Improved Titanium Dioxide Enrichment of Phosphopeptides from HeLa Cells and High Confident Phosphopeptide Identification by Cross-Validation of MS/MS and MS/MS/MS Spectra Li-Rong Yu,*,† Zhongyu Zhu,‡,§ King C. Chan,† Haleem J. Issaq,† Dimiter S. Dimitrov,§ and Timothy D. Veenstra† Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, Basic Research Program, SAIC-Frederick, Inc., NCI-Frederick, and Nanobiology Program, NCI-Frederick, P.O. Box B, Frederick, Maryland 21702 Received March 18, 2007
Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH4Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH4Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate. Keywords: Phosphoproteomics • titanium dioxide • phosphopeptide enrichment • HeLa cells • tandem mass spectrometry • neutral loss scan
Introduction Reversible phosphorylation represents a major mechanism of signal transduction in a variety of cellular functions including cell proliferation,1 apoptosis,2 cell cycle control,3,4 DNA damage response,5 etc. These basic functions are integrated within a cell to form an entire network, and hence, any perturbation could potentially trigger a series of responses in protein phosphorylation. Such a cell signaling network is even more complicated, and poorly understood, in many diseases such as cancer, AIDS, diabetes, and neuronal disorders. It would be advantageous, therefore, to investigate the whole signaling network comprised by numerous phosphoproteins (i.e., phosphoproteome) to understand the mechanisms of disease development as well as basic cellular functions at a systemic * Corresponding author. Tel.: (301) 846-7607; fax: (301) 846-6037; email:
[email protected]. † Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, Inc., NCI-Frederick. ‡ Basic Research Program, SAIC-Frederick, Inc., NCI-Frederick. § Nanobiology Program, NCI-Frederick.
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level. Although protein phosphorylation is prevalent in cells, the event typically occurs at low stoichiometry.4,6 While fulfillment of a protein’s function by multiple phosphorylation has been well-documented,4,7,8 the critical role of a single phosphorylated site is not uncommon for a particular protein or cellular function as revealed in cell cycle and drug resistance studies, for example.9-13 It is apparent that novel strategies need to be developed and improved to enrich phosphoproteins or phosphopeptides without any bias toward either singly or multiply phosphorylated species for phosphoproteomic analysis. Great efforts have been made to enrich phosphorylated proteins and peptides from biological samples. In the past several years, chemical modifications to the phosphate moiety of phosphopeptides via β-elimination in solution14-16 or a covalent capture and release approach on a solid phase17-21 have been developed. Owing to the complexity and potential side reactions of these chemical approaches, a limited number of phosphoproteins has been identified. Conventional immunoprecipitation has been applied as an alternative strategy to 10.1021/pr070152u CCC: $37.00
2007 American Chemical Society
research articles
TiO2 Enrichment and Identification of Phosphopeptides
phosphoprotein or phosphopeptide enrichment.22-26 Currently, these approaches are mainly suitable for phosphotyrosine (pTyr) analysis because of the availability of robust anti-pTyr antibodies; however, motif-based anti-phosphoserine (pSer) and anti-phosphothreonine (pThr) antibodies may also be used to pull down a subset of the phosphoproteome. Recently, an approach to analyzing early eluted fractions from strong cationexchange (SCX) chromatography has been employed as an alternative strategy to enrich phosphopeptides.6,27 Unfortunately, many phosphopeptides are eluted in the later SCX fractions as well,28 leading to the loss of a fair number of phosphopeptides with multiple net positive charges. More recently, dendrimers have been used to covalently bind phosphopeptides, which are then released via acid hydrolysis.29 Importantly, the