehydrated in 25 mM TEABC (pH 8) containing 10 ng/µL trypsin until the gel pieces were fully immersed. The digestion was carried out at 37 °C overnight. Tryptic peptides were extracted twice with 5% (v/v) FA in 50% (v/v) ACN for 30 min, dried completely under vacuum and stored at -30 °C. For cell lysate without SDS-PAGE fractionation, the protein samples were subjected to gel-assisted digestion.30 To incorporate proteins into a gel directly in the Eppendorf vial, 18.5 µL of acrylamide/bisacrylamide solution (40%, v/v, 29:1), 2.5 µL of 10% (w/v) APS, and 1 µL of 100% TEMED were then applied to the membrane protein solution. The gel was cut into small pieces and washed several times with 1 mL of TEABC containing 50% (v/v) ACN. The gel samples were further dehydrated with 100% ACN and then completely dried by Speed-Vac. Proteolytic digestion was then performed with trypsin (protein/trypsin ) 50:1, g/g) in 25 mM TEABC with incubation overnight at 37 °C. The tryptic peptides were dried completely under vacuum and stored at -30 °C. IMAC Procedure. One end of the IMAC column was first capped with a 0.5 µm frit disk enclosed in stainless steel column-end fitting. The Ni-NTA resin was extracted from spin column (Qiagen, Hilden, Germany) and packed into a 5-cm microcolumn (500 µm i.d. PEEK column, Upchurch Scientific/ Rheodyne, Oak Harbor, WA). Automatic purification of phosphopeptides was performed connected to an autosampler and an HP1100 solvent delivery system (Hewlett-Packard, Palo Alto, CA) with a flow rate of 13 µL/min. First, the Ni2+ ions were removed with 100 µL of 50 mM EDTA in 1 M NaCl. Then, the IMAC column was activated with 100 µL of 0.2 M FeCl3 and equilibrated with loading buffer for 30 min before sample loading. For optimization of the phosphopeptide enrichment, the loading/condition buffers (designated as loading buffer) at different pH and different acidic conditions were prepared by 4060

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Tsai et al. adding various concentrations (see main text) of FA, AA, HCl or TFA to HPLC-grade water. The optimal loading buffer was 6% (v/v) AA and the pH was adjusted to 3.5 with 0.1 M NaOH (pH ) 12.8). The peptide samples from trypsin digestion were reconstituted in loading buffer and loaded into the IMAC column that had been equilibrated with the same loading buffer for 20 min. Then, the unbound peptides were removed with 100 µL of washing solution consisting of 75% (v/v) loading buffer and 25% (v/v) ACN, followed by equilibration with loading buffer for 15 min. Finally, the bound peptides were eluted from the IMAC column with 100 µL of 200 mM NH4H2PO4 (pH 4.4). Eluted peptide samples were dried under vacuum and reconstituted in 0.1% (v/v) FA for LC-MS/MS analysis. LC-MS/MS Analysis. Purified phosphopeptide samples were reconstituted in 4 µL of buffer A (0.1% FA in H2O) and analyzed by LC-Q-TOF MS (either QSTAR Pulsar i from Applied Biosystems, Foster City, CA or Waters Q-TOF Premier from Waters Corp, Milford, MA). For µLC-MS/MS analysis with QSTAR Pulsar i, samples were injected into a 2 cm × 100 µm capillary trap column packed in-house (Magic C18, Michrom BioResources Inc., Auburn, CA). Peptides were separated by a 10 cm × 75 µm capillary column, also packed in-house, and eluted with a linear gradient of 10-80% buffer B for 75 min at approximately 200-300 nL/min (buffer A, 0.1% FA in H2O; buffer B, 0.1% FA in ACN). A capillary-HPLC (HP1100 solvent delivery system, Hewlett-Packard, Palo Alto, CA) was used for online sample separation and introduction into the mass spectrometer. Peptide fragmentation by collision-induced dissociation was performed automatically using InformationDependent Acquisition (IDA) method of Analyst QS v1.1 (Applied Biosystems). The acquisition method performed 1 TOF MS scan (m/z 400-1600, 1 s) and automatically switched to 3 MS/MS scans (m/z 110-1600, 1.5 s) of the three most intense ions when a target ion reached an intensity of greater than 20 counts. For LC-MS/MS analysis by Waters Q-TOF Premier, samples were injected into a 2 cm × 180 µm capillary trap column and separated by 20 cm × 75 mm Waters1 ACQUITYTM 1.7 mm BEH C18 column using a nanoACQUITY Ultra Performance LC system (Waters Corp., Milford, MA). The column was maintained at 35 °C and bound peptides were eluted with a linear gradient of 0-80% buffer B (buffer A, 0.1% FA in H2O; buffer B, 0.1% FA in ACN) for 80, 120, 180, 210 and 270 min. MS was operated in ESI positive V mode with a resolving power of 10 000. NanoLockSpray source was used for accurate mass measurement and the lock mass channel was sampled every 30 s. The mass spectrometer was calibrated with a synthetic human [Glu1]-Fibrinopeptide B solution (1 pmol/µL, from Sigma Aldrich) delivered through the NanoLockSpray source. Data acquisition was operated in the data directed analysis (DDA). The method included a full MS scan (m/z 400-1600, 0.6 s) and 3 MS/MS (m/z 100-1990, 1.2 s each scan) sequentially on the three most intense ions present in the full scan mass spectrum. Database Search and Data-Filtering. Raw data were processed using either Analyst QS 1.1 (Applied Biosystems Foster City, CA) or Proteinlynx GlobalServer 2.2.5 (Waters Corp Milford, MA). For raw data files from QSTAR Pulsar i, MS and MS/MS centroid parameters were set to 50% height percentage and a merge distance of 0.1 amu. For MS/MS grouping, the following averaging parameters were selected: reject spectra with less than five peaks or precursor ions with less than 30

