Selective Enrichment and Analysis of Acidic Peptides and Proteins

Sep 23, 2010 - The typical difficulties associated with the detection of acidic peptides (i.e., those with low isoelectric points (pI)) by matrix-assi...
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Anal. Chem. 2010, 82, 8686–8691

Selective Enrichment and Analysis of Acidic Peptides and Proteins Using Polymeric Reverse Micelles and MALDI-MS Nadnudda Rodthongkum, Yangbin Chen, S. Thayumanavan,* and Richard W. Vachet* Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003 The typical difficulties associated with the detection of acidic peptides (i.e., those with low isoelectric points (pI)) by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) represent a challenge in some proteomic analyses. Here, reverse micelle-forming amphiphilic homopolymers with positively charged interiors are synthesized and used to selectively enrich low pI peptides from complex mixtures for MALDI-MS detection. When using these polymers, acidic proteolytic peptides that are undetectable during normal MALDI-MS analysis are selectively detected. We show that enrichment of these low pI peptides allows acidic proteins to be selectively targeted for detection in multiprotein digests. In addition, the presence of the positively charged polymers during MALDI-MS analyses enhances peptide ion signals by almost an order of magnitude, thereby achieving reproducible ion signals for acidic peptides at concentrations as low as 100 fM. Concurrent detection of acidic and basic peptides was also facilitated by utilizing a sequential extraction process involving reverse micelle forming polymers with positively and negatively charged interiors. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a powerful technique for analyzing peptides and proteins and therefore plays an important role in proteomics, especially as part of peptide mass mapping experiments for protein identification.1-9 Nonetheless, proteins of interest are often present in complex samples (e.g., multiprotein digests, cell lysates, tissue extracts, etc.) that are composed of hundreds or even thousands of proteins with a wide dynamic range of concentrations. The high complexity of these samples usually exceeds the * To whom correspondence should be addressed. E-mail: rwvachet@ chem.umass.edu. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2310. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. (3) Aebersold, R.; Mann, M. Nature 2003, 422, 198–207. (4) Stults, J. T. Curr. Opin. Struct. Biol. 1995, 5, 691–698. (5) Gonnet, F.; Lemaıˆtre, G.; Waksman, G.; Tortajada, J. Proteome Sci. 2003, 1, 1–7. (6) Klenø, T. G.; Andreasen, C. M.; Kjeldal, H. Ø.; Leonardsen, L. R.; Krogh, T. N.; Nielsen, P. F.; Sørensen, M. V.; Jensen, O. N. Anal. Chem. 2004, 76, 3576–3583. (7) Sumner, L. W.; Wolf-Sumner, B.; White, S. P.; Asirvatham, V. S. Rapid Commun. Mass Spectrom. 2001, 16, 160–168. (8) Wang, S.; Wei, B.; Yang, P.; Chen, G. Proteomics 2008, 8, 4637–4641. (9) Wang, S.; Bao, H.; Zhang, L.; Yang, P.; Chen, G. Anal. Chem. 2008, 80, 5640–5647.

