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Selective Extraction and Enrichment of Multiphosphorylated Peptides Using Polyarginine-Coated Diamond Nanoparticles Chia-Kai Chang,† Chih-Che Wu,*,† Yi-Sheng Wang,‡ and Huan-Cheng Chang‡,§ Department of Applied Chemistry, National Chi Nan University, Puli, Nantou 545, Taiwan, Genomics Research Center, Academia Sinica, Nankang 115, Taiwan, and Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan Despite recent advances in phosphopeptide research, detection and characterization of multiply phosphorylated peptides have been a challenge. This work presents a new strategy that not only can effectively extract phosphorylated peptides from complex samples but also can selectively enrich multiphosphorylated peptides for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Polyarginine-coated diamond nanoparticles are the solid-phase extraction supports used for this purpose. The supports show an exceptionally high affinity for multiphosphorylated peptides due to multiple arginine-phosphate interactions. The efficacy of this method was demonstrated by analyzing a small volume (50 µL) of tryptic digests of proteins such as β-casein, r-casein, and nonfat milk at a concentration as low as 1 × 10-9 M. The concentration is markedly lower than that can be achieved by using other currently available technologies. We quantified the enhanced selectivity and detection sensitivity of the method using mixtures composed of mono- and tetraphosphorylated peptide standards. This new affinity-based protocol is expected to find useful applications in characterizing multiple phosphorylation sites on proteins of interest in complex and dilute analytes.
have been developed and successfully applied.3–14 While considerable progress has been made in the technological development in the past, the question of whether the phosphopeptides isolated by current existing methods suffice to reflect the identity of their phosphorylation sites remains unanswered.15 It is therefore essential to develop highly sensitive and selective methods for extraction and enrichment of all phosphorylated peptides in a protein sample with good reproducibility. The methods that have been developed for phosphopeptide extraction and enrichment include both chemical and affinitybased approaches. One of the chemical approaches involves β-elimination of the O-phosphate moieties from phosphopeptide residues, followed by addition of an affinity tag for quantitative measurement of the phosphorylation state of proteins.3,5,6 Another approach involves selection of serine-, threonine-, and tyrosinephosphorylated peptides via coupling of these phosphopeptides to a solid-phase support using phosphoramidate chemistry (PAC).14 Nonphosphorylated peptides are subsequently removed by washing, and the extracted phosphopeptides are selectively released and detected under acidic conditions. Affinity-based methods, on the other hand, encompass immobilized metal affinity chromatography (IMAC) using Ga(III), Fe(III), or other metal ions,7,8 IMAC with methyl esterification,9 and metal oxide chromatography (MOAC) using titania,10 zirconia,11 and alumina to serve the extraction supports.12,13
Phosphorylation is an important post-translational modification of proteins that regulates various biochemical processes such as cell growth, metabolism, and signal transduction.1 Many proteins in nature are phosphorylated, not only at one site but also at multiple sites, and each modification often has a different regulatory function. Despite its importance, the analysis of protein phosphorylation has posed substantial challenges due to the frequently low stoichiometry of phosphorylated peptides and the large number of phosphorylation sites.2 A number of techniques for extraction and enrichment of phosphopeptides prior to analysis
(3) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379–382. (4) Ishihama, Y.; Wei, F.-Y.; Aoshiman, K.; Sato, T.; Kuromitsu, J.; Oda, Y. J. Proteome Res. 2007, 6, 1139–1144. (5) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578–2586. (6) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826–6836. (7) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883–2892. (8) Raska, C. S.; Parker, C. E.; Dominski, Z.; Marzluff, W. F.; Glish, G. L.; Pope, R. M.; Borcher, C. H. Anal. Chem. 2002, 74, 3429–3433. (9) Ndassa, Y. M.; Orsi, C.; Marto, J. A.; Chen, S.; Ross, M. M. J. Proteome Res. 2006, 5, 2789–2799. (10) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jørgensen, T. J. D. Mol. Cell. Proteomics 2005, 4, 873–886. (11) Kweon, H. K.; Håkansson, K. Anal. Chem. 2006, 78, 1743–1749. (12) Wolschin, F.; Wienkoop, S.; Weckwerth, W. Proteomics 2005, 5, 4389– 4397. (13) Chen, C.-T.; Chen, W.-Y.; Tsai, P.-J.; Chien, K.-Y.; Yu, J.-S.; Chen, Y.-C. J. Proteome Res. 2007, 6, 316–325. (14) Tao, W. A.; Wollscheid, B.; O’Brein, R.; Eng, J. K.; Li, X.-J.; Bodenmiller, B.; Watts, J. D.; Hood, L.; Aebersold, R. Nat. Methods 2005, 2, 591–598. (15) Bodenmiller, B.; Mueller, L. N.; Muller, M.; Domon, B.; Aebsersold, R. Nat. Methods 2007, 4, 231–237.