traditional phosphoprotein purification method, immobilized metal affinity chromatography (IMAC),30 has been improved to enrich phosphopeptides followed by subsequent analysis using mass spectrometry.31 IMAC takes advantage of the strong affinity between metal ions and phosphate groups; however, the carboxylate groups in acidic amino acids (i.e., Glu and Asp) significantly contribute to non-specific bindings. The non-specific bindings have been reduced by converting carboxylate groups to corresponding methyl esters;31 however, incomplete esterification and accompanied side reactions28,32,33 introduce additional complexity to an already complicated peptide mixture. A recent study claimed superior conversion of carboxylate groups to their methyl esters;34 however, no data were presented to support such a claim. Moreover, the observed inconsistency in phosphopeptide recovery and enrichment efficiency between different laboratories35-37 suggests that the technique lacks robustness. Despite these drawbacks, IMAC has been extensively used to characterize phosphoproteomes qualitatively and quantitatively from a variety of biological systems.38-51 IMAC is relatively simple to perform and has great potential in large-scale analysis to achieve broad coverage of a phosphoproteome. Recently, titanium dioxide (TiO2) has been demonstrated to have a high affinity and specificity to phosphopeptides in simple peptide mixtures.33,52-54 As compared to IMAC, time would be saved in the procedure of phosphopeptide enrichment using TiO2. However, non-specific binding is an issue when complex peptide mixtures (e.g., digests of cell lysates) are applied to TiO2 columns. To minimize the non-specific bindings, Larsen et al. used a buffer containing 2,5-dihydroxybenzoic acid (DHB) to load samples as well as for subsequent washes of the TiO2 column.33 To date, however, only a few applications using TiO2 to enrich for phosphopeptides from complex mixtures have been reported,55-60 and DHB has been used to reduce non-phosphopeptides for most of the applications. Further evaluation for the performance of TiO2 (e.g., specificity, recovery, etc.) in phosphopeptide enrichment from complex peptide mixtures, especially without methylation, would provide valuable information and better application of this technology to the analysis of phosphoproteomes. The objective of the present study was to evaluate and improve the specificity of TiO2 for phosphopeptide enrichment from both simple and complex proteomic mixtures. Phosphopeptides were isolated using TiO2 chromatography and analyzed by nano-LC-MS/MS (MS2) and data-dependent neutral loss MS/MS/MS (MS3).6,61 Significant improvement in TiO2 phosphopeptide enrichment was observed using ammonium glutamate to remove non-phosphopeptides without the need for methyl esterification. Highly confident identification of
phosphopeptides from the extracted proteins of HeLa cells was obtained by cross-validation using both MS2 and MS3 data.
Materials and Methods Materials. Ammonium bicarbonate (NH4HCO3), ammonium chloride (NH4Cl), ammonium hydroxide (NH4OH, 28% solution), bovine R-casein, bovine β-casein, L-glutamic acid ammonium salt (NH4Glu), phosphatase inhibitor cocktails (I and II), protease inhibitor cocktail, and Tris were purchased from Sigma-Aldrich (St. Louis, MO). DHB, L-glutamic acid, and trifluoroacetic acid (TFA) were from Fluka (Milwaukee, WI). HPLC grade acetonitrile (CH3CN) was obtained from Fisher Scientific (Fair Lawn, NJ). A bicinchonic acid (BCA) protein assay reagent kit was purchased from Pierce (Rockford, IL). Water was purified by a Barnstead Nanopure system (Dubuque, IA). Casein Sample Preparation. A 3 mg/mL stock solution of bovine R-casein or β-casein was prepared by dissolving weighed lyophilized protein into 25 mM NH4HCO3, pH 8.3. Bovine R-casein and β-casein were mixed at a ratio of 2:1 (w/w), and the casein mixture was digested for 6 h at 37 °C with sequencing grade modified trypsin (Promega, Madison, WI) at a ratio of 50:1 (w/w, protein/trypsin). The