Anal. Chem. 2009, 81, 5046–5053
Generating Peptide Titration-Type Curves Using Polymeric Reverse Micelles As Selective Extraction Agents along with Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Detection Nadnudda Rodthongkum, Jacqueline D. Washington, Elamprakash N. Savariar, S. Thayumanavan,* and Richard W. Vachet* Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003 Amphiphilic homopolymers that self-assemble into reverse micelles in nonpolar solvents have been used by us in the context of a two-phase liquid-liquid extraction protocol to selectively extract peptides from aqueous solution for MALDI-MS detection. In this manuscript, we investigate the scope of these materials in terms of its extraction capabilities, using compounds with varying isoelectric points (pI) and pKa values over a range of aqueous solution pHs. We find that the aqueous solution pH and analyte pKa values are the major factors controlling extraction selectivity. We also find that the experimental extraction efficiencies correspond very well with the fractional compositions of species calculated using analyte pKa values, indicating that these extraction materials can be used to simultaneously generate titration-type curves for each individual peptide in a mixture. We predict that such titration curves, along with accurate mass measurements, could represent a new way of improving protein identification procedures. Analysis of peptides and proteins in complex mixtures is a critical challenge in many areas of study ranging from biology and medicine to environmental science.1-3 Although ultrasensitive detection techniques, such as mass spectrometry (MS) and fluorescence spectroscopy, have been developed and are regularly used,3-7 efficient sample preparation techniques that simultaneously eliminate interferences and selectively extract and con* To whom correspondence should be addressed. E-mail: rwvachet@ chem.umass.edu (R.W.V.);
[email protected]. (1) Yanofsky, C. M.; Bell, A. W.; Lesimple, S.; Morales, F.; Lam, T. T.; Blakney, G. T.; Marshall, A. G.; Carrillo, B.; Lekpor, K.; Boismenu, D.; Kearney, R. E. Anal. Chem. 2005, 77, 7246–7254. (2) Kamysz, W.; Okroˇj, M.; Łempicka, E.; Ossowski, T.; Łukasiak, J. Acta Chromatographica 2004, 14, 180–186. (3) Pan, C. S.; Xu, S. Y.; Zhou, H. J.; Fu, Y.; Ye, M. L.; Zou, H. F. Anal. Bioanal. Chem. 2007, 387, 193–204. (4) Xu, Y.; Bruening, M. L.; Watson, J. T. Mass Spectrom. Rev. 2003, 22, 429– 440. (5) Tang, N.; Tarnatore, P.; Weinberger, S. R. Mass Spectrom. Rev. 2004, 23, 33–44. (6) Seifrtova´, M.; Pena, A.; Lino, C. M.; Solich, P. Anal. Biochem. 2008, 391, 799–805.