* Author to whom correspondence should be addressed. E-mail: wcche@ ncnu.edu.tw. Phone: 886-49-2910960. Fax: 886-49-2917956. † National Chi Nan University. ‡ Genomics Research Center, Academia Sinica. § Institute of Atomic and Molecular Sciences, Academia Sinica. (1) Reinders, J.; Sickmann, A. Proteomics 2005, 5, 4052–4061. (2) Zhou, H.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2001, 19, 375–378. 10.1021/ac702618h CCC: $40.75 2008 American Chemical Society Published on Web 04/18/2008
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Very recently, a systematic investigation was carried out to examine the reproducibility and selective preference of these generally used extraction and enrichment methods.15 Results indicated that the PAC method exhibits a tendency toward isolation of singly phosphorylated peptides. While the IMAC follows similar tendencies, it has a slightly stronger bias toward multiphosphorylated peptides. The MOAC, similarly, tends to isolate singly phosphorylated and acidic peptides. The three methods share the same traits that identify different but partially overlapping segments of the phosphoproteome, and no single method is sufficient for a comprehensive phosphoproteomic analysis. Particularly noteworthy is that the detection of multiply phosphorylated peptides is often challenging due to their poor ionization efficiencies in the presence of nonphosphorylated peptides and monophosphorylated peptides, and a significantly large quantity of analytes is sometimes required.16,17 A number of strategies have been developed for detecting multiply phosphorylated peptides.16,18–21 For example, phosphoric acid was used as a matrix additive for improving the MALDI signals of phosphopeptides in the presence of nonphosphorylated peptides.18 Inclusion of phosphoric acid to the sample solution in LC/MS/MS was found to increase the recovery of multiply phosphorylated peptides.16 Improved detection of multiphosphorylated peptides without phosphopeptide enrichment was obtained by using proteinase K digestion instead of trypsin digestion.19 Larsen and co-workers utilized the “non-phosphopeptide-excluding compounds” in the loading buffer for titanium dioxide (TiO2) chromatography and showed that inclusion of the citric acid enhances the detection of multiphosphorylated peptides.20 Very recently, an integrated strategy termed SIMAC (sequential elution from IMAC) based on sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from biological samples was reported and found a significant increase in recovery of multiply phosphorylated peptides.21 Herein, we describe a novel affinity-based strategy for selective extraction and enrichment of multiphosphorylated peptides using polyarginine-coated diamond nanoparticles (NPs). Arginines are chosen in this study because the amino acid residues or, more specifically, the guanidine moieties are typical recognition sites in protein–protein interactions involving phosphate or sulfate moieties.22 Studies have shown that the adjacent arginine residues and their high affinity for phosphate groups are able to generate stable complexes having “covalent-like” stability.23The extent of the complex formation is closely related with the number of phosphate groups in the acidic components and the number of arginine residues in the basic components of the proteins. Accordingly, polyarginine (PA) is expected to serve as a useful tool for isolating multiphosphorylated peptides, leading to improvement of their detection sensitivity. (16) Kim, J.; Camp, D. G., II; Smith, R. D. J. Mass Spectrom. 2004, 39, 208– 215. (17) Liu, S.; Zhang, C.; Campbell, J. L.; Zhang, H.; Yeung, K. K.-C.; Han, V. K. M.; Lajoie, G. A. Rapid Commun. Mass Spectrom. 2005, 19, 2747–2756. (18) Kjellstrm ¨ , S.; Jensen, O. N. Anal. Chem. 2004, 76, 5109–5117. (19) Kim, S.; Choi, H.; Park, Z.-Y. Mol. Cell 2007, 23, 340–348. (20) Jensen, S. S.; Larsen, M. R. Rapid Commun. Mass Spectrom. 2007, 21, 3635–3645. (21) Thingholm, T. E.; Jensen, O. N.; Robinson, P. J.; Larsen, M. R. Mol. Cell. Proteomics, 2007,10.1074/mcp.M700362-MCP200. (22) Schug, K. A.; Lindner, W. Chem. Rev. 2005, 105, 67–113. (23) Woods, A. S.; Ferré, S. J. Proteome Res. 2005, 4, 1397–1402.
