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Optimized Protocol for On-Target Phosphopeptide Enrichment Prior to Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry Using Mesoporous Titanium Dioxide Anna Eriksson, Jonas Bergquist,* Katarina Edwards, Anders Hagfeldt, David Malmstro¨m, and Vı´ctor Agmo Herna´ndez* Department of Physical and Analytical Chemistry, Uppsala University, Uppsala, Sweden A novel on-target phosphopeptide enrichment method is presented that allows specific enrichment and direct analysis by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) of phosphorylated peptides. Spots consisting of a thin film of anatase titanium dioxide are sintered onto a conductive glass surface. Enrichment and analysis can be performed on the modified target with minimal sample handling. The protocol leads to an enrichment efficiency that is superior to what has been reported before for similar methods. The method was tested using β-casein as a model phosphorylated protein as well as with a custom peptide mixed with its phosphorylated form. A very low detection limit, a significantly improved phosphoprofiling capability, and a simple experimental approach provide a powerful tool for the enrichment, detection, and analysis of phosphopeptides. Phosphorylation is one of the most common post-translational modifications of proteins involved in important biological processes, such as signal transmission, cellular growth control, cellular metabolism, and molecular recognition, among others.1 It is therefore important to characterize the phosphorylated proteins using an appropriate methodology. Matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) and other MS-based techniques have been proven to be efficient methods to detect and map phosphorylation sites on proteins.2-4 The analysis, however, continues to be very difficult, mainly due to the low relative abundance of phosphorylated peptides, the signal suppression observed in the presence of nonphosphorylated peptides,5 and the difficulty of forming positive ions due to the increased acidity.6,7 To overcome these difficulties, pre-enrichment methods have been developed that allow separating the phosphorylated peptides from proteolytic digest mixtures. Han et al.8 and Dunn et al.9 have written comprehensive reviews on the topic. Immobilized metal * Address correspondence to either author. (J.B.) Phone: +46 (0) 18 471 3675. Fax: +46 (0) 18 471 3692. E-mail:
[email protected]. (V.A.H.) Phone: +46 (0) 18 471 3635. Fax: +46 (0) 18 471 3654. E-mail:
[email protected]. (1) Graves, J. D.; Krebs, E. G. Pharmacol. Ther. 1999, 82, 111–121. (2) Annan, R. S.; Carr, S. A. Anal. Chem. 1996, 68, 3413–3421. (3) Mann, M.; Ong, S. E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A. Trends Biotechnol. 2002, 20, 261–268. (4) Nita-Lazar, A.; Saito-Benz, H.; White, F. M. Proteomics 2008, 8, 4433–4443. 10.1021/ac100589j 2010 American Chemical Society Published on Web 05/05/2010
affinity chromatography (IMAC)10-13 and, more recently, metal oxide affinity chromatography (MOAC)5,14-21 are among the main techniques that are used for this purpose, the latter being more efficient in avoiding nonspecific enrichment, because the oxides used are stable in a wide pH range, allowing the use of acidic buffers that prevent nonspecific binding. Titanium dioxide (TiO2) is one of the most popular oxides in MOAC because it has a high selectivity toward phosphorylated peptides while the nonspecific binding of acidic nonphosphorylated peptides can be reduced by including 2,5-dihydrobenzoic acid (DHB) or salicylic acid, as well as high concentrations of tetrafluoroacetic acid (TFA) in the loading buffer.5,20 However, to our knowledge, no method has been able to provide a comprehensive phosphopeptide profiling capability, and each method provides different, overlapping segments of a phosphoproteome.22 Sample loss during the different steps of the enrichment procedure is also unavoidable. On TiO2 affinity chromatography, for example, not all the polyphosphorylated (5) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. D. Mol. Cell. Proteomics 2005, 4, 873–886. (6) Blacken, G. R.; Volny, M.; Vaisar, T.; Sadilek, M.; Turecek, F. Anal. Chem. 2007, 79, 5449–5456. (7) Qiao, L.; Roussel, C.; Wan, J.; Yang, P.; Girault, H. H.; Liu, B. J. Proteome Res. 2007, 6, 4763–4769. (8) Han, G.; Ye, M.; Zou, H. Analyst 2008, 133, 1128–1138. (9) Dunn, J. D.; Reid, G. E.; Bruening, M. L. Mass Spectrom. Rev. 2010, 29, 29–54. (10) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature 1975, 258, 598– 599. (11) Andersson, L.; Porath, J. Anal. Biochem. 