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Immobilized Metal Affinity Chromatography Revisited

Figure 1. Factors that affect IMAC performance. (a) Specific binding is dependent on the pH and salt in solution. (b) The performance may be complicated by competitive binding from acidic peptides and substances containing carboxylic acid group in solution.

cps. The precursor mass tolerance for grouping was set to 0.01 Da, the maximum number of cycles per group was set to 1, and the minimum number of cycles per group was set to 1. For raw data files from Waters Q-TOF Premier, the processing parameters were as follows: background subtraction, adaptative; background threshold, 35%; background polynomial, 5; smoothing type, Savitzky-Golay; smoothing iteration, 2; smoothing window, 3 channels; deisotoping type, medium; deisotoping threshold, 3%. The resulting MS/MS data set was exported to either *mgf (Analyst QS 1.1) or *pkl (Proteinlynx GlobalServer 2.2.5) data file format. We performed the peptide identification and assignment of partial post-translational modifications using an in-house version of Mascot v. 2.2 (Matrix science, London, United Kingdom). The data sets were searched against International Protein Index (IPI_human v. 3.29, 68161 sequences) using the following constraints: only tryptic peptides with up to two missed cleavage sites were allowed; 0.3 Da mass tolerances for MS and 0.1 Da mass tolerances for MS/MS fragment ions. Phosphorylation (STY) and oxidation (M) were specified as variable modifications. Peptides were considered identified if their Mascot individual ion score was higher than 39 (p < 0.05). As the vast majority of phosphopeptides identified showed significant loss of phosphoric acid, the neutral loss characteristic peak, corresponding to the loss of 49 Da from the doubly charged precursor, was annotated as an additional characteristic signal for phosphopeptides. To evaluate the protein identification false discovery rate, we repeated the searches using identical search parameters and validation criteria against a randomized decoy database created by Mascot (from the 68161 sequence). The false discovery rates with Mascot score >39 (p < 0.05) range between 0.42% and 0.94% in this study. We considered the assignment criteria are sufficient because the study aims to evaluate the IMAC performance under different experimental parameters rather than high confidence phosphorylation site determination. List of the identified phosphorylated peptides are provided in Supplementary Table 2. The MS/MS spectra and assignment for identified nondegenerate peptides were included in Supplementary Figure 4

(http://proj3.sinica.edu.tw/∼yujuchen/2008JPR_IMAC/ Supplementary_Figure_4.html). The phosphorylation site of phosphoserine, phosphothreonine, and phosphotyrosine can be determined by the characteristic mass difference of 69, 83 and 243 Da, respectively. When the exact phosphorylation site could not be assigned for a given phosphopeptide, it was annotated as ambiguous in the table. When unique peptides were identified to multiple members of a protein family, proteins having the highest sequence coverage were selected from the Mascot search output result.