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analytical capabilities of MALDI-MS. This is especially true for proteins that are expressed in very low abundance.10 Currently, up to 50% of the proteins in cells or tissues have concentrations lower than the detection limits of proteomic methodologies;11,12 however, these low abundance proteins are often key molecules for clinical biomarkers and signal transduction pathways.13,14 To simplify the complexity of these samples and increase the detection of low-abundance peptides and proteins, sample preparation methods that can selectively enrich target analytes and simultaneously eliminate interferences are greatly desired prior to gel-based separations and/or MALDI-MS analysis. In response to this challenge, numerous sample preparation techniques have been developed.15-23 Because it has been reported that peptide pI values can be used as an additional parameter to facilitate peptide mass mapping in protein identification studies by MS,24-26 numerous pI based separation methods have been used to fractionate peptides in mixtures prior to MS analysis.21,24,27,28 Recently, our group has shown that reverse micelle-forming amphiphilic homopolymers and their dendrimeric analogues can selectively extract/fractionate peptides based on their pI values for direct MALDI-MS detec(10) Karlsson, K.; Cairns, N.; Lubec, G.; Fountoulakis, M. Electrophoresis 1999, 20, 2970–2976. (11) Boschetti, E.; Righetti, P. G. Biotechniques 2008, 44, 663–665. (12) Boschetti, E.; Righetti, P. G. Proteomics 2009, 9, 1492–1510. (13) Lee, H. J. Curr. Opin. Chem. Biol. 2006, 10, 42–49. (14) Wisniewski, J. R. Arch. Pathol. Lab. Med. 2008, 132, 1566–1569. (15) Xu, Y.; Bruening, L. M.; Watson, J. T. Anal. Chem. 2003, 75, 185–190. (16) Xu, Y.; Bruening, L. M.; Watson, J. T. Mass Spectrom. Rev. 2003, 22, 429– 440. (17) Xu, Y.; Bruening, L. M.; Watson, J. T. Anal. Chem. 2004, 76, 3106–3111. (18) Tang, N.; Tornatore, P.; Weinberger, S. R. Mass Spectrom. Rev. 2004, 23, 34–44. (19) Zhao, X.; Barber-Singh, J.; Shippy, S. A. Analyst 2004, 129, 817–822. (20) Pan, C. S.; Xu, S. Y.; Zhou, H. J.; Fu, Y.; Ye, M. L.; Zou, H. F. Anal. Bioanal. Chem. 2007, 387, 193–204. (21) Ren, S. F.; Guo, Y. L. J. Am. Soc. Mass Spectrom. 2006, 17, 1023–1027. (22) Zhang, Y.; Fang, J.; Kuang, Y.; Guo, X.; Lu, H.; Yang, P. Chem. Commun. 2007, 4468–4470. (23) Wong, V. N.; Fernando, G.; Wagner, A. R.; Zhang, J.; Kinsel, G. R.; Zauscher, S.; Dyer, D. J. Langmuir 2009, 25, 1459–1465. (24) Cargile, B. J.; Bundy, J. L.; Freeman, T. W.; Stephenson, J. L. J. Proteome Res. 2004, 3, 112–119. (25) Cargile, B. J.; Sevinsky, J. R.; Essader, A. S.; Stephenson, J. L.; Bundy, J. L. J. Biomol. Tech. 2005, 16, 181–189. (26) Xie, H.; Bandhakavi, S.; Griffin, T. J. Anal. Chem. 2005, 77, 3198–3207. (27) Krijgsveld, J.; Gauci, S.; Dormeyer, W.; Heck, A. J. R. J. Proteome Res. 2006, 5, 1721–1730. (28) Lin, C. Y.; Tseng, W. L. Electrophoresis 2009, 30, 532–539. 10.1021/ac101922b  2010 American Chemical Society Published on Web 09/23/2010

tion.29-32 The pI cutoff, when extracting with these polymeric materials, can be tuned by simply adjusting the pH of the extraction solution. While successful, this previous work demonstrated reverse micelles with carboxylate interiors that are selective only for positively charged peptides.29 In addition, at low extraction pH values the extraction capacity of these reverse micelles was found to be limited because the carboxylate groups become protonated, thereby diminishing the number of negatively charged groups available to bind positively charged peptides.29,31