1986, 154, 250–254. (12) Neville, D. C. A.; Rozanas, C. R.; Price, E. M.; Gruis, D. B.; Verkman, A. S.; Townsend, R. R. Protein Sci. 1997, 6, 2436–2445. (13) Zhang, X.; Ye, J.; Jensen, O. N.; Roepstorff, P. Mol. Cell. Proteomics 2007, 6, 2032–2042. (14) Pinkse, M. W. H.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R. Anal. Chem. 2004, 76, 3935–3943. (15) Kweon, H. K.; Hakansson, K. Anal. Chem. 2006, 78, 1743–1749. (16) Zhou, H.; Tian, R.; Ye, M.; Xu, S.; Feng, S.; Pan, C.; Jiang, X.; Li, X.; Zou, H. Electrophoresis 2007, 28, 2201–2215. (17) Klemm, C.; Otto, S.; Wolf, C.; Haseloff, R. F.; Beyermann, M.; Krause, E. J. Mass Spectrom. 2006, 41, 1623–1632. (18) Chen, C.; Chen, Y. Anal. Chem. 2005, 77, 5912–5919. (19) Han, L.; Shan, Z.; Chen, D.; Yu, X.; Yang, P.; Tu, B.; Zhao, D. J. Colloid Interface Sci. 2008, 318, 315–321. (20) Thingholm, T. E.; Jorgensen, T. J. D.; Jensen, O. N.; Larsen, M. R. Nat. Protoc. 2006, 1, 1929–1935. (21) Thingholm, T. E.; Larsen, M. R.; Ingrell, C. R.; Kassem, M.; Jensen, O. N. J. Proteome Res. 2008, 7, 3304–3313. (22) Bodenmiller, B.; Mueller, L. N.; Mueller, M.; Domon, B.; Aebersold, R. Nat. Methods 2007, 4, 231–237.
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peptides are recovered during elution because they have a high affinity for the material.23 A method combining IMAC and MOAC (referred to as SIMAC: sequential elution from IMAC) has been proposed to get complementary information from both techniques,23 but sample loss still poses a problem. One approach that minimizes the handling of the sample between digestion and analysis is the direct on-target enrichment of phosphopeptides prior to MALDI-MS.7,24-28 In general, this approach consists of modifying a MALDI target plate with spots of a material known to enrich phosphopeptides. In principle, materials similar to those used in IMAC and MOAC can be employed. The enrichment is performed directly on the analysis target, and the nonspecifically bounded peptides can be removed by using appropriate washing protocols. As with the chromatographic methods, TiO2 has proven to provide one of the best substrates to achieve specific enrichment.7,24,25 Qiao et al.,7 for example, identified all five phosphorylated sites of β-casein using TiO2 spots sintered to a MALDI stainless steel target. The approach, however, still fails to detect some of the phosphorylated digestion products. In this example, even though all the expected phosphorylation sites were identified, they were detected only on three peptide fragments of β-casein, meaning that some of the well-known overlapping phosphopeptide sequences obtained from tryptic digestion of the protein were not detected. In more complex mixtures, one should be able to detect all phosphorylated fragments to characterize the whole phosphoproteome. Furthermore, the lack of control and full characterization of the structure of the TiO2 spots present as well a drawback of this approach. Torta et al.24 used pulsed laser deposition (PLD) to achieve a higher level of control on the morphology and structure of TiO2 films deposited on a MALDI target. However, the PLD method is rather complex, and the results obtained for phosphopeptide enrichment do not represent any improvement over other available methods, with several known phosphopeptides not appearing in the analysis. A simpler approach that provides a very well characterized and controlled TiO2 morphology was recently described by Niklew et al.,29 who synthesized nanoparticles of the material via the sol-gel route, producing, after sintering on an adequate surface, a mesoporous film consisting of anatase titania with small traces of brookite. Although the results obtained are similar to those reported using previous approaches, the simplicity of the method and the high level of film structure control that it provides make it a very attractive option for on-target phosphopeptide enrichment. In the present work, we show that by introducing some modifications into the film preparation (23) Thingholm, T. E.; Jensen, O. N.; Robinson, P. J.; Larsen, M. R. Mol. Cell. Proteomics 2008, 7, 661–671. (24) Torta, F.; Fusi, M.; Casari, C. S.; Bottani, C. E.; Bachi, A. J. Proteome Res. 2009, 8, 1932–1942. (25) Tan, F.; Zhang, Y.; Wang, J.; Wei, J.; Qin, P.; Cai, Y.; Qian, X. Rapid Commun. Mass Spectrom. 