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This report presents the results of our applications of PA-coated diamond NPs as high-affinity probes to selectively extract and enrich multiphosphorylated peptides from the tryptic digestion products of R-casein, β-casein, and nonfat milk. The advantage of using these NP probes is that the extracted phosphopeptides can be directly analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) without prior removal of the solid supports, which greatly simplifies the analytical procedures.24 The significantly enhanced detection capability for multiphosphorylated peptides and the good reproducibility of the detection method are demonstrated with an analysis of a mixture composed of both mono- and tetraphosphorylated peptide standards. EXPERIMENTAL METHODS Chemicals. Synthetic abrasive diamond powders of sizes in the range of 100–500 nm were obtained from Kay Industrial Diamond (Boca Raton, FL). R-Casein and β-casein (from bovine milk), horse cytochrome c, equine myoglobin, urea, poly-L-arginine hydrochloride (molecular weights ranging from 15000 to 70000 Da), ammonium bicarbonate, sodium hydroxide, trypsin (from bovine pancreas, TPCK treated), phosphopeptide positive control sets from bovine casein (P9615), and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) were purchased from Sigma (St. Louis, MO). 2,5-Dihydroxybenzoic acid (DHB) and boric acid were obtained from Fluka (Seelze, Germany). Acetonitrile (ACN), trifluoroacetic acid (TFA), and phosphoric acid were obtained from J. T. Baker (Phillipsburg, NJ). Dithiotreitol (DTT) was purchased from Merck (Darmstadt, Germany), and iodoacetic acid (IAA) was obtained from Amersham Biosciences (Piscataway, NJ). All chemicals were used without further purification. Phos-trap Phosphopeptide Enrichment Kit (Cat. # PRT301001KT) was purchased from Perkin-Elmer (Shelton, CT). Phosphopeptide Capturing Kit MB-IMAC Fe (#221865) was purchased from Bruker Daltonics (Leipzig, Germany). Deionized water was from a Milli Q plus system (Billerica, MA). Nonfat milk was purchased from a local grocery store. Microwave-Assisted Surface Functionalization of Diamond Nanoparticles. Diamond powders were surface-functionalized with carboxyl and carbonyl groups in strong oxidative acids under microwave heating. This was done by adding diamond powders slowly to a solution containing concentrated H2SO4 and HNO3 (3:1, v/v). The particle suspension was then placed in a 100 W microwave reactor (Model Discover, CEM, USA). The temperature was ramped from room temperature to 100 °C in 5 min, and microwave-assisted heating was carried out at this temperature for 3 h. After cooling to room temperature, the resulting carboxylated/oxidized diamond nanoparticles were thoroughly washed with deionized water and separated by centrifugation with a Kubota 3700 centrifuge at 12000 rpm. The recovered precipitate was resuspended in deionized water to form a stock solution with a concentration of 1 mg/mL. Preparation of Polyarginine-Coated Diamond Nanoparticles. PA-coated diamond NPs were prepared via an EDCmediated coupling reaction following previously described procedures.24 Briefly, polyarginine was immobilized on diamond (24) Kong, X. L.; Huang, L. C. L.; Liau, S.-C. V.; Han, C.-C.; Chang, H.-C. Anal. Chem. 2005, 77, 4273–4277.
surfaces by mixing an EDC solution (22 mg/mL, 63.7 µL), a PA solution (4 mg/mL, 155 µL), and the carboxylated/oxidized diamond nanoparticles (1 mg) together in H3BO3/NaOH buffer (5 mM, pH 8.5). The mixture was shaken gently at room temperature for 2 h and then centrifuged at 12000 rpm for 10 min. The supernatant was removed by pipetting, and the PA-coated diamond NP precipitates were thoroughly washed with deionized water and prepared as a suspension with a concentration of 1 mg/ mL. Tryptic Digestion of Phosphoproteins. Phosphoproteins (Rcasein and β-casein) and trypsin were prepared in 50 mM ammonium bicarbonate (pH 8.2) solution. The protein (1 mg/ mL, 200 µL) and the enzyme (0.1 mg/mL, 40 µL) were first mixed at a weight ratio of 50:1, and the mixed solution was diluted to a total volume of 240 µL. The enzymatic digestion was carried out at 37 °C for 18 h. The digest product was then diluted to a concentration of 1 pmol/µL with 0.15% TFA aqueous solution to stop enzyme digestion and adjust the value of pH. Similarly, nonfat milk to be analyzed (0.2 mL) was first mixed with 50 mM ammonium bicarbonate solution (0.2 mL), followed by addition of trypsin (0.1 mg/mL, 80 µL) to the solution. The mixture was incubated at 37 °C for 18 h. One