Results Rationale and Experimental Design. IMAC takes advantage of the phosphate groups as electron donors that chelate metal ion (Fe3+-NTA in this study) to preferentially retain phosphopeptides. Although the simple and routinely used protocol yields satisfactory results for simple phosphoprotein mixtures, the results for proteome-wide analysis are far from optimal. As shown in Figure 1, an efficient enrichment protocol should yield specific purification (defined as selection of phosphopeptide, Figure 1a) and contamination-free purification (defined as lack of nonspecific competitive binding and release from other constituents, Figure 1b). The pH effect has been the most studied parameter that controls the binding and elution of phosphopeptides in IMAC protocol.12,21,23,25,26 In the first step toward full sample recovery in IMAC column, pH will critically determine the extent of phosphopeptides bound to Fe3+-NTA. Only at pH values above the pKa of the phosphate groups, the phosphopeptides will become completely deprotonated and chelate Fe3+-NTA. In addition, the presence of buffer components that form ion pairs with phosphopeptides can reduce recovery. On the other hand, competitive binding may arise from several factors and cause nonspecific copurification (Figure 1b). Under high pH conditions, the chelating affinity between carboxylic acid group and Fe3+-NTA may cause copurification of glutamic acid- and aspartic acidcontaining peptides, which greatly reduces the recovery and specificity of phosphopeptide enrichment. Furthermore, the presence of chelating substances, such as acid used in the loading buffer, may interact with Fe3+-NTA and inhibit phosJournal of Proteome Research • Vol. 7, No. 9, 2008 4061

research articles phopeptide binding or even strip bound phosphopeptides off the immobilized metal ion. Proper selection of strong chelating acids may effectively remove nonphosphorylated peptides. By the study on the intricate affinities between metal ion and phosphopeptides, nonphosphorylated peptides, and the components in the loading/elution buffer, we hypothesize that a single-step IMAC protocol can be tailored for full sample recovery with minimum contamination from nonphosphorylated peptides. Rational design of the experimental protocol can be performed by evaluating the effects of the factors used during the digestion and IMAC procedure on binding capacity, recovery and selectivity. In this study, the automatic purification of phosphopeptides was performed using a micro column immobilized with Fe3+-NTA-silica. Selection of Digestion Buffer. The cell lysate was digested with trypsin by our recently reported gel-assisted digestion.30 In brief, the digestion protocol incorporates protein into a polyacrylamide gel matrix in a glass tube without electrophoresis. Any interfering components, including EGTA, SDS and reduction/alkylation reagents, can be removed by subsequent in-gel washing steps. In addition, the selection of digestion buffer is critical in IMAC performance. Ammonium bicarbonate (ABC) is the most commonly used buffer for trypsin digestion. However, performing digestion and IMAC consecutively may cause significant loss of phosphopeptides because excess ammonium ions will form ion pairs with phosphate groups.31 Most studies have applied desalting procedure prior to IMAC.6,9,32 To confirm the hypothesis and examine resulting losses in binding capabilities, we compared the IMAC performance in five different buffers including two ammonium salts and two triethylammonium salts; triethylammonium has an additional bulky triethyl group as compared with ammonium. Total proteins extracted from H1299 cells were digested, desalted by a C18 column and then divided into five equal parts: (1) in 25 mM triethylammonium bicarbonate; (2) in 25 mM triethylammonium formate; (3) in 25 mM ammonium bicarbonate; (4) in 25 mM ammonium formate. As a control experiment, the last part was in salt-free condition. The purified phosphopeptides under these buffer/salt-free conditions were subjected to purification using an IMAC column in 0.5%(v/v) acetic acid (AA) (pH 3.0). As shown in Figure 2, the number of identified phosphopeptides in triethylammonium bicarbonate (322 ( 6 phosphopeptides, FDR ) 0.3%) and formate (330 ( 36 phosphopeptides) were clearly greater than those purified in ammonium bicarbonate (284 ( 19 phosphopeptides) and formate (253 ( 26 phosphopeptides), confirming that the use of ammonium salt may interfere the binding between phosphate group and metal ion. The observation also explained that desalting step after protein digestion was critical in most previously published protocols.6,9,32 Without the need of additional desalting step, the use of triethylammonium bicarbonate identified slightly lower number of phosphopeptides to those under salt-free conditions (356 ( 38 phosphopeptides). Therefore, we conclude that the use of triethylammonium salt demonstrated to be an IMAC-compatible buffer without additional desalting step prior to loading the sample onto the IMAC column. pH Controls Recovery and Specificity of IMAC. In principle, the proportion of negative charges on phosphorylated groups is mainly controlled by the buffer pH. On the basis of the relationship between pH value and known pKa values for aspartic acid (3.65), glutamic acid (4.25), and phosphorylated residues (1.52),33,34 Figure 3a shows the correlation between 4062