2007, 21, 2407–2414. (26) Ekstro ¨m, S. E.; Wallman, L.; Helldin, G.; Nilsson, J.; Marko-Varga, G.; Laurell, T. J. Mass Spectrom. 2007, 42, 1445–1452. (27) Dunn, J. D.; Igrisan, E. A.; Palumbo, A. M.; Reid, G. E.; Bruening, M. L. Anal. Chem. 2008, 80, 5727–5735. (28) Wang, W.; Bruening, M. L. Analyst 2009, 134, 512–518. (29) Niklew, M.-L.; Hochkirch, U.; Melikyan, A.; Moritz, T.; Kurzawski, S.; Schlu ¨ ter, H.; Ebner, I.; Linscheid, M. W. Anal. Chem. 2010, 82, 1047– 1053.
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and phosphopeptide enrichment procedures employed by Niklew et al.,29 the use of mesoporous anatase as a substrate for on-target enrichment allows identifying most phosphorylated protein digestion products, thus providing a complete or comprehensive phosphopeptide profile. EXPERIMENTAL SECTION Materials. Kemptide (LRRASLG) and its phosphorylated form (LRRA-pS-LG) were obtained from GenScript (Piscataway, NJ). Bovine serum albumin (BSA), β-casein (from bovine milk, 90%), 2,-5-dihydrobenzoic acid (DHB), acetonitrile (ACN), trifluoroacetic acid (TFA >99%), phosphoric acid (85%), ammonium hydroxide solution (25-28%), ammonium bicarbonate, 1,4-dithiothreitiol (DTT), and iodoacetamide (IAA) were obtained from SigmaAldrich (Schnelldorf, Germany). Sequence grade trypsin was a product of Roche Diagnostics (Basel, Switzerland). All reagents were used as received. All aqueous solutions were prepared in deionized water (18.2 MΩ cm) obtained from a Milli-Q system (Millipore, Bedford, MA). Traces (up to 10%) of other milk proteins, such as R- and κ-casein, have been previously detected in the β-casein sample employed (e.g., by Keller et al.30). The latter can therefore be used as a model for complex mixtures with some components present in substoichiometric concentrations. Preparation of the TiO2 Nanoparticle Suspension. The preparation of TiO2 nanoparticles, with a diameter of about 20 nm and pure anatase crystal structure, followed the so-called acidic route described in detail by Wang et al.31 A 10 g portion of acetic acid was added dropwise to 48.8 g of titanium iso-propoxide under stirring at room temperature. The modified precursor was stirred for about 15 min and poured into 240 mL water as fast as possible with vigorous stirring (700 rpm). A white precipitate was instantaneously formed. One hour of stirring was performed to achieve a complete hydrolysis reaction. After adding 4.5 mL of 65% nitric acid, the mixture was heated from room temperature to 78 °C within 40 min and peptized for 75 min, then water was added to the cooling liquid mixture to adjust the volume to be 310 mL. The resultant mixture was kept in a 470 mL titanium autoclave and heated at 250 °C for 12 h. After that, 2.0 mL of 65% nitric acid was added and dispersed with an ultrasonic titanium probe. The resultant colloidal solution was concentrated with a rotary evaporator to contain 18% TiO2. Finally, it was centrifuged to remove nitric acid and washed with ethanol three times to produce a colloidal solution containing 40% TiO2 and 4% water in ethanol. To prepare a fluid paste, the TiO2 colloid in ethanol was mixed with terpineol and a solution of ethyl cellulose in ethanol. The weight proportion of TiO2/terpineol/ethyl cellulose was 1:4: 2.2. After removing the ethanol and water with a rotary evaporator, a paste consisting of 16.2% 20-nm-sized TiO2 and 4.5% ethyl cellulose in terpineol was prepared. The paste was further diluted with three parts of terpineol before use. Preparation of the Modified MALDI Target Plate. An array of TiO2 spots on a microscope glass slide (75 mm × 25 mm) coated with a conductive indium-tin oxide (ITO) layer was prepared by dropping small volumes of the paste (1-2 µL) (30) Keller, A.; Purvine, S.; Nesvizhskii, A. I.; Stolyar, S.; Goodlett, D. R.; Kolker, E. Omics 2002, 6, 207–212. (31) Wang, P.; Zakeeruddin, S. M.; Comte, P.; Charvet, R.; Humphry-Baker, R.; Gratzel, M. J. Phys. Chem. B 2003, 107, 14336–14341.