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centrate the desired analytes from biological samples (e.g., multiprotein digests, cell lysates, etc.) are greatly desired. Numerous techniques have been created for the enrichment of analytes from complex samples.8,9 While promising, these techniques also have a few shortcomings that necessitate improvements or the investigation of new extraction approaches. Recently, we have been investigating the extraction capabilities of a new type of amphiphilic homopolymer based on styrene monomers with carboxylic and alkyl substituents (Figure S1, Supporting Information) that can form reverse micelles in nonpolar solvents. The reverse micelles formed by this polymer are composed of hydrophobic exteriors and hydrophilic interiors, which allow them to solubilize polar analytes in nonpolar solvents.10-12 These polymers and dendrimeric analogues have been used by our group to selectively extract peptides for direct detection by matrix assisted laser desorption ionization (MALDI)MS.13,14 Several features of the extraction and detection were noteworthy: (i) the extraction selectivities of the polymers were found to depend on peptide pI values; (ii) the peptides with pI values above the solution pH were extracted, but those with pI values below the solution pH were not; (iii) an unexpected enhancement in the MALDI-MS signal for extracted peptides ionized in the presence of the polymer was observed, which allowed us to obtain reproducible ion signals for some peptides at concentrations as low as 1 pM. Given the promising attributes of these polymeric materials, we have been interested in testing their scope and further extending their applicability. Specifically, we are interested in selectively fractionating molecules ac(7) Jorge, I.; Casas, E. M.; Villar, M.; Perez, I. O.; Ferrer, D. L.; Ruiz, A. M.; Carrera, M.; Marina, A.; Martinez, P.; Serrano, H.; Canas, B.; Were, F.; Gallardo, J. M.; Lamas, S.; Redondo, J. M.; Dorado, D. G.; Vazquez, J. J. Mass Spectrom. 2007, 42, 1391–1403. (8) Cichna-Markl, M. J. Chromatogr., A 2006, 1124, 167–180. (9) Pawliszyn, J. Anal. Chem. 2003, 75, 2543–2558. (10) Basu, S.; Vutukuri, D. R.; Shyamroy, S.; Sandanaraj, B. S.; Thayumanavan, S. J. Am. Chem. Soc. 2004, 126, 9890–9891. (11) Basu, S.; Vutukuri, D. R.; Thayumanavan, S. J. Am. Chem. Soc. 2005, 127, 16794–16795. (12) Savariar, E. N.; Aathimanikandan, S. V.; Thayumanavan, S. J. Am. Chem. Soc. 2006, 128, 16224–16230. (13) Combariza, M. Y.; Savariar, E. N.; Vutukuri, D. R.; Thayumanavan, S.; Vachet, R. W. Anal. Chem. 2007, 79, 7124–7130. (14) Escudero, A. G.; Azagarsamy, M. A.; Theddu, N.; Vachet, R. W.; Thayumanavan, S. J. Am. Chem. Soc. 2008, 130, 11156–11163. 10.1021/ac900661e CCC: $40.75 2009 American Chemical Society Published on Web 05/21/2009
cording to the acid/base characteristics (i.e., pKa values) of their functional groups. We believe that a correlation between the extent of fractionation and functional group pKa values should significantly enhance molecular identification. For example, in the analysis of complex peptide mixtures (e.g., from multiprotein digests), fractionation and/or separation of peptides is, of course, very important to reduce the complexity of samples. If the peptides can be separated in a way, though, that also provides additional information about their physical or chemical properties, then this additional information can be used to increase confidence levels in protein identification studies.15-17 It has been reported that when peptide properties such as pI value15,18,19 and retention time20,21 from reversed-phase LC separations are combined with accurate peptide mass information, false positive rates for protein identifications can be decreased. In the present study, we investigate whether or not polar molecules such as peptides can be extracted from aqueous solutions of varying pH in a way that reflects the acid/base characteristics of their constituent functional groups. In effect, we seek to determine if a predictable trend, such as a titration curve, can be generated for molecules upon extracting them with these polymers. Before using these polymeric materials to separate peptide mixtures, however, we first test the performance of these materials on compounds with well-defined pKa values: a series of fluorescent pI markers. Such markers are