Here, a new amphiphilic homopolymer with a positively charged interior is used to selectively enrich peptides with low pI values over a wide range of extraction pHs. This homopolymer is based on a styrene monomer with quaternary ammonium and alkyl substituents (I). We predicted that this polymer would be effective at extracting low pI peptides, which would be useful for identifying proteins with low pI values in peptide mass mapping experiments. MALDI ion signals of low pI peptides are often much lower than positively charged peptides either because of ionization suppression by positively charged peptides33 or the lack of basic residues to carry the necessary positive charge.34 In this work, we show that peptides with low pI values, which are not detectable in complex mixtures, can be selectively enriched and readily detected using reverse micelles of polymer I. In addition, the presence of the polymer during the MALDI-MS analysis leads to significant signal enhancement of the extracted peptides, allowing for very sensitive analyses. We expect that the positively charged reverse micelles of these polymers will improve the identification of low pI proteins when used as part of peptide mass mapping approaches by increasing the number of detectable peptides. These materials may then have real promise for the detection of low pI proteins, which are important markers of brain tumors (e.g., Glial fibrillary acidic protein, GFAP)35-38 or acidic proteins that are useful for distinguishing strains of pathogenic bacteria.39-41 (29) Combariza, M. Y.; Savariar, E. N.; Vutukuri, D. R.; Thayumanavan, S.; Vachet, R. W. Anal. Chem. 2007, 2007, 7124–7130. (30) Gomez-Escudero, A.; Azagarsamy, M. A.; Theddu, N.; Vachet, R. W.; Thayumanavan, S. J. Am. Chem. Soc. 2008, 130, 11156–11163. (31) Rodthongkum, N.; Washington, J. D.; Savariar, E. N.; Thayumanavan, S.; Vachet, R. W. Anal. Chem. 2009, 81, 5046–5053. (32) Rodthongkum, N.; Chen, Y.; Thayumanavan, S.; Vachet, R. W. Anal. Chem. 2010, 82, 3686–3691. (33) Ballard, J. N. M.; Lajoie, G. A.; Yeung, K. K. C. J. Chromatogr., A 2007, 1156, 101–110. (34) Olumee, Z.; Sadeghi, M.; X., T.; Vertes, A. Rapid Commun. Mass Spectrom. 1995, 9, 744–752. (35) Yung, W. A.; Luna, M.; Borit, A. J. Neuro. Oceanol. 1985, 3, 35–38. (36) Quintanar, J. L.; Franco, L. M.; Salinas, E. Parasitol. Res. 2003, 90, 261– 263. (37) Notturno, F.; Caporale, C. M.; Lauretis, A. D.; Uncini, A. Muscle Nerve 2008, 38, 899–903. (38) Notturno, F.; Capasso, M.; Delauretis, A.; Carpo, M.; Uncini, A. Muscle Nerve 2009, 40, 50–54. (39) Moline, H. E.; Johnson, K. S.; Anderson, J. D. Phytopathology 1983, 73, 224–227. (40) Ochiai, K.; Uchida, K.; Kawamoto, I. Int. J. Syst. Bacteriol. 1993, 43, 69– 76.

EXPERIMENTAL SECTION Reagents. Kinetensin (MW 1172, IARRHPYFL), angiotensin I (MW 1296, DRLVYIHPFHL), and preproenkephalin (MW 1955, SSEVAGEGDGDSMGHEDLY) were purchased from American Peptide Company (Sunnyvale, CA). Cytochrome c (horse heart), lysozyme (chicken egg white), myoglobin (equine skeletal muscle), bovine serum albumin (BSA), toluene, trifluoroacetic acid (TFA), R-cyano-hydroxycinnamic acid (R-CHCA), Tris(hydroxymethyl)aminomethane (Tris), Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), dithiothreitol (DTT), dimethylformamide (DMF), dichloromethane (DCM), triethylamine, ethyl chloroformate, N,N-dimethylethylenediamine, iodomethane, and acetonitrile (ACN) were obtained from Sigma-Aldrich (St. Louis, MO). Human β2m was purchased from Fitzgerald Industries International, Inc. (Concord, MA). Trypsin and chymotrypsin were acquired from Promega (San Luis Obispo, CA). Tetrahydrofuran (THF) was obtained from Fisher Scientific (Pittsburgh, PA) and then distilled over Na/Ph2CO before use. All other chemicals were used as provided. The water used in preparation of all solutions was obtained from a Milli-Q water purification system (Millipore, Bedford, MA). Reverse Micelle Formation. Amphiphilic homopolymer I was synthesized as described in the Supporting Information. Reverse micelles of polymer I were prepared at a 1.0 × 10-4 M concentration by dissolving 10 mg of the polymer in 3.57 mL of toluene and adding 2 equiv of water per equiv of quaternary ammonium group. The equivalents of water are important to form a water pool inside the reverse micelle. This solution mixture was sonicated until a visibly clear solution was obtained and then was used for the liquid-liquid extraction. Proteolytic Digestion. For the single protein digests, 20 µL of the stock protein solution (5.0 × 10-4 M) was prepared in 40 µL solution of 50 mM Tris and 1 mM of CaCl2. To this solution, 24 µL of milli Q water and 1 µL of DTT (1 M) were added. This solution was left at 37 °C for 45 min and 5 µL of ACN was added to the solution. Then, the mixture solution was heated at 60 °C for 10 min to denature the protein, and 10 µL of trypsin (∼5 µg) in a solution of 50 mM Tris and 1 mM of CaCl2 was mixed with denatured protein. The resulting solution was incubated at 37 °C for 15 h. The digestion was stopped by filtering the solution through a 10K MWCO Centricon filter and centrifuged at 12 000 rpm for 15 min. The filtrates were kept in the freezer until they were extracted and analyzed. For the β2m digestion, an enzyme mixture of trypsin and chymotrypsin (50:50) was used, whereas the other single proteins were digested by 100% trypsin. For the multiprotein digests, equal concentrations of cytochrome c, lysozyme, myoglobin, and BSA were digested together by trypsin. Other reagents were calculated and added to the protein mixture solution using the same protein/enzyme ratio described above. After the digestion was complete, an equal concentration of digested β2m was added. Liquid-Liquid Extraction Procedure. A two-phase liquidliquid extraction protocol previously described by our group was used.29 The peptide mixtures were dissolved in a solution of 50 mM Tris/Tris-HCl and adjusted to the desired pH by using 0.5 (41) Wynne, C.; Fenselau, C.; Demirev, P. A.; Edwards, N. Anal. Chem. 2009, 81, 9633–9642.