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Figure 2. The effect of digestion buffer on IMAC performance. Comparison between salt-free condition with triethylammonium salt and ammonium salt buffers on the number of enriched phosphopeptides from 300 µg of H1299 cells using IMAC.

pH and the theoretical degree of dissociation/ionization (%) (i.e., the ratio of RCOO-/ [RCOOH + RCOO-] and RHPO4/[RH2PO4 + RHPO4-]) at pH 1∼5. As the pH increases from 2 to 3, the degree of ionization of the phosphorylated group is significantly increased from 75% to 97%, whereas the degrees of ionization of glutamic acid and aspartic acid are concomitantly increased from 1% to 5% and 2% to 18%, respectively. When the pH is elevated above 3.0, the increased degree of ionization for glutamic acid and aspartic acid will generate significantly more negative carboxylic acid groups (15∼95%); however, only 3% more negative phosphopeptides will be capable of binding to Fe3+-NTA. Therefore, the optimal pH range for phosphopeptide purification should fall between pH 2.5∼3.0, where nearly all phosphate groups are negatively charged to ensure complete binding onto Fe3+-NTA. Meanwhile, less than 5% of negatively charged glutamic acid and aspartic acid ions will be present to cause nonspecific binding. To experimentally confirm the above inference, we compared the IMAC purification performance using smaller pH intervals between 2.5 and 3.5 to determine an optimal pH. Figure 3b shows the total number of identified peptides, phosphopeptides and their corresponding percent specificity (number of identified phosphopeptides/total number of identified peptides). As expected, the extraction specificity (right y-axis in Figure 3b) is highly pH-dependent. When different concentration of acetic acid was used, selectivity of 61, 82, 86 and 97% were observed at pH 3.5, 3.0, 2.7 and 2.5, respectively, demonstrating a concurrent increase in nonspecific binding with elevated pH. At low pH, the number of nonspecifically bound nonphosphopeptides was minimized with enhanced selectivity, but the number of phosphopeptides decreased due to incomplete deprotonation of phosphate group. Although nearly 100% selectivity was obtained at pH 2.5 (Figure 3b), the number of identified phosphopeptides (n ) 136) was less than that obtained at pH 2.7 (n ) 190) and pH 3.0 (n ) 166). On the other hand, a higher pH (pH 3.5) also dramatically decreased the number of phosphopeptides obtained (n ) 114), which may be due to competitive binding and ion suppression from

Immobilized Metal Affinity Chromatography Revisited

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Figure 3. The effects of pH and acid concentration on enrichment recovery and specificity of phosphopeptids from H1299 cell lysate using IMAC. (a) Correlation between pH and theoretical degree of dissociation/ionization (%) for the ratio of RCOO-/ [RCOOH + RCOO-] and RHPO4-/[RH2PO4 + RHPO4-] at pH 1∼5. (b) The number (bar graph and left y-axis) and specificity (line graph and right y-axis, number of phosphopeptide/total number of identified peptides) of identified phosphopeptides were affected by loading buffer at different pH (2.5∼3.5) and different AA concentrations (0.1∼6%); (c) number of identified phosphopeptide and specificity at different concentrations of AA (0.5∼6%) at constant pH (pH ) 3.0); and (d) number of identified phosphopeptide and specificity at different pH (pH ) 2.5∼3.5) under constant AA concentration (6%).