digestion reaction was terminated by adding 0.2% TFA, and the sample was lyophilized and stored at -80 °C. Cell Culture and Proteome Sample Preparation. The human cervical epithelial cancer cell line HeLa (ATCC, Rockville, MD) was cultured in Dulbecco’s modified Eagle’s medium (Quality Biologicals, Gaithersburg, MD) supplemented with 10% cosmic calf serum (CCS) (HyClone, Logan, UT), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (DMEM-10). Cell cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere to reach 90% confluence. The cultured cells were harvested and lysed in a lysis buffer of 8 M urea and 50 mM Tris-HCl, pH 8.3, with protease (40× dilution) and phosphatase inhibitor cocktails (100× dilution). The lysate was centrifuged at 18 000g for 15 min at 4 °C, and the supernatant was collected. The protein concentration was determined by BCA assay. The protein sample was digested for 8 h at 37 °C with sequencing grade modified trypsin (Promega, Madison, WI) at a ratio of 40:1 (w/w, protein/ trypsin). The resulting peptide mixture was desalted using high capacity C18 cartridges (Alltech Associates Inc., Deerfield, IL) and lyophilized. Trypsin-Catalyzed 16O-/18O-Labeling. For 18O-labeling, 50 µL of 50% CH3CN in 18O-enriched water (95%, Isotec, Miamisburg, OH) was first added to each 300 µg of dried peptides from casein digestion or the digested HeLa cell lysates, followed by the addition of 100 µL of 18O-water. Sequencing grade modified trypsin (Promega, Madison, WI) dissolved in 18O-water was added to the samples at a ratio of 30:1 (w/w, protein/trypsin), and the mixtures were incubated at 37 °C for 15 h. The reactions were quenched by boiling the samples for 10 min in a water bath, followed by addition of 0.2% TFA. An identical procedure was carried out in parallel for 16O-labeling of the same amounts of peptides in which the regular 16O-water was used instead of 18O-water. The 16O- and 18O-labeled samples were lyophilized separately and stored at -80 °C for further analysis. Phosphopeptide Isolation Using TiO2 Columns. To evaluate phosphopeptide isolation efficiency for simple peptide mixtures, TiO2 microcolumns were slurry packed using 5 µm TiO2 particles (GL Sciences Inc., Tokyo, Japan) against the PEEK Journal of Proteome Research • Vol. 6, No. 11, 2007 4151
research articles tubings with inner diameters (i.d.) of 0.5 and 1.0 mm. The casein digest was dissolved in 0.1%TFA, and 3-600 µg of the digest was loaded onto 0.5 mm i.d. × 50 mm or 1.0 mm i.d. × 50 mm TiO2 microcolumns. To compare the efficiency of removing non-phosphopeptides, the 0.5 mm i.d. × 50 mm TiO2 columns were washed with 15 bed volumes of one of the following solutions: 0.1% TFA, 50-200 mM DHB in 80% CH3CN/0.1% TFA, 80% CH3CN/0.1% TFA, or 50-200 mM NH4Glu, pH 2.0 (pH was adjusted using HCl). Phosphopeptides were eluted using 10 bed volumes of 0.5-1.5% NH4OH and lyophilized. To enrich phosphopeptides from complex peptide mixtures, the HeLa cell lysate digestate was dissolved in 50% CH3CN/0.1% TFA, and 400 µg of the peptides was loaded onto a 4 mm i.d. × 20 mm TiO2 column (GL Sciences Inc., Tokyo, Japan) followed by a column wash with 20 bed volumes of 0.1% TFA or 100-200 mM NH4Glu, pH 2.0. Phosphopeptides were eluted using 10 bed volumes of 1.5% NH4OH and lyophilized. To assess the phosphopeptide recovery during TiO2 enrichment, 3 µg of 16O-labeled casein peptides was loaded onto a 0.5 mm i.d. × 50 mm TiO2 column, followed by a column wash of one of the following solutions: 0.1% TFA; 50, 100, or 200 mM NH4Glu, pH 2.0; or 130 mM DHB in 80% CH3CN/ 0.1% TFA. Phosphopeptides were eluted using 1.5% NH4OH and lyophilized. The enriched phosphopeptides were dissolved in 0.1% TFA and mixed with 3 µg of 18O-labeled casein peptides prior to LC-MS2-MS3 analysis. Equivalent amounts of 16Olabeled and 18O-labeled whole casein digests (without TiO2 enrichment) were combined as well and subjected to LC-MS2MS3 analysis for normalization purposes. Nano-RPLC-MS2-MS3 of Peptides. Nano-flow RPLC separation of peptides