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Figure 1. MALDI mass spectrum of a 3.3 × 10-6 M solution of PP (left) and MALDI mass spectrum of the organic phase after extraction of a 1.0 × 10-7 M solution of PP with the positively charged polymeric reverse micelles from an aqueous phase solution at pH 7.0 (right).

M HCl or 0.5 M NaOH. The liquid-liquid extraction was performed by first mixing 200-400 µL of toluene solution of the polymeric reverse micelles (1.0 × 10-4 M) with 1 mL of aqueous sample solution and then vortexing this mixture. The peptide or protein digest concentrations in the sample solutions were between 1.0 × 10-7 and 5.0 × 10-7 M. Centrifugation at 12 000 rpm for 30 min was then used to break the resulting emulsion, and the two phases were separated. The aqueous phase was removed, and the organic phase was dried to obtain a solid residue. The dried residue was then redissolved into 10 µL of distilled THF and mixed with 20 µL of an R-CHCA matrix solution (0.16 M in 60:40:0.3% THF/H2O/TFA) for a final volume of 30 µL, and 1 µL of this solution was directly spotted on a stainless steel MALDI target for MALDI-MS analysis. Also, 10 µL of the remaining aqueous phase was mixed with 10 µL of an R-CHCA matrix solution (0.16 M in 60:40:0.3% THF/H2O/ TFA) and analyzed by MALDI-MS. Instrumentation. A Bruker Omniflex MALDI time-of-flight mass spectrometer or a Bruker Autoflex III MALDI time-of-flight mass spectrometer was used to perform the MALDI-MS analysis. When the Omniflex was used, all mass spectra were obtained in the reflectron mode and represent an average of 150 shots acquired at 13% laser power; the accelerating voltage was set to 20 kV. When the Autoflex III was employed, all mass spectra were acquired in the reflectron mode and represent an average of 100 shots acquired at 15% laser power. The accelerating voltage was set to 19 kV. To prepare the sample for analysis, 1 µL of the sample/matrix solution was spotted on a stainless steel MALDI target using the dried droplet method.42 Then, the hot spots (∼100-150 µm in size) on the MALDI target surface, which can be observed by the microscope on the MALDI instrument, were irradiated by the laser pulse to acquire the peptide ion signals. 1 H NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer using residual proton resonance of the solvents as an internal standard. Chemical shifts are reported in parts per million (ppm). The peptide pI values were calculated using the software named Innovagen’s Peptide Property Calculator, which is available at the following Web site: http://www. innovagen.se/custom-peptide-synthesis/peptide-property-calculator/ peptide-property-calculator.asp. (42) Kussmann, M.; Nordhoff, E.; Rahbek-Nielsen, H.; Haebel, S.; Rossel-Larsen, M.; Jakobsen, L.; Gobom, J.; Mirgorodskaya, E.; Kroll-Kristensen, A.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 593–601.