nonphosphopeptides. Among the identified nonphosphopeptides, we observed a significantly higher occurrence of glutamic acid and aspartic acid residues at pH 3.0 (see Supplementary Figure 1). On the basis of the above observations, we learned that the pH control (by different concentration of acids, v/v) adapted in most literatures only causes different extent of compromise between recovery and specificity. A 100% specificity can be achieved at very low pH values accompanied by an inevitable loss in recovery, whereas full deprotonation of phosphopeptides can be obtained only at pH values above 3.0. To reduce the nonspecific binding at higher pH, we hypothesized that the addition of molecules containing a carboxylic acid groups may compete with the nonspecific binding of peptides containing carboxylic acid groups. Therefore, we next investigated whether high concentrations of acids could inhibit nonspecific binding to improve selectivity at higher pH. As shown in Figure 3c, the number of captured phosphopeptides was independent of the amount of acetic acid35 at fixed pH (pH ) 3, 160-167 phosphopeptides). What is affected by acid concentration under fixed pH is the degree of selective enrichment of phosphopeptides. As high as 95% selectivity was achieved in 6% (v/v) AA compared with 82.2% selectivity in 0.5% (v/v) AA and 82.1% selectivity in 3% (v/v) AA, confirming that the nonphosphorylated peptides can be effectively removed in the presence of high concentrations of AA. To achieve maximum recovery under the above optimized acidic concentration, we adjusted the sample loading buffer to pH 2.5, 2.7, 3.0 and 3.5 in 6% AA (v/v) using NaOH (Figure 3d). Compared to the results obtained in Figure 3b, loading buffers pH at 2.5, 2.7, 3.0 and 3.5 surprisingly exhibited a similar selectivity, ranging from 94 to 97%. Furthermore, we observed tendency of increased number of phosphopeptides retained

at higher pH; 180 phosphopeptides at pH 3.5 compared with 133 phosphopeptides at pH 2.5. These results strongly demonstrated that the number of identified phosphopeptides is critically dependent on the pH; the pH must be high enough for all the phospho-amino acids to carry negative charges, thus, ensuring complete binding to the IMAC resin. Therefore, relatively high pH as well as high concentrations of AA are essential for high selectivity and recovery of phosphopeptides. Structure of Acids Determine Elution Efficiency. Different acids such as 2,5-dihydroxybenzoic acid (DHB)21 and acid containing hydroxyl groups24 or carboxylic acid groups23 have been reported to inhibit the nonspecific binding of acidic peptides to metal oxide chromatography. To assess how IMAC releases the bound phosphopeptides and to study whether the acid composition is responsible for this intriguing competition effect, we compared loading buffer containing three different organic acids, acetic acid (AA, pKa: 4.76), formic acid (FA, pKa: 3.75), trifluoroacetic acid (TFA, pKa: 0.3), and one inorganic acid, HCl. The three organic acids have one common carboxylic acid and different alkyl groups. Figure 4a compares the number of isolated phosphopeptides in loading buffer containing different acids (pH ) 3.0) and eluted by 200 mM ammonium phosphate. The use of HCl, TFA and AA identified approximately the same number of phosphopeptides, among which only AA had nearly 100% specificity, whereas HCl had lowest specificity (75%). The low specificity in HCl may be attributed to the lack of a carboxylic group in HCl for chelating Fe3+ to effectively compete with nonphosphopeptide binding. We originally hypothesized that TFA would yield the highest selectivity due to its low pKa, where a greater number of negative carboxyl groups could better inhibit nonphosphopeptides binding. Interestingly, TFA exhibited a lower selectivity (83%) than AA, which strongly suggests that either electron Journal of Proteome Research • Vol. 7, No. 9, 2008 4063

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Figure 4. The structure of acid determines elution efficiency. (a) Number (bar graph and left y-axis) and specificity (line graph and right y-axis, number of phosphopeptide/total number of identified peptides) from H1299 cell lysate in loading buffer containing 6% HCl, 6% TFA, 6% AA or 6% FA (pH ) 3.0). (b) The distribution percentages of identified monophosphopeptide, polyphosphopeptide, and nonphosphopeptide under sequential elution conditions. Phosphopeptides were first bound in 6% HCl loading buffer. The bound phosphopeptides and nonphosphopeptides were then sequentially eluted by different buffers in the following order: 6% TFA, 6% AA, 6% FA and then 200 mM NH4H2PO4.