using a robotic x-y-z-axis motion system (Bai Probot, LCPackings, Sunnyvale, CA). The spots were allowed to dry at room temperature. Afterward, the spots were sintered onto the surface following a well-defined temperature program (10 min ramp to a final temperature of 180 °C, hold for 10 min; second 10 min ramp to a final temperature of 220 °C, hold for 10 min; third 10 min ramp to a final temperature of 390 °C, hold for 10 min; final 15 min ramp to a temperature of 450 °C, hold for 30 min) and subsequently cooled down before being stored in a desiccator. The obtained spots had a diameter of ≈1.5 mm. The spots were characterized by scanning electron microscopy (SEM) using a LEO 1530, Gemini SEM instrument, and by a DEKTAK 3 profilometer (Veeco, Mannheim, Germany). Model Peptide Mixtures. Stock solutions (1 mg/mL) of kemptide and its phosphorylated form were prepared in a DHB buffer (20 mg/mL DHB in ACN/water/TFA 50/49.9/0.1 (v/v)). To perform enrichment experiments, the stock solutions were mixed and diluted with DHB buffer to obtain 1/1000, 1/100, and 1/10 (mol/mol) mixtures of phosphopeptide/peptide at a nonphosphorylated peptide concentration of 5 µg/mL. Digestion of Proteins. One milligram of the desired protein (β-casein or BSA) was dissolved in 0.5 mL of 100 mM ammonium bicarbonate aqueous solution. Forty microliters of 10 mM DTT was added, and the sample was incubated at 50 °C for 15 min. After cooling down, 40 µL of 20 mM IAA was added, and the solution was kept in the dark for 15 min at room temperature. Finally, the protein was digested with trypsin (2.5% w/w) overnight at 37 °C. On-Target Enrichment of Phosphopeptides. Model Peptide Mixtures. The phosphorylated peptides were enriched on the spots according to the procedure reported in a previous publication.7 A 0.5 µL portion of each of the prepared phospho-kemptide/ kemptide mixtures, corresponding to ≈3 pmol of total peptide content, was loaded onto the TiO2 spots, and the system was kept in a humidity chamber at room temperature for 30 min. The spots were subsequently washed several times with DHB buffer. A 0.5 µL portion of 400 mM ammoniac aqueous solution was then added to desorb the enriched phosphorylated peptides from the TiO2 surface. Finally, 0.3 µL of 2% (v/v) aqueous TFA solution was added, and the spots were left to dry at room atmosphere. Before the MALDI-MS experiments, 0.5 µL of a DHB matrix solution (20 mg/mL DHB in ACN/water/TFA 50/ 49.9/0.1 (v/v)) was added to the spots. The enrichment of the phosphorylated peptides on the TiO2 modified target plate was analyzed by MALDI-TOF MS. The reproducibility was examined by repeating all enrichment experiments at least twice. Reference measurements on TiO2 omitting the washing procedure, were also performed. For the mixtures containing a low relative amount of phosphokemptide (1/100 and 1/1000 molar ratios), no clear signal was obtained after following the enrichment procedure described above. In those cases, the matrix solution used was modified by replacing the TFA with 1% phosphoric acid (20 mg/mL DHB in ACN/water/phosphoric acid 50/49/1 (v/v)). The latter acid is known to improve the phosphorylated peptides’ ion response, probably because the peptides are incorporated in the matrix lattice with higher charged states.32 The corresponding reference (32) Kjellstrom, S.; Jensen, O. N. Anal. Chem. 2004, 76, 5109–5117.