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RESULTS AND DISCUSSION Selective Enrichment of Negatively Charged Peptides. To test the extraction selectivity of the positively charged reverse micelles of polymer I, a mixture of the peptides preproenkephalin (PP, pI 3.6, m/z 1955), angiotensin I (Ang I, pI 7.7, m/z 1296), and kinetensin (KT, pI 11.1, m/z 1172) was extracted from an aqueous solution at pH 6.4 (Figure S1 in the Supporting Information). The results indicate that only the negatively charged peptide, PP, is extracted into the organic phase, whereas the positively charged peptides, Ang I and KT, are not extracted and thus still remain in the aqueous phase. Interestingly, the ion abundance of PP is very low in the MALDI mass spectrum (Figure S1a in the Supporting Information) prior to extraction due to the difficulty of detecting such negatively charged peptides at low concentrations in the presence of other peptides that are readily ionized. Once this peptide is selectively enriched and separated from the positively charged peptides, it is readily detected by MALDI-MS. MALDI-MS Signal Enhancement of Low pI Peptides after Extraction by the Positively Charged Reverse Micelles. Extraction of the negatively charged peptides from the aqueous phase into the organic phase is accompanied by a theoretical concentration factor of 33-fold (1000 µL/30 µL) after the organic phase is dried and the sample is reconstituted in the matrix (see the Experimental Section). Even so, the MALDI ion signal of the peptide PP after extraction by the positively charged reverse micelles is noticeably enhanced compared to the expected concentration factor. A comparison of the MALDI mass spectrum of an unextracted sample of PP at 3.3 × 10-6 M, which is the expected concentration after extraction of a 1.0 × 10-7 M solution, with a MALDI mass spectrum of a 1.0 × 10-7 M solution after extraction shows that the MALDI signal enhancement is approximately 6-fold (Figure 1). In previous work using negatively charged analogues of polymer I, we demonstrated that signal enhancements of approximately one-order of magnitude were possible after the extraction of positively charged peptides.32 In this recent study, we determined that this signal enhancement is caused by coalescence of polymer-peptide conjugates into “hotspots” on the MALDI target surfaces. Visual inspection of the polymer-peptide conjugates on the MALDI target indicates that the same phenomenon is occurring with polymer I. Because of the MALDI signal enhancement, using our extraction protocol along with MALDI-MS detection allows us to obtain reproducible ion signals for PP and other negatively charged

Figure 2. (a) MALDI mass spectrum of a BSA digest (2.0 × 10-7 M) before extraction. (b) MALDI mass spectrum of the organic phase after extraction of a BSA digest (2.0 × 10-7 M) by the positively charged reverse micelles at a pH of 7.5. The numbers above the peaks correspond to the calculated peptide pI values.

peptides at concentrations as low as 100 fM (Figure S2 in the Supporting Information), whereas the lowest detectable concentration of PP and other negatively charged peptides using the same MALDI-MS instrumentation without extraction is around 1 nM. To put this in context, detection of a 100 fM peptide solution after extraction results in approximately 6.45 × 10-18 g of peptide loaded on the target for analysis. Selective Extraction of Negatively Charged Peptides from Protein Digests. Tryptic digests of several proteins, including cytochrome c (pI, 9.6), β-2-microglobulin (pI 6.1), lysozyme (pI 11.1), and BSA (pI 4.7), were extracted by the positively charged reverse micelles and subsequently analyzed by MALDI-MS. MALDI mass spectra of a BSA digest before and after extraction are shown in Figure 2. The m/z ratios of each measured BSA peptide fragment are shown in Table S1 in the Supporting Information. The results obtained from other protein digests are shown in Figures S3-S5 and Tables S2-S4 in the Supporting Information. The data in Figure 2 and Table S1 in the Supporting Information illustrate several features of the extractions using the positively charged polymers. First, all of peptides with pI values below 7.5, which is the extraction pH, that are detected before extraction are also detected after extraction. Second, none of the peptides with pI values above 7.5 that are detected before extraction are detected after extraction. Together these two observations demonstrate the exquisite selectivity of these materials to extract only negatively charged peptides. A third noteworthy