withdrawal or the steric hindrance effect of the fluoride groups of TFA may play a more important role in competing with nonphosphopeptides for coordination with Fe3+. The purification selectivity of FA (99%) was comparable to that of AA (98%), but the number of identified phosphopeptides in FA (n ) 30) dramatically decreased as compared with AA (n ) 159). FA has a low pKa and reduced steric hindrance, which facilitates coordination with Fe3+. Therefore, our results suggest that FA not only competes with nonphosphopeptides, but also hinders binding of phosphopeptides and results in low recovery. This hypothesis can be further confirmed by the occurrence of identified mono- and polyphosphorylated peptides and nonphosphopeptides using different acidic loading buffers (Supplementary Figure 2). The use of FA identified mostly polyphosphorylated peptides with only 9% monophosphorylated peptides; most monophosphorylated peptides were lost in FA-containing loading buffer. On the contrary, the use of TFA, AA and HCl resulted in mostly monophosphorylated peptides. This result strengthens our hypothesis that the commonly used FA buffer is not appropriate because it competes with phosphopeptides for binding to Fe3+, greatly diminishing their recovery. The above result further inspired us to compare the competitive affinity between phosphopeptides, AA, TFA, FA and NH4H2PO4 with Fe3+-NTA. The peptides from the H1299 lysate were first bound to the IMAC column with 6% HCl loading buffer, then the trapped phosphopeptides were sequentially 4064

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Tsai et al. eluted by TFA, AA, and FA followed by NH4H2PO4. As shown in Figure 4b, both TFA (first elution) and AA (second elution) eluted mostly weakly bound nonphosphopeptides (TFA: n ) 166; AA: n ) 22) and a small percentage of monophosphorylated peptides (TFA: n ) 23; AA: n ) 13), suggesting that TFA and AA do not effectively compete with phosphopeptides. In the third sequential elution by FA, the eluted peptides were predominantly monophosphopeptides (n ) 78) with negligible nonspecific binding. Fourth elution with NH4H2PO4 yields most effective release of phosphopeptide including 30 mono- and 44 polyphosphopeptides. On the basis of the above results, we concluded that the order of competition with phosphopeptide for binding to Fe3+ is NH4H2PO4 > FA > phosphopeptides > AA > TFA > HCl. Sample Recovery of the Optimized IMAC System. In previous IMAC protocols, high selectivity is often accompanied with low sample recovery, or vice versa.10,14 On the contrary, we expect that the pH/acid-controlled IMAC protocol developed in this study can simultaneously achieve high purification selectivity with low sample loss. To quantitatively achieve a trustworthy estimation of capacity and recovery in IMAC column, we compared the peak areas of extracted ion chromatogram (XIC) of the purified phosphopeptide mixture after first-round and second-round of IMAC purification. Minimum sample loss, that is, unchanged area of XIC for every phosphopeptides, is expected for a good IMAC protocol with high sample recovery. The H1299 cell lysate (200 µg) with two spiked internal standard proteins, β-casein and R-casein, were digested with trypsin using TEABC buffer, and then subjected to the IMAC purification in 6% (v/v) AA at pH ) 3.5. The purified phosphopeptides from β-casein, R-casein and cell lysate were desalted and divided into two parts. It is noted that desalting was performed to avoid interference of the elution buffer, ammonium phosphate, to the following second-round IMAC purification. One part was directly analyzed by triplicate LCMS/MS as a control set, whereas the other part was subjected to second-round IMAC purification, desalted and then analyzed by triplicate LC-MS/MS runs. The recovery of this IMAC column was computed by the peak area ratio of XIC from the two parts of purified phosphopeptides. On the basis of three standard phosphopeptides, FQpSEEQQQTEDELQDK, VPQLEIVPNpSAEER and TVDMEpSTEVFTK from β-casein and R-casein, we estimated that the optimized IMAC procedure has about 100 ( 16% recovery (Supplementary Table 1). Even for complex phosphopeptide in H1299 cell lysate, an average of 81 ( 18% recovery can be achieved for the identified 150 phosphopeptides from H1299 cell lysate (data not shown). The sequence of the major phosphopeptides, FQSEEQQQTEDELQDK, has multiple glutamic acids and aspartic acids. To exclude the possibility that the above high recovery arose from the high affinity of acidic peptide (FQSEEQQQTEDELQDK) with IMAC, we further compared the IMAC enrichment before and after dephosphorylation procedure. By Mascot database search, only phosphopeptides (FQpSEEQQQTEDELQDK) was identified whereas the dephosphorylated counter peptide was not identified. The computed XIC area of dephosphorylated (XIC area ) 34) was negligible compared with the phosphopeptide (XIC area ) 2280), demonstrating the enrichment specificity by pH-acid controlled method. The collective data demonstrated the low sample loss of our IMAC protocol. Finally, the reported protocol

