Ethylenediaminetetraacetic Acid Increases Identification Rate of Phosphoproteomics in Real Biological Samples Tatsuji Nakamura,†,§ Khin Than Myint,†,§ and Yoshiya Oda*,‡,§ Eisai Company, Ltd., Biomarkers and Personalized Medicine Unit, Tsukuba, Ibaraki 300-2635, Japan, Eisai Company, Ltd., Biomarkers and Personalized Medicine Unit, Andover, Massachusetts 01810, and Core Research for Evolutional Science and Technology, Japan Science and Technology, Saitama 332-0012, Japan Received October 12, 2009
We have developed a novel approach to enhance phosphopeptide identification in liquid chromatography/mass spectrometry (LC/MS)-based phosphoproteomics. After enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO2) microcolumns, samples were coinjected with ethylenediaminetetraacetic acid (EDTA) into LC/MS. This procedure decreased the MS peak intensity of nonphosphorylated peptides, but not that of phosphopeptides, and as a result, the number of identified phosphopeptides was increased. EDTA appeared to have no effect on liquid chromatographic separation of phosphorylated and nonphosphorylated peptides. Although the mechanism of the positive effect of EDTA on identification of phosphopeptides is unknown, and we have never observed metal ion adduct peaks in LC/MS spectra, coinjection of EDTA seemed to enhance phosphopeptide recovery from the LC/MS system. This simple technique was successfully applied to the identification of phosphopeptides in mouse brain (2938 phosphopeptides), human plasma (127 phosphopetides), and human cerebrospinal fluid (CSF) (123 phosphopeptides). We also identified nonphosphopeptides in the same samples using a two-dimensional (2D) LC/MS-based shotgun approach. The results overall indicated that 20-25% of brain proteins were phosphorylated, while only 1-2% of proteins in plasma and CSF were phosphorylated. These ratios were almost constant throughout the range of protein expression levels. In addition, EDTA-enhanced phosphoproteomics could identify low-abundance proteins in the samples, because nonphosphoproteins corresponding to more than one-third of the identified phosphoproteins could not be identified by 2D-LC/MS. Finally, we were able to find that the newly developed approach was very effective for the phosphoproteome analysis in Alzheimer disease model mice brain. Keywords: Phosphoproteomics • Phosphorylation • Mass spectrometry • Immobilized metal affinity column • Titanium oxide • EDTA • Alzheimer’s Disease
Introduction Protein phosphorylation plays a significant role in a wide range of cellular processes, including growth, metabolism and differentiation,1,2 and identification of phosphorylation sites is important for elucidating signaling pathways, for molecular classification of diseases, and for profiling of kinase inhibitors.3 Therefore, monitoring the phosphoproteomic profiles of cells in different physiological states is important for cell biology. The identification of phosphorylation sites is usually accomplished by mass spectrometry (MS) owing to its high sensitivity, specificity and speed, compared with conventional biochemical methods.4-6 However, complete characterization of phosphorylation profiles is still challenging due to both biological and analytical limitations. Many phosphoproteins are present in low abundance, and their relative abundance may * To whom correspondence should be addressed. E-mail, yoshiya_oda@ eri.eisai.com; tel., +1-978-837-4926; fax, +1-978-837-4863. † Eisai Co. Ltd., Japan. § Japan Science and Technology. ‡ Eisai Co., Ltd., Andover, MA. 10.1021/pr900918h
2010 American Chemical Society
change very quickly.7 In addition, the presence of large amounts of nonphosphopeptides can suppress ionization of phosphopeptides in positive-mode MS detection or mask phosphopeptides, preventing their identification. In addition, phospho-Ser/Thr peptides intrinsically tend to undergo neutral loss of phosphate during collision-induced dissociation, producing neutral loss peak-dominated MS/MS spectra. Therefore, product ions of low-intensity precursors of phosphopeptides often show MS/MS spectra that are too poor in quality to allow sequence identification, and this results in many false-negative as well as false-positive identifications.8 To overcome these difficulties, it is essential to develop highly sensitive and selective methods. Many techniques for phosphopeptide enrichment prior to MS analysis have been described, and these include immunoprecipitation with phospho-specific antibodies,9,10 chemical modifications such as β-elimination,11 immobilized metal affinity chromatography (IMAC)12,13 strong-cation exchange (SCX) chromatography,14 and titanium dioxide (TiO2) chromatography.15-17 It is currently possible to identify thousands of phosphopeptides by using Journal of Proteome Research 2010, 9, 1385–1391 1385 Published on Web 01/25/2010
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Nakamura et al. 18,19
combinations of these methods. However, most of the reported methods show sometimes poor reproducibility among experiments/laboratories. Here we report a simple, reproducible procedure employing ethylenediaminetetraacetic acid (EDTA), which is frequently used in biological experiments as a chelating agent to remove metal ions and to inactivate metaldependent enzymes. EDTA is also used to remove trace metal ion contaminants from tubing and columns in liquid chromatography (LC) systems. We found that a larger number of phosphopeptides could be reproducibly identified in a LC/MS system by coinjection of EDTA with phosphopeptide-enriched samples obtained from both IMAC/C18 and TiO2/C18 microcolumns, compared with the same samples in the absence of EDTA.