Figure 3. MALDI mass spectrum of the organic phase after extraction of a BSA digest (2.0 × 10-7 M) by the positively charged reverse micelles at a pH of (a) 4.5 and (b) 10.5. The numbers above the peaks correspond to the calculated peptide pI values.

observation is that an additional seven low pI peptides and three midrange (6.3-7.4) pI peptides, which were undetectable before the extraction, are now detected after extraction. This result indicates that extraction with the positively charged reverse micelles enables the detection of negatively charged peptides that are not directly detectable by MALDI-MS. Overall, 53% of the possible negatively charged peptides above m/z 800 (assuming no missed cleavages) are detected after extraction, whereas only 24% of these peptides are detected before extraction. Effect of Extraction pH on the Number of Detectable Peptides from a Protein Digest. To investigate the effect of aqueous solution pH on extraction selectivity, BSA digests (2.0 × 10-7 M) were prepared and extracted by the positively charged reverse micelles at the different aqueous solution pH values (Figure 3 and Table S6 in the Supporting Information). The extraction selectivity can be tuned by simply adjusting the aqueous solution pH. It is clear from a comparison of the results in Figures 2b and 3 that the number of detectable peptides increases at higher aqueous solution pH values. These results are expected based on the fact that the number of negatively charged peptides in solution increases as the aqueous solution pH increases. Interestingly, these data suggest that the pH inside the reverse micelles is controlled by the aqueous solution pH. The same phenomenon was observed in previous work using negatively charged reverse micelles.29,31,32 A very striking set of observations is the collection of peptides extracted at pH 4.5 (Figure 3a and Table S6a in the Supporting Information). Again, only peptides with pI values below the Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

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aqueous phase pH are extracted, but in this case almost all the peptides now detected were undetectable before extraction. Indeed, 85% of the possible negatively charged peptides above m/z 800 (assuming no missed cleavages) are detected after extraction at pH 4.5, whereas only 5% of these peptides are detected before extraction. Evidently, the removal of the more positively charged and thus more easily detected peptides eliminates the ion suppression normally experienced by these very negatively charged peptides. Consistent with this explanation is the fact that some of the peptides that are detected after extraction at a pH of 4.5 are no longer detected at the higher extraction pHs where more and more positively charged peptides are now extracted. Another important observation from the BSA experiments is the increase in sequence coverage that accompanies the extractions at different pHs. The sequence coverage of the BSA digest before extraction is only about 29%, but this increases to 35%, 38%, and 47% after extraction by reverse micelles at the aqueous pHs of 4.5, 7.5, and 10.5, respectively. If all the detected peptides are considered from the three extraction experiments, then the sequence coverage is 71%. These results suggest that extracting protein digests at several different pH values using the positively charged reverse micelles would be a straightforward way to increase protein sequence coverage, and therefore the confidence level of protein identifications, in peptide mass mapping experiments. Extractions of Multiprotein Digests. We further investigated the extraction capabilities of the positively charged reverse micelles on a multiprotein digest consisting of cytochrome c (pI, 9.6), β-2-microglobulin (pI 6.1), lysozyme (pI 11.1), myoglobin (pI 6.8), and BSA (pI 4.7) (Figure 4 and Table S7 in the Supporting Information). After extraction at pH 5.4 (Figure 4b), most of the detected peptides (77%) come from BSA and β-2-microglobulin, which are the low pI proteins. Again, many of the peptides detected after extraction at this pH are undetectable before extraction (Figure 4a). In addition, very few peptides (12%) from cytochrome c and lysozyme are observed, which is not surprising because these proteins have higher pI values and thus more peptides with pI values that would not be enriched by the reverse micelles. As was observed for the BSA digest in Figures 2 and 3, the number of detectable peptides in the multiprotein digest increases when the extraction pH is increased to pH 7.4, which is consistent with more peptides being negatively charged at the higher aqueous solution pH. Overall, these results indicate that selective enrichment and detection of acidic proteins in protein mixtures, via their acidic peptides, is possible with these materials. This approach might be beneficial for selective identification of low pI proteins in the context of peptide mass mapping experiments. Sequential Extraction of Multiprotein Digests Using Positively Then Negatively Charged Polymeric Reverse Micelles. Because peptides from low pI proteins can be readily enriched with the positively charged reverse micelles, we decided to see if we could sequentially enrich peptides from low pI proteins and then high pI proteins that are present in the same solution. To do this, we first extracted the same multiprotein digest with the positively charged reverse micelles at a pH of 4.8 and then removed the organic phase containing the positively charged reverse micelles and the extracted low pI peptides (i.e., peptides 8690