microliter of the mixture was acidified and diluted with 0.15% TFA in acetonitrile/water (1:2, v/v) solution to a total volume of 100 µL. On the basis of the value provided by the milk company, 100 mL of nonfat milk contains 3 g of proteins. Furthermore, the percentage of R- and β-caseins in the milk protein is about ∼66%. Thus, we estimated that the concentration of caseins used for obtaining Figure 5a is ∼2.5 × 10-6 M. The sample solution was further 100-fold and 1000-fold diluted with 0.15% TFA in acetonitrile/water (1:2, v/v) solution prior to phosphopeptide extraction experiment with PA-coated diamond NPs. Extraction and Enrichment of Phosphopeptides Using PA-Coated Diamond NPs. Tryptic digests (1 pmol/µL) were acidified and diluted with 0.15% TFA in acetonitrile/water (1:2, v/v) solution to a final concentration of 0.1–10 fmol/µL. The diluted solution (50 µL or 100 µL) was then mixed with a suspension of PA-coated diamond NPs (1 mg/mL, 20 µL). After incubation for 90 s, the diamond nanoparticles were collected by centrifugation at 12000 rpm for 3 min, washed twice with the same 0.15% TFA solution (100 µL), and mixed with 0.7 µL of DHB (30 mg/mL) containing 5% phosphoric acid. The resulting slurry (∼0.7 µL) was deposited directly on a MALDI sample plate for MS analysis. FT-IR Measurements. The types of functional groups on diamond NP surfaces were characterized by infrared spectroscopy. Prior to the spectroscopic measurements, both the acid-treated diamond NPs and the PA-coated diamond NPs were made to water suspensions. An aliquot (typically 0.1 mL) of each suspension was deposited on a single-polished Si(111) wafer and dried in air to form a thin film.20 The film-containing wafer was then mounted on a sample holder and positioned in a small evacuated chamber to eliminate adsorbed and atmospheric water. A N2-purged Fourier-transform infrared (FT-IR) spectrometer (Bomem MB154), equipped with a liquid nitrogen cooled MCT detector, acquired the spectra at room temperature. Absorbance spectra were obtained by ratioing the sample spectra with the background
Figure 1. Infrared spectra of 100 nm diamond particles, obtained after (a) pretreatment with strong acids and (b) coating with poly-Larginines. Polynomial fits were used for baseline subtraction of each spectrum.
spectra taken for a pure Si(111) wafer. The spectra were typically acquired at 100 scans and an instrumental resolution of 4 cm-1. Mass Spectrometry and Data Analysis. Mass spectra were obtained with a linear TOF mass spectrometer (Voyager-DE PRO, Applied Biosystems, USA) equipped with a 1 m flight tube. It was operated in a positive ion mode at an acceleration voltage of 20 kV. Desorption/ionization of the analyte was achieved by using a 337 nm nitrogen laser with a pulse width of ∼3 ns. The laser power was adjusted to a value slightly above the desorption/ionization threshold, and each mass spectrum was obtained by averaging 100 laser shots scanned across the sample surface unless otherwise mentioned. The Data Explorer software (Version 4.0) from Applied Biosystems was used to create peak lists, and the PeptideMass tool (www.us.expasy.org) was used as a search engine to identify peptides. Search parameters used: UniProt Knowledgebase [Swiss-Prot (Release 54.2 of 11-Sep-2007) and TrEMBL (Release 37.2 of 11-Sep-2007)]. Digest used: Trypsin. Maximum of missed cleavages: 2. Variable modification: methionine oxidation and serine, threonine and tyrosine phosphorylation. RESULTS AND DISCUSSION Figure 1a presents the FT-IR absorption spectrum of dried diamond nanoparticles after the microwave-assisted oxidative acid treatment. Similar to the results reported previously,25 a broad absorption band was observed at ∼3500 cm-1, which is ascribable to the O-H stretching vibrations of adsorbed water and/or surface carboxylic groups. The most prominent feature at 1782 cm-1 is characteristic of the CdO stretching vibrations of carbonyl and/ or carboxyl groups.26 The lowest-frequency feature at 1301 cm-1 has been attributed to the ether-like groups on the oxidized diamond powder surfaces.27 The results indicate that the diamond nanoparticles have been properly surface-functionalized with carbonyl and carboxyl groups. It supports the suggestion that microwave-assisted heating, in combination with the strong (25) Huang, L.-C. L.; Chang, H.-C. Langmuir 2004, 20, 5879–5884. (26) Tu, J.-S.; Perevedentseva, E.; Chung, P.-H.; Cheng, C.-L. J. Chem. Phys. 2006, 125, 174713. (27) Ando, T.; Yamamoto, K.; Ishii, M.; Kamo, M.; Sato, Y. J. Chem. Soc. Faraday Trans. 1993, 89, 3635–3640.