Figure 1. SEM picture of the TiO2 surface used to enrich phosphorylated peptides. The bar represents 200 nm.
measurements on untreated TiO2 were also performed using the modified matrix solution. β-Casein Tryptic Digest. The tryptic digest of β-casein was diluted in the DHB buffer to a final concentration of 120 µg/mL (≈5 µM). This buffer was used to lower the pH and because the inclusion of substituted aromatic acids in the loading solution is known to avoid the nonspecific binding of nonphosphorylated acidic peptides.5 A 0.5 µL portion of the solution was loaded onto the TiO2 spots, and the same enrichment and washing procedure described above was followed. Before the MALDI-MS measurements, 0.5 µL of the DHB matrix with 1% phosphoric acid was added. For comparison, a 0.5 µL droplet of the β-casein digest diluted in DHB buffer was deposited on a MALDI steel target and on a TiO2 spot and analyzed by MALDI-MS without further treatment. Protein Mixtures. Tryptic digests of β-casein and BSA were mixed at 1/1, 1/10, 1/100, and 1/1000 β-casein/BSA molar ratios and diluted in DHB buffer to a constant BSA concentration of 330 µg/mL (≈5 µM). In total, 0.5 µL of the different mixtures was loaded, enriched, and prepared for analysis on the TiO2 modified target plates as described above. As a complementary model for complex mixtures, the tryptic digest of β-casein was also mixed with an excess of kemptide to a final composition of 5 µM β-casein and 650 µM kemptide in DHB buffer. The mixture was enriched and analyzed following the same procedure as for the other samples. MALDI-TOF/TOFMS. Mass data were acquired with an Ultraflex II MALDI-TOF/TOF (Bruker Daltonics) in reflector positive mode. A mass range of 700-4000 Da was analyzed. MALDI-TOF/TOF tandem MS analysis was performed in LIFT mode by software-controlled data acquisition. RESULTS AND DISCUSSION Modified MALDI Target Plate. The TiO2 spots sintered on the ITO coated glass slides present a mesoporous structure, as shown in Figure 1. Figure 2 shows the height profile of a representative spot with an average film thickness of 2.9 µm and a root-mean-square (rms) roughness of 155 nm. The small difference between the highest peak (3.04 µm) and the deepest valley (2.5 µm) in the height profile shown in Figure 2, as well as the relatively low value of the rms roughness, contrast with the rather irregular topology of the TiO2 films used by Niklew et Analytical Chemistry, Vol. 82, No. 11, June 1, 2010
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Figure 2. Height profile of a TiO2 spot sintered on an ITO-coated glass microscope slide, as measured using a contact stylus surface profiler.