commonly used as reference compounds in isoelectric focusing.22,23 Initially studying these molecules is useful because these compounds also allow us to quantify the extraction via fluorescence spectroscopy. Analysis of these molecules by fluorescence is needed because MALDI-MS of peptides is semiquantitative at best. In addition, these pI markers provide another set of molecules, besides peptides, to test the scope of pKa-based fractionation. Following the study with the pI markers, we demonstrate that these polymeric reverse micelles can be used to extract peptides in a way that generates titration-type curves when combined with MALDI-MS detection. We envision that peptide “titration curves” should be even more characteristic of a given peptide than a single pI value, and so such information could provide an even better constraint for database searches in protein identification studies. EXPERIMENTAL SECTION Reagents. The fluorescent pI markers, methanol, toluene, trifluoroacetic acid (TFA), R-cyano-hydroxycinnamic acid (RCHCA), Tris/Tris-HCl, and cytochrome C were purchased from Sigma-Aldrich (St. Louis, MO). The chemical names, structures, pI and pKa values of the nine markers22 are shown in the (15) Cargile, B. J.; Stephenson, J. L. Anal. Chem. 2004, 76, 267–275. (16) Krijgsveld, J.; Gauci, S.; Dormeyer, W.; Heck, A. J. R. J. Proteome Res. 2006, 5, 1721–1730. (17) Lam, H. T.; Josserand, J.; Lion, N.; Girault, H. H. J. Proteome Res. 2007, 6, 1666–1676. (18) Cargile, B. J.; Bundy, J. L.; Freeman, T. W.; Stephenson, J. L. J Proteome Res. 2004, 3, 112–119. (19) Cargile, B. J.; Talley, D. L.; Stephenson, J. L. Electrophoresis 2004, 25, 936–945. (20) Smith, R. D.; Anderson, G. A.; Lipton, M. S.; Tolic, L. P.; Shen, Y.; Conrads, T. P.; Veenstra, T. D.; Udseth, H. R. Proteomics 2002, 2, 513–523. (21) Kawakami, K.; Tateishi, K.; Yamano, Y.; Ishikawa, T.; Kuroki, K.; Nishimura, T. Proteomics 2005, 5, 856–864. (22) Horka´, M.; Willimann, T.; Blum, M.; Nording, P.; Friedl, Z.; Sˇlais, K. J. Chromatogr., A 2001, 916, 65–71.
Supporting Information (Table S1). OVA peptide 323-339 (MW 1773, ISQAVHAAHAEINEAGR), angiotensin I (MW 1296, DRVYIHPFHL), ACTH 1-13 human (MW 1624, SYSMEHFRWGKPV), kinetensin (MW 1172, IARRHPYFL), and bradykinin (MW 1060, RPPGFSPFR) were acquired from the American Peptide Company (Sunnyvale, CA). Trypsin was purchased from Promega (San Luis Obispo, CA). Potassium phosphate and potassium hydrogen phthalate were acquired from Fisher Scientific (Fair Lawn, NJ). Potassium chloride, sodium borate, and sodium chloride were obtained from Mallinckrodt (Paris, KY). Tetrahydrofuran (THF) was obtained from Fisher (Pittsburgh, PA) and was freshly distilled over Na/Ph2CO. All other chemicals were used as provided. Polymer Synthesis and Reverse Micelle Formation. The styrenic monomer was synthesized according to our previously reported procedure.11 The polymer was synthesized using reversible addition-fragmentation chain transfer polymerization of the styrene based monomer that has tert-butyl ester and n-decyl groups with ether linkages at meta positions relative to the olefinic group. 2-Cyanoisopropyl dithiobenzoate was used as the chain transfer agent, and tert-butyl ester was hydrolyzed after polymerization.11,12 The number average molecular weight (Mn) of the polymer, which was determined using size exclusion chromatography before ester hydrolysis, was found to be about 25 kDa with a polydispersity index (PDI) of 1.32. The reverse micelle solution of the polymer was prepared by using our reported procedure.11 In this experiment, 10 mg of the polymer was dissolved in toluene to obtain a concentration of 1 × 10-4 M, and 2 equivalents of water per monomer were added to the toluene to generate the water pool in the reverse micelle interiors. The solution was sonicated for 6 h or until a visibly clear solution was obtained. This solution was then used for the liquid-liquid extraction. Proteolytic Digestion. For the cytochrome C digestion, 100 µL of a cytochrome C solution (5.4 × 10-4 M) in Tris/CaCl2 was mixed with 50 µL of MeOH. The solution was heated at 60 °C for 15 min in order to denature the protein. The trypsin solution that contains 2 µg/450 µL of protein, 50 mM of TRIS buffer, and 1 mM of CaCl2 was prepared, and 150 µL of this solution was added to the denatured protein sample. The resulting solution