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Immobilized Metal Affinity Chromatography Revisited

Figure 5. Increase of number of identified phosphoproteins from 550 µg of H1299 cell lysate by extended gradient in LC-MS/MS. The number of identified phosphoproteins is shown in bar graph (left y-axis) with phosphopeptides indicated within each bar. The enrichment specificity is shown in line graph (right y-axis). The analysis was performed using the pH/acid-controlled IMAC procedure and single LC-MS/MS analysis. The LC gradients were performed at 80, 120, 180, 210 and 270 min.

has maximum capacity to adsorb approximately 1 mg of lysate using a 10 cm microcolumn (i.d.: 500 µm). Large Scale Phosphoproteome Profiling of Non-SmallCell Lung Cancer Cell. To evaluate the performance of the optimized IMAC on a proteomic scale, H1299 cell lysate was digested with trypsin using TEABC buffer, subjected to the IMAC purification and analyzed by a single LC-MS/MS run. To increase the identification coverage of the phosphoproteome, we first attempted to increase the LC gradient to identify more phosphopeptides. As shown in Figure 5, single LC-MS/ MS analysis of 550 µg lysate (the protein concentration was determined by BCA assay) using a 80-, 120-, 180-, 210-, or 270min gradient identified 244 phosphoproteins/361 phosphopeptides, 258 phosphoproteins/403 phosphopeptides, 286 phosphoproteins/442 phosphopeptides, 386 phosphoproteins/637 phosphopeptides and 376 phosphoproteins/596 phosphopeptide, respectively. The extension of LC gradient effectively increased the number of phosphoproteins. We have provided a list of all the detailed information regarding the identification confidence and phosphorylation sites as well as MS/MS spectra of unique peptides in Supplementary Table 2 and Supplementary Figure 4. The false discovery rates as determined by decoy database search ranged from 0.42% to 0.94%. Most interestingly, nearly 100% enrichment specificity (96∼97%) from the cell lysate was observed under all conditions. We were also interested in evaluating any bias toward global enrichment of different phosphoamino acids from the whole cell phosphoproteome. The observed proportion of phosphotyrosine (7%) was higher than the expected percentage of F5 ≈ 40 kDa) than in the lower molecular-weight gel bands (Figure 6b). Taking into account the nondegenerate phosphopeptides (indicated in each bar Figure 6b) and the corresponding number of phosphoproteins in each gel band, the average number of phosphopeptides per protein ranged from 1.5 to 2.2 phosphopeptides. For all gel slices, monophosphorylation sites represented the major group (>76%). However, doubly phosphopeptides and poly phosphopeptides ranged from 4.8 to 24.3% (Figure 6c). Supplementary Table 2 lists all the detailed information regarding the identification confidence and phosphorylation sites and MS/ MS spectra of nondegenerate peptides are provided in Supplementary Figure 4. These results demonstrate that simple onestep IMAC in combination with SDS-PAGE and LC-MS/MS provides a large-scale and highly specific strategy for comprehensive profiling of phosphoproteomes.