Materials and Methods Acetonitrile, methanol, trifluoroacetic acid (TFA), acetic acid, formic acid, urea, dithiothreitol (DTT), iodoacetamide, and Lys-C were obtained from Wako Pure Chemical Co. (Osaka, Japan). Sequence-grade modified trypsin was obtained from Promega (Madison, WI). The water used for preparing the mobile phases was purified using a Milli-Q SP TOC (Millipore, Marlborough, MA). NTA-agarose was obtained from Quiagen (Hilden, Germany). Titania (titanium dioxide; particle size: 10 µm) was obtained from GL Science (Tokyo, Japan). Protease inhibitor cocktail and phosphatase inhibitor cocktail were purchased from Roche Diagnostics (Basel, Switzerland) and Sigma-Aldrich Co. (St. Louis, MO), respectively. Human plasma and human cerebrospinal fluid (CSF) were purchased from PrecisionMed. Inc. (San Diego, CA). Other reagents of analytical grade were used without further treatment. Preparation of PC-9 Cell Cytoplasmic Protein. PC-9 cells were grown in RPMI-1640 medium, supplemented with 10% fetal bovine serum plus antibiotics. For each experiment, one dish (5 × 107 cells/15-cm-diameter dish) of confluent cells was used. Protein in phosphate buffer saline (PBS) samples were obtained from PC-9 cell homogenate which were prepared with a Teflon Potter-type homogenizer in the presence of a protease inhibitor cocktail and phosphatase inhibitor cocktails, and the soluble fraction was collected as the supernatant after centrifugation (100 000g) with a Hitachi Himac CS150GXL (Tokyo, Japan). Animals. Four, 15, and 18 month old Tg2576 mice (Taconic Farms, Inc. Germantown, NY) were used in this study which contained human APP695 bearing the double mutation K670N, M671L that was found in a large Swedish family with early onset Alzheimer’s disease with wild-type C57B6 mice as controls. Preparation of Mouse Brain Protein. Whole brains from Tg2576 and C57B6 mice were homogenized with PBS in a Teflon Potter-type homogenizer in the presence of a protease inhibitor cocktail and phosphatase inhibitor cocktails, and centrifuged at 710g for 10 min to separate the nuclear fraction. The supernatant was centrifuged at 100 000g for 1 h to separate the membrane fraction and cytosol fraction. Preparation of Human Plasma and Cerebrospinal Fluid (CSF) Protein. Human plasma and CSF were concentrated through ultrafiltration membrane cartridge (Ultrafree, cutoff: 5K, Millipore, Marlborough, MA) after adding of 10 times volume of 50 mM ammonium bicarbonate. Protein Digestion. Protein amounts were measured using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). The pH of protein samples was adjusted by adding 500 mM Tris-HCl buffer (pH 8.5) and the solutions were made up 1386
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to 8 M urea (final concentration). These mixtures were reduced with DTT, alkylated with iodoacetamide, and digested with Lys-C (Wako, Osaka, Japan) after having been desalted on a PD-10 column (GE Healthcare, Buckinghamshire, U.K.) to remove phosphorylated small molecules, such as nucleotides and phospholipids. Then, after 4-fold dilution with 50 mM ammonium bicarbonate, trypsin was added and the mixtures were kept at 37 °C overnight. The digested solutions were desalted and concentrated using an