was kept at 37 °C for 24 h in order to digest the protein. After that, the digestion was stopped by filtering the solution through a 10K MWCO Centricon filter and then centrifuged at 12 000 rpm for 20 min. The filtrates were then stored in the freezer before extraction. Liquid-Liquid Extraction Procedure. A two-phase liquidliquid extraction protocol that was previously developed and reported by our research group was employed.13 An aqueous solution of each pI marker was prepared at a given pH using an appropriate buffer. The extraction was performed by first mixing a toluene solution (100 µL) of the polymeric reverse micelles (1 × 10-4 M) with an aqueous solution (500 µL) of a given pI marker (1 × 10-7 M), buffered to the desired pH. The mixture was vortexed and then centrifuged at 12 000 rpm for 30 min to break the resulting emulsion. The top (organic) layer was removed, and the bottom (aqueous) layer was analyzed by fluorescence spectroscopy. The extractions by the polymeric Analytical Chemistry, Vol. 81, No. 12, June 15, 2009
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reverse micelles were carried out in triplicate for each marker at each pH value. Extractions of the individual peptides, peptide mixture, and cytochrome C digest were done in a manner similar to described above. The aqueous peptide solution was prepared in 50 mM Tris/ Tris-HCl solution at the desired pH using 0.5 M HCl or NaOH for pH adjustment. Initially, a 200 µL toluene solution of 1 × 10-4 M polymeric reverse micelles was mixed with a 1000 µL of an aqueous solution of peptide (1 × 10-7 M). The mixture was vortexed and then centrifuged for 30 min. The top (organic) layer was quickly dried and redissolved in 10 µL of THF. This solution was then mixed with 20 µL of 0.16 M R-CHCA matrix solution (in 60:40:0.3% THF/H2O/TFA) for a final volume of 30 µL, and this solution was immediately analyzed by MALDIMS. Also, the lower (aqueous) phase from the peptide extraction was analyzed by MALDI-MS. An equal amount of aqueous phase solution (5 µL) and 0.16 M R-CHCA matrix solution (in 60:40:0.3% THF/H2O/TFA) were mixed together, and 1 µL of the resulting solution was analyzed immediately. Fluorescent Measurements for pI Markers. The concentration of the pI marker left in the bottom (aqueous) layer after extraction was determined on a Photon Technology International fluorometer (Ontario, Canada). Analysis of the organic phase was difficult because the spectrum of the polymer in this phase was very broad and in some cases overlapped with the signal from the pI markers. The excitation and emission wavelengths for each pI marker were chosen to maximize the fluorescence signal. Standard solutions with concentrations ranging from 1 × 10-8 to 5 × 10-6 M were prepared for each marker at different pH values to construct calibration curves. The fluorescence intensity of the aqueous phase after a given extraction was measured, and in each case the fluorescence intensity was used to determine the concentration of the pI marker remaining in the aqueous phase after extraction. The amount of pI marker that was extracted by the reverse micelles was then calculated by subtracting the remaining aqueous phase concentration from the initial concentration (1 × 10-6 M). The measurements were repeated 3 times for each sample, and the average is reported. MALDI-MS analysis for peptides. MALDI-MS analysis was performed on a Bruker Reflex III time-of-flight mass spectrometer. This mass spectrometer has a nitrogen laser (337 nm), a 1.0 m flight tube, and a stainless steel sample target. MALDI mass spectra were obtained in the reflectron mode using 16 kV voltage with 60 shots at 45% laser power. To prepare the samples for analysis, 1 µL of the sample/matrix solution was spotted on a stainless steel target using the dried droplet method. Theoretical Calculations. To better understand the extraction behavior by polymeric reverse micelles, theoretical calculations of species present in solution at different pH values were performed for pI markers and peptides. All of the pI markers are diprotic; therefore, simple equations can be used to calculate the fractional composition of each species in aqueous solutions. The chemical equilibrium of diprotic compounds (H2A) are K1
H2A+ y\z H+ + HA (23) Chmelik, J.; Mazanec, K.; Slais, K. Electrophoresis 2007, 28, 3315–3323.