Discussion Identification of large numbers of phosphopeptides with high specificity, reproducibility and recovery is critical in phosphoproteomics. Despite the long history of IMAC development and its advantages for enrichment of all phosphorylation types, phosphotyrosine, phosphoserine and phosphothreonine, the use of one-step IMAC has gradually lost popularity because acidic residues are typically copurified with phosphopeptides and lead to a reduction in both selectivity and recovery. To address the drawback, many alternative methods have rapidly evolved to replace IMAC purification, such as chemical modification, metal oxide chromatography and tandem purification. Many groups reported that methylation prior to IMAC purification efficiently enhances selectivity.15,41 However, robust purification may not be achieved due to the additional chemical reaction; a wide range of selectivity (60-87%) was reported in literature. Furthermore, inevitable sample loss16,17 will reduce the number of purified phosphoproteins. In our experience, more than 50% sample loss was observed after methyl esterification (data not shown). In other one-step phosphopeptide purification methods, most of the enrichment specificity is also unsatisfactory (3.5). This observation also explains why most of the previous protocols that use pH 3.0 and under have less satisfactory enrichment recovery.14,4 Under higher pH, however, the accompanying lower selectivity has to be overcome because both phosphopeptides and nonphosphopeptides bind to the Fe3+-NTA (Figure 7c). The release of bound nonphosphopeptides and retaining of phosphopeptides can be controlled by selection of the structure and concentration of acid. We demonstrated that use of high concentrations of AA (6%) with more carboxylic acid groups provides the most prominent inhibition for nonspecific binding by effective competition predominantly with nonphosphorylated peptides for binding sites on IMAC (Figure 7d, bottom part). The use of FA runs the risk of significantly reducing sample recovery. Conceivably, the physically smaller and

negatively charged carboxylic acid groups in FA should have stronger coordination with the Fe3+,47 and naturally repel nonphosphopeptides along with existing monophosphopeptides. The different binding affinities indicated that AA and TFA can be used as washing buffers, whereas the stronger affinity of FA for Fe3+ makes it more appropriate for an elution buffer rather than a washing buffer. In the elution step shown in Figure 7d (top part), the NH4H2PO4 (pH 4.4) achieves the best elution efficiency, where the phosphate groups effectively repel not only mono- phosphopeptides but also the poly phosphopeptides from the Fe3+NTA IMAC system. The sequential elution based on different affinity was reported very recently by Thingholm et al.; the multiphosphopeptides were bounded on iron ion stronger than monophosphopeptides.25 They demonstrated monophosphopeptides could be first eluted by low pH (the optimal pH was 1.0, 1% TFA) and the remaining bound multiphosphopeptides were eluted by basic condition (NH4OH, pH 11.3). Even under same pH, however, we found that eluent structures play important role for elution of phosphopeptides. On the basis of the observed order of binding affinity with iron metal ion, phosphopeptides with different number of phosphorylation sites may be eluted to different extent depending on the structures of organic acid (at same pH) or phosphate salt. Sequential elution by the order of TFA, AA, FA and NH4H2PO4 may potentially fractionate the phosphopeptides and increase the number of identified phosphopeptides toward a comprehensive profile of the complex phosphoproteome. Journal of Proteome Research • Vol. 7, No. 9, 2008 4067

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Conclusions Efficient and simple enrichment is a prerequisite for profiling or quantitation of transiently phosphorylated proteins. The presented IMAC protocol demonstrated enrichment with high specificity and low sample loss without an additional esterification and desalting step. This procedure may be applicable to a variety of materials such as tissue, cell and body fluid. Given the high sample recovery and large number of phosphopeptides demonstrated by the present IMAC protocol, it is interesting to compare the complimentary role in phosphopeptide enrichment with other methods. In the future, the combination of this protocol with either stable isotope tagging or a label-free technique may be employed for large-scale comparative proteomic studies to decipher the dynamic and heterogeneous phosphoproteome.

Acknowledgment. This work was supported by Academia Sinica and the National Science Council in Taiwan. We thank Chuan-Yih Yu and Chih-Chiang Tsou from the Institute of Information Science, Academia Sinica, for developing MIB (MS/MS Identification Information Builder) to generate the annotated MS/MS spectra and preparing the supplementary data.

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Supporting Information Available: The amino acid

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composition of the identified nonphosphopeptides is shown in Supplementary Figure 1. Supplementary Figure 2 shows the percentage of identified mono- and polyphosphorylated peptides in different acidic conditions. The recovery of IMAC and distribution of identified phosphoserine, phosphothreonine and phosphotyrosine are shown in Supplementary Table 1 and Figure 3. The annotated MS/MS spectra of identified nondegenerate phosphopeptide in the large-scale profiling of H1299 cells are shown in the Supplementary Figure 4 (http://proj3.sinica.edu.tw/∼yujuchen/2008JPR_ IMAC/Supplementary_Figure_4.html). A complete list of identified phosphopeptides and phosphoproteins along with detailed information are in Supplementary Table 2. This material is available free of charge via the Internet at http:// pubs.acs.org.

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