was conducted using a 10 cm long × 75 µm i.d. fused silica capillary electrospray ionization (ESI) column62 that was coupled online to a 3-D ion-trap (IT) MS (LCQ Deca XP, Thermo Electron, San Jose, CA) for MS/MS analysis or to a 2-D linear IT (LIT) MS (LTQ, Thermo Electron, San Jose, CA) for MS/MS/MS analysis (nano-RPLC-MS2-MS3). For the analysis of 16O-/18O-labeled samples, a 7 T hybrid linear IT-Fourier transform ion cyclotron resonance MS (LTQ-FT, Thermo Electron, San Jose, CA) was employed. The ESI column was slurry packed with 5 µm, 300 Å pore sized Jupiter C18 RP particles (Phenomenex, Torrence, CA) against a 10 cm × 75 µm i.d. fused-silica capillary (Polymicro Technologies, Phoenix, AZ) with a flame-pulled fine i.d. (i.e., 5-7 µm) tip. Mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in CH3CN) were delivered by an Agilent 1100 nano-flow LC system (Agilent Technologies, Palo Alto, CA). Trypsin digests or TiO2-enriched peptides were loaded in 30 min while the column was maintained with 2% solvent B at a flow rate of ∼250 nL/min. The peptides were eluted using a step gradient: 2-42% solvent B in 40 min, 42-98% solvent B for 10 min, and 98-98% solvent B for 5 min. The mass spectrometers were operated in a data-dependent mode to automatically switch between MS and MS2 for the 3-D IT-MS and switch between MS, MS2, and neutral loss-dependent MS3 for the 2-D LIT-MS and LTQ-FT MS. Following the MS survey scan with m/z 3501800, MS2 spectra were sequentially and dynamically acquired for the three or seven most intense peptide molecular ions in the MS scan for the 3-D IT-MS or 2-D LIT-MS and LTQ-FT MS, respectively. For LTQ-FT MS, the MS survey scan was performed in the Fourier transform ion cyclotron resonance (FTICR) portion with a resolution of 5 × 104, and the MS2 and MS3 scans were acquired in the LTQ portion. Data-dependent neutral loss MS3 was triggered when a neutral loss of phos4152
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phoric acid (H3PO4) was detected with a loss of m/z 98, 49, or 32.7 among the seven most intense fragment ions in the MS2 scan. The normalized collision energy was 35% for both MS2 and MS3 collision-induced dissociation (CID). The electrospray voltage was set at 1.6 kV, and the voltage and temperature for the ion source capillary were set at 45 V and 160 °C, respectively. Peptide Identification. The raw MS2 and MS3 data were searched using TurboSEQUEST (Thermo Electron, San Jose, CA) on a Beowulf 18-node parallel virtual machine cluster computer, against a protein database including bovine R-S1casein, R-S2-casein, β-casein, and κ-casein for the casein samples or against the human protein database (37 542 protein sequence entries) downloaded from the European Bioinformatics Institute (EBI) (http://www.ebi.ac.uk/proteome/index.html) for the samples from HeLa cells. A peptide mass tolerance of 2.0 Da for IT-MS data and 0.08 Da for FTICR-MS data and a fragment ion tolerance of 1.0 Da were allowed with tryptic specificity allowing two missed cleavages. Dynamic phosphorylation of Ser, Thr, and Tyr by mass addition of 79.9663 Da was set in a single search when searching MS2 data, while dynamic phosphorylation of Ser, Thr, and Tyr (+79.9663 Da) as well as dynamic loss of water (-18.0106 Da) from Ser and Thr were set when the MS3 data were searched. A dynamic 4.0085 Da modification on the C-terminus was also set for the 16 O-/18O-labeled samples. In addition, dynamic oxidation of Met by the addition of one oxygen (+15.9949 Da) and two oxygens (+31.9898 Da) was included when searching the MS2 and MS3 data from the casein samples. To evaluate false positive peptide identifications,63 the MS2 and MS3 data from HeLa cells were also searched against the reversed human protein database using the same searching parameters as those used for searching the normal database. The SEQUEST filtering criteria for initial identification of fully tryptic peptides from MS2 data were Xcorr g1.7 for [M + H]1+ ions, g2.1 for [M + 2H]2+ ions with a molecular weight (Mw)