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Figure 4. (a) MALDI mass spectrum of a multiprotein digests (5.0 × 10-7 M) before extraction. (b) MALDI mass spectrum of the organic phase after extraction of a multiprotein digest (5.0 × 10-7 M) by the positively charged reverse micelles at a pH of 5.2. (c) MALDI mass spectrum of the organic phase after extraction of a multiprotein digest (5.0 × 10-7 M) by the positively charged reverse micelles at a pH of 7.4. The numbers above the peaks correspond to the calculated peptide pI values.

with pI values below 4.8). Next, we increased the pH of the remaining aqueous phase solution to a value of 8.9, added a fresh organic phase containing negatively charged reverse micelles (polymer AP, see synthesis in Supporting Information),29,31,32 and extracted the high pI peptides (i.e., peptides with pI values above 8.9). The two resulting organic phases, one with low pI peptides and the other with high pI peptides, were then separately analyzed by MALDI-MS (Figure 5 and Table S8 in the Supporting Information). It is clear from Figure 5a that the initial extraction results in the detection of peptides mostly from BSA. This result is expected given the low pI value (4.7) of this protein. About 60% of the peptides come from BSA, and peptides from β2m, which has the next lowest pI value (6.1), have the next highest occurrence. Only

Supporting Information), fewer peptides are detected than that shown in Figure 5b. In other words, prior separation by the positively charged reverse micelles simplifies the mixture so that about 35% more peptides are detected. Clearly, increasing the number of detectable peptides increases protein sequence coverage, which would improve protein identification by peptide mass mapping. Sequential extractions like this at just the right pH values might represent a powerful approach for targeting the detection of desired proteins.

Figure 5. (a) MALDI mass spectrum of the organic phase after extraction of a multiprotein digest (5.0 × 10-7 M) by the positively charged reverse micelles at a pH of 4.8. (b) MALDI mass spectrum of the organic phase after sequential extraction with the negatively charged reverse micelles of the remaining aqueous phase of the multiprotein digest at a pH of 8.9. The numbers above the peaks correspond to the calculated peptide pI values.

5 peptides are seen for cytochrome c (pI ) 9.6), myoglobin (pI ) 6.8), and lysozyme (pI ) 11.1) combined. In contrast, when the pH of the remaining aqueous phase is increased to 8.9 and extracted by the negatively charged reverse micelles, peptides from cytochrome c and lysozyme dominate the MALDI mass spectrum (Figure 5b). About 75% of the peptides come from these two basic proteins, while only four peptides come from acidic BSA, even though BSA has more possible tryptic fragments than all the other four proteins combined. Interestingly, when the same multiprotein digest is only extracted by the negatively charged reverse micelles at pH 8.9 (Figure S6 and Table S9 in the

CONCLUSIONS In summary, we have shown that (i) reverse micelle-forming amphiphilic homopolymers with positively charged interiors can selectively enrich low pI peptides that are undetectable or barely detectable during regular MALDI-MS analysis; (ii) MALDI ion signals for these extracted peptides are significantly enhanced, which allows us to achieve reproducible ion signals for acidic peptides at concentrations as low as 100 fM; (iii) the extraction selectivity can be tuned by changing the aqueous solution pH; (iv) the positively charged reverse micelles can be used to facilitate the analysis of acidic proteins via the detection of their component peptides; and (v) the positively charged reverse micelles can be used in a sequential extraction protocol with negatively charged reverse micelles to improve the analysis of acidic and basic proteins from the same sample. Overall, these positively charged polymers should be very beneficial for improving the detection of acidic proteins, which are important biomarkers of brain tumors and are often species-differentiating gene products in pathogenic bacteria. ACKNOWLEDGMENT We thank the Office of Naval Research (Grant N000140510501) and the National Science Foundation Center for Hierarchical Manufacturing (Grant CMMI-0531171) for support of this work. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review July 24, 2010. Accepted September 10, 2010. AC101922B

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