Empore C18-SD disk cartridge (3M, MN) to remove hydrophilic phosphorylated molecules such as ATP and GTP. Preparation of IMAC Resin. Five milliliters of NTA-agarose suspended in 30% ethanol (50% v/v slurry) was added to 45 mL of 100 mM FeCl3 in 0.6% acetic acid and the mixture was incubated for 24 h at 4 °C under rotation. The resin was then washed 4 times with 20 mL of 0.6% acetic acid and finally resuspended in 5 mL of 0.6% acetic acid as a 33% slurry. Optimized Phosphopeptide Enrichment Procedure. Phosphopeptide enrichment was performed using custom-made biphasic chromatographic (IMAC/C18 or TiO2/C18) microcolumns according to the protocol described previously8,13 with some modification (Figure 1). Briefly, C18 Empore disk was packed into a 200 µL micropipet tip, and then 20 µL of IMAC resin or 25 µL of TiO2 suspended in methanol was loaded. Sample loading, washing, and elution were performed by centrifugation at 100g for 1-10 min. Acetonitrile/water/TFA (50/50/0.3, v/v/v, 200 µiL) was used to equilibrate the microcolumns. TiO2/C18 microcolumns were prewashed 3 times with 200 µL of acetonitrile/water/phosphoric acid (5/95/1, v/v/v) before equilibration. Protein samples of less than 0.5 mg were dissolved in 200 µiL of acetonitrile/water/TFA (50/50/0.3, v/v/ v). After sample loading, the microcolumns were washed with 100 µiL of acetonitrile/water/TFA (50/50/0.3, v/v/v) and then with 100 µL of acetonitrile/water/TFA (5/95/0.3, v/v/v). After that, acetonitrile/water/phosphoric acid (5/95/1, v/v/v) was added to transfer phosphopeptides from IMAC/TiO2 to the C18 Empore disk. After washing with 200 µL of acetonitrile/water/ TFA (5/95/0.1, v/v/v), phosphopeptides were eluted with 10 µL of acetonitrile/water/TFA (75/25/0.1, v/v/v). The eluted phosphopeptides were dissolved in 25 µg/mL EDTA, acetonitrile/water/TFA (5/95/0.1, v/v/v). In the mice brain extract, human plasma and human CSF studies, phosphopeptides were enriched from 1 mg of protein samples using IMAC/C18 and TiO2/C18 sequentially. Nano LC/MS. LC/MS was performed using an Ultimate 3000 nano LC pump (Dionex Co., Sunnyvale, CA), a HTC-PAL autosampler (CTC Analytics, Switzerland) and a LTQ-orbitrap (Thermo Electron, San Jose, CA) with an in-house-built nanosprayer (100 µm inner diameter, 6 µm opening, 150 mm length). ReproSil-Pur C18 materials (3 µm, 120 Å, Dr. Maish, Ammerbuch, Germany) were packed into the nanosprayer. The mass spectrometer was operated in the data-dependent mode to automatically switch between MS full scan and MS2 at a spray voltage of 2400 V. The mobile phases A and B for ODS separation at LC/MS consisted of 0.2% acetic acid in water and in water/acetonitrile (1:4), respectively. The gradient was 5% B (0-5 min), 5-40% B (5-80 min), 40-100% B (80-85 min), 100% B (85-95 min), and 5% B (95-115 min) at a flow rate of 500 nL/min. In the mice brain extract, human plasma and human CSF studies, three LC-MS/MS analysis per each sample were executed. 2D-LC/MS-Based Shotgun Proteomics. The flow-through fractions of mouse brain, human CSF and human plasma
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EDTA Increases Identification Rate of Phosphoproteomics
Figure 1. Flowchart of phosphopeptide enrichment strategy for phosphoproteomics.