al.29 The smoother surface can be an effect of the dilution of the TiO2 paste in terpineol, which causes a better wetting of the ITO surface and, therefore, a more regular distribution of the nanoparticles on the substrate. Although in principle, the larger surface area found in a more irregular surface may be considered an advantage, the production of a film with a well-controlled height profile allows more tuning capabilities to optimize the material performance for phosphopeptide enrichment and the subsequent MALDI analysis. On-Target Enrichment of Model Phosphorylated Peptides. Figure 3a, c, and e shows the MALDI-TOF mass spectra obtained on the TiO2-modified target plate for mixtures of phosphokemptide (852 g/mol)/kemptide (772 g/mol) at 1/10, 1/100, and 1/1000 molar ratios, respectively, when the washing step is omitted. Only the signal corresponding to the nonphosphorylated peptide is visible in all cases. Figure 3b, d, and f shows, on the other hand, that following the enrichment procedure described above, the phosphorylated peptide is clearly detected. In Figure 3f, an unidentified signal is detected at m/z ) 917, which may correspond either to a coenriched impurity or to a product of the oxidation of the two arginine and the two leucine residues of phospho-kemptide. Addition of phosphoric acid to enhance the phosphopeptide signal on the washed spots was not necessary for the 1/10 mixture. However, lower amounts of phosphopeptide were clearly detected only when the acid was included, confirming the role of the latter as a signal enhancer. The results shown above demonstrate the on-target phosphopeptide enrichment capability of the method proposed. By using a mixture of a peptide and its phosphorylated isoform, we make sure that the observed selectivity arises due only to the presence of the phosphoryl group. Furthermore, by having a well-defined and controlled stoichiometry, the enrichment capabilities can be evaluated. As seen in Figure 3, even a substoichiometric ratio of phosphorylated peptides of 1/1000 can be unequivocally enriched and identified from a very small peptide sample (≈3 pmol of total peptide content, corresponding to ≈3 fmol of phosphopeptide). On-Target Enrichment of β-Casein Phosphopeptides. Figure 4 shows the MALDI-TOF mass spectra obtained on TiO2 spots for the β-casein tryptic digest without any pre-enrichment and after 30 min on-target enrichment. A comparison between Figure 4a and b clearly shows enhancement of the phosphopep4580
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tide signals after enrichment. Of all the peaks found in Figure 4a, only one (peak 10) corresponds to a phosphopeptide, and its intensity is rather low when compared with the other peaks recorded. In Figure 4b, on the other hand, most signals arising from nonphosphorylated peptides have been eliminated, and 13 signals corresponding to phosphorylated peptides are detected. Five of the expected phosphopeptides obtained from tryptic digestion of β-casein and five from the substoichiometric R-S1and R-S2-casein digestion products are identified, as listed in Table 1. Three of the β-casein phosphopeptides were also detected as doubly charged ions (peaks 1, 2, and 4 in Figure 4) as deduced by a separation between the isotope peaks of 0.5 m/z. The only nonphosphorylated peptides detected correspond to peaks 7 (m/z ) 1753.2, HPHPHLSF-Mox-AIPPKK from κ-casein) and 11 (m/z ) 2202.4, D-Mox-PIQAFLLYQEPVLGPVR from β-casein). Remarkably, both correspond to methionine oxidized (-Mox-) peptides. In terms of the number of different phosphopeptides detected, it can be seen from Figure 4 and Table 1 that the enrichment capabilities of the modified target plate and the enrichment method presented here represent an improvement when compared to those reported by Qiao et al.,7 Torta et al.,24 and Niklew et al.29 using similar approaches for on-target enrichment. The use of a mesoporous film consisting almost solely of anatase titania combined with a previously optimized enrichment protocol is a key factor for optimizing the performance of the modified target plate. To prove the method using more complex systems, the enrichment of phosphopeptides from BSA/β-casein and kemptide/ β-casein mixtures was performed using the same protocol. Figure 5a-c shows the MALDI-TOF mass spectra obtained before and after on-target enrichment of 1/1 and 1/10 β-casein/BSA mixtures. For the 1/1 mixture, two β-casein and three R-casein phosphorylated peptides are clearly identified. Some of the reported phosphorylation sites on BSA are also detected (BSA-1, m/z ) 1751, corresponding to VPQV-pS-pT-P-pT-LVEVSR; BSA-2, m/z ) 1880, corresponding to KVPQV-pS-pT-P-pT-LVEVSR). For the 1/10 mixture, one phosphorylated peptide of β-casein is still clearly identified. However, smaller amounts of β-casein do not allow identification of any of its phosphorylation sites because the mass spectrum is dominated by the signals arising from the BSA phosphorylated peptides (data not shown). In all cases, signals are detected at m/z ) 1480 and 1567, corresponding to the BSA fragments LGEYGFQNALIVR and DAFLGSFLYEYSR containing the acid residues E and D. Although this could be an indication of coenrichment of acidic peptides, it is observed that the ratio of the signals arising from the phosphorylated peptides and those arising from the mentioned acidic peptides increases significantly after carrying on the enrichment procedure (in the 1/1 mixture, from ≈0.03 to ≈1.12 for the case of the β-casein phosphorylated peptide at m/z ) 2062 and the BSA nonphosphorylated peptide at m/z ) 1480), showing a large selectivity toward the phosphorylated peptides. Because the mentioned signals from nonphosphorylated peptides dominate the spectrum of the mixture also prior to enrichment, the obtained results suggest that its presence after the enrichment is mainly due to the difficulty of washing away the peptides found in large excess. To further support this argument, Figure 5d presents the mass spectrum obtained from a mixture of 2.5 pmol of β-casein and
Figure 3. MALDI-TOF mass spectra normalized with respect to the largest occurring signal obtained for (a, c and e) mixtures 1/10, 1/100 and 1/1000 (mol/mol), respectively, of phospho-kemptide (p-K) /kemptide (K) without pre-enrichment and (b, d, and f) after 30 min on-target enrichment.