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Figure 2. (a) Direct MALDI mass spectrum of a tryptic β-casein digest. The concentration of the protein used for the digestion was 1 µM (1 µL). (b-d) MALDI mass spectra of tryptic β-casein digests extracted by PA-coated diamond NPs at concentrations of (b) 1 × 10-8 M (50 µL), (c) 1 × 10-10 M (100 µL), and (d) 1 × 10-10 M (50 µL) for the protein. Phosphopeptides in spectrum a are denoted by Arabic numerals.
oxidative acid treatment, is an effective approach in oxidizing diamond surfaces and a significant increase in the number of surface carboxyl groups can be resulted. Our previous study has quantified the density of carboxyl groups on the acid-treated diamond nanoparticle surfaces. A density of 7% was determined by conductometric titration.28 This density is sufficient for immobilization of biopolymers such as PA and polylysines24 containing amino groups for covalent linkages with the diamond surfaces. Figure 1b shows the FT-IR spectrum of the PA-coated diamond NPs. A spectrum entirely different from that of the acid-treated NPs was obtained. We assign the absorption bands at 2929 and 2856 cm-1 to the C-H stretching vibrations and the most prominent feature at 1670 cm-1 to the amide vibrations of PA.29 The observations confirm coating of polyarginines onto the diamond nanoparticle surfaces. Most likely, the biopolymers are attached to the diamond substrate in an extended configuration.25 Figure 2a presents a direct MALDI-TOF mass spectrum of the tryptic digest of β-casein at a concentration of 1 µM without any pretreatment. In the spectrum, the signals of phosphopeptides are strongly suppressed by those of nonphosphorylated peptides that are more abundant in the mixture. Only peaks at m/z 2061.9, 2556.1, and 3122.0 belong to phosphopeptides (labeled with β1, β2, and β4, respectively), and their sequences are displayed in Table 1. With the use of PA-coated diamond NPs for selective extraction and enrichment, the detection sensitivity for phosphopeptides is greatly improved (Figure 2b). Only peaks arising from phosphopeptide ions are present in the spectrum, and additionally, the intensity of the tetraphosphorylated peptides (β4) is higher than those of the singly phosphorylated peptides (β1 and β2). The detection sensitivity of phosphopeptides from β-casein reported in previous literatures ranged from low-picomole to low-femtomole (28) Nguyen, T.-T.-B.; Chang, H.-C.; Wu, V. W.-K. Diamond Relat. Mater. 2007, 16, 872–876. (29) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic: Boston, MA, 1991.
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levels in which the sample concentration ranged from 1 µM to 0.9 nM when affinity-based MALDI MS was used.7–13,30–32 In those reports, detection of the singly phosphorylated peptide (β1) from a concentration of 9 × 10-10 M (50 µL injected, 45 fmol) was reported, but the detection of the tetraphosphoryalted peptide (β4) required a much higher concentration (9 × 10-7 M, 50 µL).31 Highconcentration criteria are usually required for detection of multiphosphorylated peptides owing to their poor ionization efficiency. Therefore, when the concentration is below 1 × 10-9 M, no peak derived from multiphosphorylated peptides has ever been identified. However, with the use of our protocol, the detection limit for tetraphosphorylated peptide (β4) is achieved, for the first time, at a concentration of 1 × 10-10 M (5 fmol, 50 µL) (Figure 2d). This detection sensitivity of multiphosphorylated peptides is markedly lower than that of the commonly used methods such as IMAC beads,8 but it is comparable to the lowest detection limit (∼5 fmol) reported in the literature.20,30 Additionally, the concentration limit of tetraphosphorylated peptide is significantly lower by a few orders of magnitude than that of the common phosphopeptide isolation methods. This low-femtomole detection sensitivity is attributable to the high trapping capacity of the PA-coated diamond NPs as well as the high specificity of the arginine residues toward multiphosphopeptides. Furthermore, the sample loss due to elution is minimized when using this solid-phase extraction and elution approach. In summary, the results demonstrate our protocol for phosphopeptide enrichment using PAcoated diamond NPs provides a markedly superior concentration limit (1 × 10-10 M) while offering comparable or slightly better detection sensitivity. We demonstrate further that these PA-coated diamond nanoparticles can be used as high-affinity probes to extract and enrich phosphopeptides from a tryptic digest mixture containing high concentrations of denaturant, salt, and nonphosphoproteins. The mixture of β-casein, myoglobin, and cytochrome c at a molar ratio of 1:120:120 was prepared in 1 mL of denaturing buffer containing 8 M urea and 50 mM ammonium bicarbonates and incubated at 38 °C for 30 min. DTT (100 mM, 50 µL) was added to open the disulfide bond, and IAA (200 mM, 50 µL) was added to carbamidomethylate the resulting cysteine residues. In order to test if the presence of urea, DTT, and IAA as well as the abundant nonphosphoproteins in a complex sample interferes with the trapping selectivity of phosphopeptides by PA-coated diamond NPs, the resulting protein digests were subjected to phosphopeptide analysis by MALDI following the same protocol of β-casein digest. As shown in Figure 3a, no signal can be detected at all. The tetraphosphorylated peptide signal (β4), in contrast, can be easily recovered after pretreatment using PA-coated diamond NPs (Figure 3b). Acetylation of tetraphosphorylated peptide (β4) is also revealed by a mass shift of 44 Da. Considering the concentration of phosphopeptides is 2 orders of magnitude lower than that (30) Zhang, H.; Zhang, C.; Lajoie, G. A.; Yeung, K. K.-C. Anal. Chem. 2005, 77, 6078–6084. (31) Lo, C.-Y.; Chen, W.-Y.; Chen, C.-T.; Chen, Y.-C. J. Proteome Res. 2007, 6, 887–893. (32) Zhou, H.; Tian, R.; Ye, M.; Xu, S.; Feng, S.; Pan, C.; Jiang, X.; Li, X.; Zou, H. Electrophoresis 2007, 28, 2201–2215. (33) Wolschin, F.; Weckwerth, W. Rapid Commun. Mass Spectrom. 2006, 20, 2516–2518. (34) Pan, C.; Ye, M.; Liu, Y.; Feng, S.; Jiang, X.; Han, G.; Zhu, J.; Zou, H. J. Proteome Res. 2006, 5, 3114–3124.