samples obtained from the phosphopeptide enrichment procedure were analyzed by 2D-LC/MS-based shotgun proteomics as we reported before.20 Briefly, the flow-through fraction of each digested protein sample was fractionated on a polymerbased reversed-phase column using an acetonitrile gradient in an alkaline mobile phase as first-dimensional LC separation. Then, all the fractions from the first-dimensional separation were subjected to nano LC/MS as described above. Data Analysis. Mass ++ data analysis software was used to obtain a peak list, peak intensities and mass chromatograms (available at http://groups.google.com/group/massplusplus). Phosphopeptides were identified via automated database searching using Mascot v2.1 (Matrix Science, London. U.K.) against the NCBInr database, with a mass tolerance of 10 ppm for precursor ions, 0.4 Da for product ions and strict trypsin specificity. Carbamidomethylation was treated as a fixed modification of Cys residues, whereas oxidation of Met residues was allowed as a variable modification. One missed cleavage of trypsin was allowed. In the MS/MS spectral searches, phosphorylations of Ser, Thr, and Tyr were considered as variable modifications. MS/MS spectra of phospho-Ser/Thr peptides each showed a peak corresponding to the neutral loss of 98 Da from the precursor ion. Only peptides with clear mass spectra with a Mascot score of >99% reliability were considered in this study. Phosphorylated sites were determined based on Mascot scores of peptides. In phosphorylated sites, determinations by Mascot were not correct or ambiguous. MS/MS spectra were manually validated. Pseudo internal standard (PIS)21) was used for label-free quantitation of identified peptides. Briefly, the relative peak intensities of peptides of interest are compared with those of PIS. These standards are determined during a proteomics MS experiment and are represented by peaks that are present in a 1:1 ratio between samples. For analyses, PIS within a 1 min retention time interval of a target peak are chosen for comparisons.
Results and Discussion Optimization of Sample Preparation and Analysis. EDTA is often used as a column pretreatment agent to remove metal
Figure 2. Effects of EDTA and phosphoric acid as prewashing reagents for TiO2/C18 microcolumns. The soluble protein fraction of PC-9 cells were reduced, alkylated, and then digested with Lys-C and trypsin, and phosphopeptides were enriched on TiO2/ C18 microcolumns which had been prewashed with 25 µg/mL EDTA or 0.1% phosphoric acid. The enriched phosphopeptide samples were analyzed by nanoLC-MS. Each value represents the mean ( SD of three repeated enrichment procedures followed by LC-MS/MS analysis.
ions by chelation before chromatography. On the other hand, it has been reported that aliphatic hydroxy acids reduce the interaction between nonphosphorylated peptides and TiO2 via a chelating effect,16,22 and as a consequence, phosphopeptides are enriched more effectively with the aid of the acids. We found that when TiO2/C18 microcolumns were prewashed overnight with 25 µg/mL EDTA or 1% phosphoric acid, the number of identified nonphosphorylated peptides was dramatically reduced and more phosphopeptides were identified from digested PC-9 cell cytoplasmic protein, especially in the case of the phosphoric acid protocol (Figure 2). Therefore, prewashing of TiO2/C18 microcolumns with 1% phosphoric acid was employed in all subsequent phosphopeptide enrichment procedures. Figure 3 compares the numbers of phosphopeptides identified with and without coinjection of EDTA into the nano-LC/ MS system. The number of identified peptides after phosphoJournal of Proteome Research • Vol. 9, No. 3, 2010 1387
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Figure 3. Comparison of numbers of identified phosphopeptides in the presence and in the absence of EDTA as a additive during nanoLC-MS analysis. The phosphopeptide samples were prepared from the soluble protein fraction of PC-9 cells after reduction, alkylation, enzymatic digestion and enrichment on TiO2/C18 microcolumns. Each value represents the mean ( SD of four repeated enrichment procedures followed by LC-MS/MS analysis.