Figure 4. Mass spectra of the β-casein tryptic digest obtained on a TiO2 modified MALDI target plate (a) without enriching and (b) after 30 min enrichment. Nonphosphorylated peptides in part b are marked with an asterisk.
325 pmol of kemptide. It is seen that, even though kemptide is not acidic, its signal and those from its oligomers (m/z ) 1177,
1384, and 1526, confirmed by MS/MS), although strongly diminished, persist after the enrichment procedure due only to Analytical Chemistry, Vol. 82, No. 11, June 1, 2010
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Table 1. MALDI-MS Peaks Corresponding to Phosphorylated Peptides Identified for the β-casein Tryptic Digest after 30 min On-target Enrichment no. 1 2 3 4 5 6 8 9 10 12 13 14 15 a
[M + H]+ a
1031.03 1278.20a 1466.80 1561.24a 1594.91 1661.02 1952.20 1977.73 2062.05 2432.31 2556.39 2984.50b 3122.47
phosphorylations
position
peptide sequencec
1 1 1 4 1 1 1 1 1 1 1 4 4
β (33-48) β (33-52) R-S2 (153-164) β (1-25) R-S2 (153-165) R-S1 (121-134) R-S1 (119-134) R-S2 (40-56) β (33-48) β (30-48) β (33-52) β (2-25) β (1-25)
FQ-pS-EEQQQTEDELQDK FQ-pS-EEQQQTEDELQDKIHPF TVDME-pS-TEVFTK RELEELNVPGEIVE-pS-L-pS-pS-pS-EESITR TVDME-pS-TEVFTKK VPQLEIVPN-pS-AEER YKVPQLEIVPN-pS-AEER NMAINP-pS-KENLCSTFCK FQ-pS-EEQQQTEDELQDK IEKFQ-pS-EEQQQTEDELQDK FQ-pS-EEQQQTEDELQDKIHPF ELEELNVPGEIVE-pS-L-pS-pS-pS-EESITR RELEELNVPGEIVE-pS-L-pS-pS-pS-EESITR
Doubly charged ions. b Water adduct, verified by MALDI-MS/MS. c Sequences cited from the UniProt Knowledgebase.
Figure 5. Mass spectra of (a) a 1/1 mixture (2.5 pmol each) of BSA/β-casein tryptic digests obtained on a TiO2-modified MALDI target plate without enriching, (b) the same after 30 min of enrichment, (c) a mixture of BSA (2.5 pmol)/β-casein (250 fmol) after 30 min of enrichment, and (d) a kemptide (325 pmol)/β-casein (2.5 pmol) mixture after 30 min of on-target enrichment. Numbered peaks correspond to Table 1. BSA phosphorylated peptides are labeled as BSA-#. Kemptide and its oligomers are labeled as K-#.
their large excess. However, even at these conditions, most β-casein phosphorylated peptides can still be identified, showing that the enrichment and analysis protocol here described is highly efficient. Potentially, an even harsher washing protocol could reduce the unspecific interaction with the enrichment material. The higher performance of anatase can be related to its increased surface basicity33,34 and optimized surface area when compared with brookite and rutile. The advantages of anatase titania over anatase-brookite-rutile mixtures concerning phosphopeptide enrichment have been described and exploited previ(33) Spanos, N.; Georgiadou, I.; Lycourghiotis, A. J. Colloid Interface Sci. 1995, 172, 374–382. (34) Morimoto, T.; Sakamoto, M. Bull. Chem. Soc. Jpn. 1963, 36, 1369–1370.