Table 1. Phosphopeptides Identified in the MALDI Mass Spectra of Tryptic β-Casein Digests after Extraction and Enrichment with PA-Coated Diamond NPs
a
no.
calcd m/z
phosphorylation site
phosphopeptide sequencesa
β1 β2 β3 β4
2061.8 2556.1 2966.2 3122.3
1 1 4 4
FQpSEEQQQTEDELQDK(48∼63) FQpSEEQQQTEDELQDKIHPF(48∼67) ELEELNVPGEIVEpSLpSpSpSEESITR(17∼40) RELEELNVPGEIVEpSLpSpSpSEESITR(16∼40)
The pS denotes phosphorylated serine.
Figure 3. MALDI mass spectra of a tryptic digest mixture containing β-casein (8.3 × 10-8 M), myoglobin (1.0 × 10-5 M), and cytochrome c (1.0 × 10-5 M) at the molar ratio of 1:120:120 prepared in a highly contaminated solution having 8 M urea, 100 mM DTT, and 200 mM IAA: (a) 1 µL, without prior enrichment; (b) on-particle analysis of phosphopeptides obtained when using PA-coated diamond NPs to selectively isolate multiphosphorylated peptides from the above tryptic digest mixture, 100 µL.
of nonphosphopeptides, the trapping selectivity of PA-coated diamond NPs is quite high. These results indicate that the PAcoated NPs have high affinity for phosphopeptides in the presence of highly abundant nonphosphopeptide as well as in a highly contaminated solution. The generality of this method is demonstrated with the tryptic digest of R-casein, which is composed of S1 and S2 units. A direct MALDI-TOF mass spectrum of the tryptic digest of this protein without any pretreatment is shown in Figure 4a. Compared to the MALDI spectrum of β-casein, the signals of the phosphopeptides are considerably more suppressed by the nonphosphorylated peptide ions. For example, five phosphorylated peptide ions from R-S1-casein only appeared as weak signals at m/z 1661.3, 1926.9, 1952.0, 2703.8, and 2720.7 No feature of phosphopeptide–ions from R-S2-casein was detected in the spectrum. The situation dramatically changed when the PA-coated diamond NPs were used for extraction and enrichment (Figure 4b,c). Signals from R-S1-casein and R-S2-casein can both be revealed (Table 2 for corresponding peptide sequences). It can be also found that multiply phosphorylated peptide peaks generally dominate the mass spectra at whatever concentrations of R-casein are used. More interestingly, one of the dominant peaks at m/z 2720.7 (denoted as R7) derived from the pentaphosphorylated peptide residue still remains noticeable as a concentration as low as 1 × 10-9 M. It evidence the fact that the PA-coated diamond NPs preferentially extracts multiphosphorylated peptides.
Figure 4. (a) Direct MALDI mass spectrum of a tryptic R-casein digests. The concentration of the protein used for the digestion was 1 µM (1 µL). (b-d) MALDI mass spectra of tryptic R-casein digests extracted by PA-coated diamond NPs at concentrations of (b) 1 × 10-8 M (50 µL) and (c) 1 × 10-9 M (50 µL) for the protein. Phosphopeptides in spectra a are denoted by Arabic numerals, whereas the phosphopeptides containing oxidized methionine residues are marked with # in spectra b and c.