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K2
HA y\z H+ + A-
where K1 is the acid dissociation constant of H2A+ and K2 is the acid dissociation constant of HA. In a diprotic system, we designate the fraction in the form H2A+ as RH2A+, the fraction in the form HA as RHA, and the fraction in the form A- as RA-. These fractional compositions can be calculated by the following equations. [H2A+] [H+]2 ) + 2 F [H ] + [H+]K1 + K1K2
(1)
RHA )
K1[H+] [HA] ) F [H+]2 + [H+]K1 + K1K2
(2)
RA- )
K1K2 [A-] ) + 2 F [H ] + [H+]K1 + K1K2
(3)
RH2A+ )
RH2A+ + RHA + RA- ) 1
(4)
For the pI markers, the fractional compositions were converted to percentage fractional composition in order to compare with the percentage extractions obtained from the experiment. Fractional composition calculations were also performed for the peptides. Since the amino acid sequences and pKa of amino acid for each peptide are known, the fractional composition at different pH values can also be calculated. The calculation procedure is similar to the pI markers but involves more acidic/ basic functional groups and is expected to be less accurate because the pKa values of amino acid residues can change depending on the identity of nearby amino acids. Table S2 in the Supporting Information shows an example of the calculation for a peptide. RESULTS AND DISCUSSION Extraction of the pI Markers by the Reverse Micelles. Before application of the polymeric reverse micelle-like nanoassemblies to selectively extract peptides in the complex mixtures, the performance of these polymeric materials were investigated on a series of fluorescent pI markers. Fluorescence spectroscopy is inherently more quantitative than MALDI-MS, so these markers offer the opportunity to assess the quantitative nature of our approach. Because our reverse micelles have carboxylate groups in their interiors, the positively charged compounds are expected to be extracted due to Coulombic attraction. Upon extraction, the concentrations of the pI markers left in the aqueous phase were determined. As an example of the data from the aqueous solution, the fluorescence spectra of pI marker 2, before (solid line) and after extraction (dashed line) by the reverse micelles at an aqueous pH of 1.9, are shown in Figure 1. The decrease in fluorescence intensity after extraction indicates that most of the pI marker is extracted and transferred into the organic phase. This pI marker has a pI value of 3.0, and at an aqueous pH of 1.9, most molecules of this compound are positively charged and therefore interact favorably with the negatively charged interiors of the reverse micelles.
Figure 1. Fluorescence spectra of pI marker 2 in aqueous solution before (solid line) and after (dashed line) extraction by the polymeric reverse micelles at an aqueous pH of 1.9.
Figure 3. A comparison of experimentally observed and theoretically predicted extraction percentages for pI marker 6 as a function of aqueous pH. The solid line represents the experimentally observed data, the lowest dashed line represents the fractional composition of the positively charged species (MH2+) calculated from the pKa values, the middle dotted line represents the sum of the calculated fractional composition of MH2+ and the percentage of the marker experimentally extracted by toluene, and the highest dashed line represents the sum of the calculated fractional composition of MH2+, the percentage of the marker experimentally extracted by toluene, and the calculated fractional composition of the neutral zwitterionic form (MH) arbitrarily multiplied by 30%. Table 1. Percentages Contributed by the Neutral Zwitterionic Species Providing the Best Fit with Fractional Composition Calculation of the Nine pI Markers
Figure 2. Extraction percentages as a function of aqueous phase pH for pI markers 2 (pI ) 3.0), 3 (pI ) 4.0), 5 (pI ) 6.2), 6 (pI ) 7.2), and 9 (pI ) 10.3).