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ously by Niklew et al.29 However, these authors employed loading (5 mM ammonium bicarbonate) and washing (100 mM acetic acid) solutions that do not prevent the nonspecific binding of acidic peptides. In fact, the mentioned authors detected two signals arising presumably from coenriched acidic peptides. The presence of nonphosphorylated peptides is known to reduce the ionization efficiency of phosphorylated peptides, and therefore, small signals may be lost. Consequently, their enrichment efficiency was significantly lower than what was obtained in the present report. Previous research5 has shown that acidic (pH ≈ 1.9) loading and washing solutions containing a high concentration of a substituted aromatic carboxylic acid, such as DHB, avoid the nonspecific binding of acidic peptides while the enrichment of
phosphorylated peptides remains unaltered. As shown in Figure 4, following a procedure consistent with the mentioned optimal conditions for phosphopeptide enrichment, the signals from acidic peptides are completely avoided, and more phosphorylated peptides are detected. Interestingly, however, signals arising from methionine oxidized peptides are observed. One possibility is that they coenrich due to an affinity for the TiO2 surface stronger than that of their corresponding nonoxidated isoforms. However, it is not clear how the formation of sulfoxides may increase the affinity for the TiO2. A second possibility is that the methionine residues of the corresponding peptides are oxidated in situ during the MALDI-MS measurement due to the high photooxidation capacity of the TiO2 nanoparticles. Similar reactions have already been reported to occur during MALDI-MS experiments on a mesoporous TiO2 surface,35 and it is therefore not unlikely that the oxidated peptides are generated during the measurement. The results reported in Figure 5 indicate the potential of the described protocol for the analysis of more complex mixtures. Even though some phosphorylated peptides go undetected in the analysis, the results obtained still represent a clear improvement when compared to previously reported protocols. Loading as little as 250 fmol of β-casein, and despite the protein’s being mixed with a larger protein in 10-fold molar excess (∼30-fold mass excess), one of the phosphorylated peptides of β-casein is still clearly detected. The roughness of the surface on the micrometer level, considered a large advantage in the paper by Niklew et al.,29 does not seem to play such an important role when considering the more uniform profile of the films used in this work. Also for a macroscopically flat spot, the surface area of the mesoporous film is apparently high enough to provide an excess of binding sites for the phosphorylated peptides. As stated above, the ability to control and tune the thickness of a flat film may, in fact, be an (35) Qiao, L.; Roussel, C.; Wan, J.; Kong, J.; Yang, P.; Girault, H. H.; Liu, B. Angew. Chem., Int. Ed. 2008, 47, 2646–2648.
advantage when optimizing the conditions for phosphopeptide enrichment. CONCLUSIONS This study demonstrates the advantages of using thin mesoporous TiO2 films for the selective enrichment of phosphorylated peptides as well as the importance of selecting the right enrichment protocol. The results show that very low and substoichiometric amounts of phosphorylated peptides can be detected, down to 1/1000 molar ratio and a total amount of ≈3 fmol phosphopeptide. Further, as compared with previous approaches for on-target enrichment, a clear improvement in the enrichment of casein phosphopeptides is observed, obtaining a complete or comprehensive phosphoprofile for the majority β-casein component (five phosphorylation sites identified on five partially overlapping peptide sequences) and detecting five phosphorylated peptide sequences from the minority R-casein, leading to the identification of three phosphorylation sites. If the β-casein is mixed in substoichiometric quantities with another protein, some of its phosphorylated peptides can still be detected. In addition to the higher binding capacity of anatase titania, the use of appropriate loading and washing solutions is of great importance to achieve the reported high enrichment efficiencies. The procedure to print mesoporous TiO2 spots of well-defined and relatively uniform thickness on a conductive glass is simple and produces robust platforms for phosphopeptide enrichment. In this way, a new, fast, simple, and potentially comprehensive method for phosphoproteome profiling is presented. ACKNOWLEDGMENT Financial support from the Swedish Research Council is acknowledged. Received for review March 4, 2010. Accepted April 23, 2010. AC100589J
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