In the mass spectra shown in Figure 4b,c, we also found evidence for the oxidation of the methionine residues in the extracted phosphorylated peptides (peaks labeled with R2#, R4#, R7#, and R8#).29,30 The oxidized methionine-containing phosphopeptides differ from the unoxidized methionine-containing phosphopeptides by 16.0 Da in mass. It is of importance to note that the peaks associated with the pentaphosphorylated peptide ions still can be found in the spectra at a concentration as low as 1 × 10-9 M. The lowest detection limit that could be achieved in this experiment for multiphosphorylated peptides of R-casein is at the low-femtomole level. To demonstrate a real-world application of this method for a complex mixture, the PA-coated diamond NPs were used to extract and enrich phosphopeptides from the tryptic digests of nonfat milk, which contains abundant phosphoproteins including R- and β-caseins. Figure 5a presents a direct MALDI mass spectrum of the tryptic digest of this nonfat milk with an estimated casein concentration of 2.5 × 10-6 M (1 µL). Among these peaks, only four of them (marked with R2, R3, β1, and β3) derive from phosphopetide ions. Upon the extraction and enrichment, a large number of peaks belonging to the phosphopeptide residues from either R-casein or β-casein are identified in the mass spectra (Figure 5b and Table 1 and Table 2). The dominant peak appearing at m/z 2966.3 corresponds to the tetraphosphorylated peptide ion (β3). Again, no nonphosphorylated peptide peak was observed in the mass spectra after the extraction and enrichment Analytical Chemistry, Vol. 80, No. 10, May 15, 2008
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Table 2. Phosphopeptides Identified in the MALDI Mass Spectra of Tryptic r-Casein Digests after Extraction and Enrichment with PA-Coated Diamond NPs
a
no.
calcd m/z
phosphorylation site
phosphopeptide sequencesa
R1 R2 R2# R3 R4 R4# R5 R6 R7 R7# R8 R8# R9 R10
1660.8 1927.7 1943.7 1952.0 2618.9 2634.9 2703.5 2678.0 2720.9 2736.9 2935.2 2951.2 3008.0 3087.3
1 2 2 1 1 4 1 3 5 5 3 3 4 3
VPQLEIVPNpSAEER(S1/121∼134) DIGpSEpSTEDQAMEDIK(S1/58∼73) DIGpSEpSTEDQAoMEDIK(S1/58∼73) YKVPQLEIVPNpSAEER(S1/119∼134) YKVPQLEIVPNpSAEER(S1/119∼134) NToMEHVpSpSpSEESIIpSQETYK(S2/17∼36) LRLKKYKVPQLEIVPNpSAEERL(S1/114∼135) VNELpSKDIGpSEpSTEDQAMEDIK(S1/52∼73) QMEAEpSIpSpSpSEEIVPNpSVEQK(S1/74∼94) QoMEAEpSIpSpSpSEEIVPNpSVEQK(S1/74∼94) EKVNELpSKDIGpSEpSTEDQAMEDIK(S1/50∼73) EKVNELpSKDIGpSEpSTEDQAoMEDIK(S1/50∼73) NANEEEYSIGpSpSpSEEpSAEVATEEVK(S2/61∼85) pSTpSEENSKKTVDMEpSTEVFTKKTKL(S2/144∼168)
pS and oM denote phosphorylated serine and oxidized methionine, respectively.
Figure 5. (a) Direct MALDI mass spectrum of the tryptic digest of nonfat milk with an estimated casein concentration of 2.5 × 10-6 M (1 µL). (b, c) MALDI mass spectra of the same sample obtained using PA-coated diamond NPs to extract and enrich phosphopeptides from the 100 µL tryptic digest product after (b) 100-fold and (c) 1000-fold dilution. The phosphopeptides without oxidation are denoted by Arabic numerals, while the phosphopeptides containing oxidized methionine residues are marked with # in spectra b and c.
treatment. Additionally, the oxidation of the methionine residues in the phosphorylated peptides was also identified from the peaks (denoted by R2#, R4#, and R7#) showing a mass shift of 16.0 Da. The consistency of the results we obtained for R-casein, β-casein, and nonfat milk highlights the strength of our affinity-based extraction and enrichment protocol, which is capable of differentiating between singly and multiply phosphorylated peptides. As shown in Figure 6a for the MALDI mass spectrum of the mixture (1:1 molar ratio), the peak heights (Imono and Itetra) of these two peptide–ions are about the same initially, but the intensity ratio Itetra/(Imono + Itetra) increases dramatically to >90% after the enrichment. Kjellström and Jensen18 have previously reported that the phosphoric acid in combination with the DHB matrix has an effect of enhancing the phosphopeptide signals. However, no noticeable preference for multiphosphorylated peptides was observed in the MALDI mass spectra. In the present case, with the assistance of PA-coated diamond NPs for extraction and enrichment, the tetraphosphorylated peptide–ions clearly domi3796
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Figure 6. MALDI mass spectra of the phosphopeptide standard mixture containing 1 pmol each of mono- and tetraphosphorylated peptides obtained (a) without and (b) with enrichment with PA-coated diamond NPs.