peptide enrichment on TiO2/C18 microcolumns was significantly increased (about 1.7-fold) by coinjection of EDTA, even though we have never observed metal ion adduct peaks in mass spectra taken in the absence of EDTA. It was found that the effect of the EDTA remained in subsequent LC/MS analysis. We have also compared the efficiency of phosphoproteome analysis using metal-rich LC system which has metal suction filters and metal-less LC system which has metal suction filters removed. Although the beneficial effect of EDTA coinjection in phosphoproteome analysis using metal-less systems was less than in the case of metal-rich pumps, the metal-less systems allowed the maximum number of phosphopeptides to be identified from both IMAC/C18 and TiO2/C18 microcolumns (Figure 4). The average numbers of identified phosphopeptides were 166 per LC/MS analysis of IMAC enrichment samples and 122 per LC/MS analysis of TiO2 enrichment samples. When 25 µg/mL of EDTA was coinjected into LC/MS, the numbers of identified phosphopeptides were increased to 172 from IMAC enrichment samples and 169 from TiO2 enrichment samples. On the other hand, the number of identified nonphosphorylated peptides was dramatically decreased. As a result, the ratio of the number of identified phosphopeptides to all identified peptides from both IMAC and TiO2 enrichment samples was statistically significantly increased, as shown in Figure 4. In our standard phosphoproteome analysis (Figure 1), we employed three different column chromatographic steps, (1) isolation of protein using a PD-10 column before digestion, (2) desalting step after the digestion using an Empore C18-SD disk cartridge, and (3) IMAC/C18 and TiO2/C18 enrichment of phosphopeptides, before the LC/MS step. Therefore, we examined at what point it would be most effective to add EDTA. Neither prewashing of each column with EDTA, nor EDTA addition to samples before the three different column steps had a positive effect on phosphopeptide identification (data not shown). Only the LC/MS system among the four chromatographic steps partly involved metal components, and this may be the reason why the maximum effect of EDTA was seen on addition at the LC/MS step, even though we were never able to detect any metal adduct ions in the mass spectra in 1388
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Figure 4. Comparison of numbers of identified phosphopeptides and nonphosphorylated peptides in the presence and in the absence of EDTA as a additive during metal-less nanoLC-MS analysis. The phosphopeptide samples were prepared from the soluble protein fraction of PC-9 cells after reduction, alkylation, enzymatic digestion and enrichment on TiO2/C18 or IMAC/C18 microcolumns. Each value represents the mean ( SD of four repeated enrichment procedures followed by LC-MS/MS analysis.
the absence of EDTA. Since phosphoric acid has a positive effect in prewashing of the TiO2/C18 microcolumn (Figure 2), and it has been reported that phosphoric acid improves detection of multiply phosphorylated peptides,23 we examined the effect of adding phosphoric acid to LC/MS samples. Addition of phosphoric acid (final concentration of 0.1-0.5% v/v) also improved phosphopeptide identification from samples obtained in both the IMAC and TiO2 enrichment procedures (supplementary 1 in Supporting Information). Although the dates of cell preparation, sample pretreatment and LC/MS analysis of phosphoric acid-added samples were different from those of the experiment on the effect of EDTA, the positive effects of phosphoric acid and EDTA appeared to be about the same, based on the ratio of phosphopeptide identification to all identified peptides. Phosphoric acid, however, often corrodes the MS ion source, so we concluded that EDTA is the best additive for phosophopeptide analysis, and therefore used EDTA for further phosphoproteome analysis. Investigation of the Impact of EDTA on LC/MS Analysis. Sample recovery from the pretreatment, including phosphopeptide enrichment, was not affected by EDTA addition (data not shown). As regards the LC/MS step, there was very little change in LC/MS peak width (supplementary 2 in Supporting Information) or in retention times (supplementary 3 in Supporting Information) of the identified phosphopeptides, as well as nonphosphopeptides, in the case of EDTA coinjection, as compared with no coinjection. Thus, EDTA appeared to have little effect on the chromatographic separation of peptides. Interestingly, the average peak intensity of the phosphopeptides was at least maintained, or even increased by up to 20%, when EDTA was coinjected, whereas the average peak intensity of the nonphosphorylated peptides was decreased by 40-70% (Table 1). Since EDTA is nonvolatile and negatively charged, it is not suitable for general proteome analysis using LC/MS, and it may suppress overall ionization of peptides. We speculate that the increased recovery of phosphopeptides counterbal-
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EDTA Increases Identification Rate of Phosphoproteomics Table 1. Effect of EDTA on Peak Intensity enrichment method
phosphopeptides
IMAC TiO2 a
n
nonphosphopeptide
n ) 160 n ) 121
a
1.2 1.0a
a
0.6 0.3a
n
p-value
n ) 21 n ) 139