nate the mass spectrum (Figure 6b). The result is consistent with our earlier observations that the tetraphosphorylated peptide has a higher binding affinity for the polyarginine molecules than the monophosphorylated peptides. As a result, the multiphosphorylated peptides can be more effectively extracted and enriched. The strong preference of the PA-coated diamond NPs for tetraphosphorylated peptides over monophosphorylated peptides was further examined over 30 different sample spots for the 1:1 mixture. Excellent shot-to-shot reproducibility in the intensity ratio Itetra/(Imono + Itetra) was found. The mean relative peak height of the tetraphosphorylated peptide versus the total height of the phosphopeptide–ions in the mass spectrum was 92.3 ± 3.8%. In contrast, the ratio was 51.3 ± 5.4% without the extraction and enrichment treatment. (see Tables S1 and S2 in the Supporting Information for details) The high degree of similarity between the mass spectra indicates that the unique extraction and enrichment protocol presented here can reproducibly isolate multiply phosphoryalted peptides from a complex mixture. A comparison of the ability of three phosphopeptide isolation methods including immobilized metal affinity chromatography, titanium dioxide, and polyarginine-coated diamond nanoparticles to isolate phosphopeptides from tryptic β-casein digests is displayed in Figure 7. Phosphopeptide enrichment in spectra a and
Figure 7. Comparison of the performance of (a) IMAC beads, (b) TiO2 beads, and (c) PA-coated diamond NPs for selective enrichment of phosphopeptides from a tryptic β-casein digest at a concentration of 1 × 10-8 M (100 µL).
b of Figure 7 was performed using a phosphopeptide capturing kit (MB-IMAC Fe, Bruker Daltonics) and a phosphopeptide enrichment kit (Phos-trap, PerkinElmer), respectively. After enrichment by IMAC beads and TiO2 beads, both peaks derived from mono- and tetraphosphorylated peptides appear in the mass spectra (Figure 7a,b). However, only peaks derived from tetraphosphoryalted peptide are identified in mass spectrum after enrichment by the PA-coated diamond NPs. The results clearly showed that the PA-coated diamond NPs have an excentionally strong bias toward multiply phosphorylated peptides. The improved detection of multiply phosphorylated peptides after the pretreatment with PA-coated diamond NPs is attributed to the strong arginine-phosphate interaction. A control experiment using bare diamond NPs to isolate phosphopeptides from trypic β-casein digest was performed, and the result showed that the bare diamond NPs exhibit no selectivity toward phosphopeptides (cf. Figure S1 in the Supporting Information) Efficient multipoint interaction between multiphosphorylated peptides and guanidinum groups of polyarginines is capable of providing a significant contribution to binding between host and guest.
Compared to the enrichment of phosphopeptides using IMAC and TiO2 chromatography, the enrichment of phosphopeptides using PA-coated diamond NPs was validated to increase specificity and binding affinity toward multiply phosphorylated peptides through the incorporation of multiple and flexible functional binding partners. In conclusion, a novel method has been applied, for the first time, to selectively extract and enrich phosphorylated peptides in complex mixtures such as the proteolytic digests of R-casein, β-casein, and nonfat milk. The uniqueness of this protocol is that it uses PA-coated diamond NPs as the solid-phase extraction support, which possesses an exceptionally high selectivity for multiphosphorylated peptides. The present work demonstrates that phosphopeptides from the digests of R-casein and β-casein can be isolated and analyzed by MALDI-TOF-MS at the lowfemtomole level without time-consuming and tedious procedures. This NP-coupled mass spectrometric approach is rapid, sensitive, easy to use, and most importantly, it is highly effective in identifying multiphosphorylated peptides. The new protocol leads to enhanced response of multiphosphorylated peptide ion signals in MALDI-MS, which are immediately amenable to peptide sequencing by MALDI-MS/MS, and should find many applications in the rapidly growing field of phosphoproteomics. ACKNOWLEDGMENT Financial support was provided by the National Science Council (Grant NSC 95-2113-M-260-011-MY2) of Taiwan, Republic of China. This work was partially supported by the Institute of Atomic and Molecular Sciences, Academia Sinica. We thank Dr. X. L. Kong and Dr. C.-C. Han for insightful discussions. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 26, 2007. Revised manuscript received for review March 11, 2008. Accepted March 12, 2008. AC702618H
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