Previously, we had shown for a series of peptides that charge complementarity exerts an important influence on the extraction process with these amphiphilic homopolymers.13 Does the importance of analyte charge mean that the aqueous solution pH directly controls extraction efficiency? While aqueous solution pH definitely controls the charge of the pI markers in the aqueous phase, it is not obvious that aqueous solution pH will control the extraction process because the analyte is transferred out of the aqueous phase and into the organic phase. To test the effect of aqueous solution pH on extraction efficiency, each pI marker was extracted over a range of pH values spanning its pI value. The extraction percentages of five of the pI markers as a function of aqueous phase pH are shown in Figure 2. These plots demonstrate that aqueous phase pH is a crucial factor controlling extraction selectivity and efficiency. All of the pI markers exhibit the same trend; they are extracted well at low pH values and poorly at high pH values. For every pI marker, the fraction of molecules that are positively charged is greatest at the lower pH values, and so the extraction efficiency is greatest. At higher pH values, a lower fraction of the molecules are positively charged, and thus the extraction efficiency is much lower. Moreover, we found that the
pI marker
1
2
3
4
5
6
7
8
9
% neutral contribution
5
20
20
20
30
30
30
20
20
small extraction percentage at pH values well above their pI values is mostly controlled by the marker’s inherent affinity for the toluene phase. To better understand the extraction behavior with these polymeric reverse micelles, the experimental data were compared with the fractional composition of species present in solution at different aqueous pH values. As an example, the best fit with the experimental data of pI marker 6 was obtained from the sum of the fractional composition of positively charged species (MH2+), the percentage of the marker 6 extracted by toluene and the calculated fractional composition of the neutral form (MH) arbitrarily multiplied by 30% of the total percentage, as illustrated in the highest dashed line (Figure 3). A small fraction of the neutral zwitterionic species appears to be extracted by the negatively charged reverse micelles probably because it contains a positively charged amine group capable of interacting with the carboxylate groups of the reverse micelle interiors. When the same type of curve fitting is done for other pI markers, a similar degree of agreement is achieved; however, the percent contribution of the neutral species extracted by the polymeric reverse micelles is slightly different for each marker (Table 1) with an average value of 21 ± 7.9%. Interestingly, the best curve fittings for almost all of the pI markers are obtained when the percentage contribution of neutral species is about 20-30%. There is only one notable outlier, pI marker 1, which Analytical Chemistry, Vol. 81, No. 12, June 15, 2009
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Figure 4. The normalized ion abundances of the organic phase after extraction (solid line) and the fractional composition of the positively charged species (dotted line) as a function of aqueous pH for the OVA peptide, angiotensin I, ACTH, and kinetensin.
has a neutral contribution of 5%. The exact reason for this outlier is not clear, but one possibility is suggested. The close proximity of the positively charged and negatively charged group may not allow the negatively charged micelle interior to interact optimally with the positively charged group in the zwitterionic form of marker 1. Extraction of Individual Peptides by the Reverse Micelles. Because extraction selectivity and efficiency depend on the analyte charge, which is controlled by the aqueous solution pH, extraction of peptides that have different charges over a range of aqueous solution pH might allow one to generate titration-type curves for peptides. To initially test this idea, individual peptide with different pI values, including OVA peptide 323-339 (pI 6.00), angiotensin I (pI 7.70), ACTH 1-13 (pI 9.30), and kinetensin (pI 11.10) were extracted by the reverse micelles over a range of aqueous pH values and then analyzed by MALDI-MS. The normalized ion abundances of the organic phase that represent the amount extracted by the polymers were plotted as a function of aqueous solution pH and compared to the fractional composition (Figure 4). The fractional compositions that provide the best fit with the experimental data for all of the individual peptides are comprised by only the positively charged species without having to include any percentage of the peptides extracted by toluene or any percentage due to neutral zwitterionic forms of the peptides. These results are therefore different from the results obtained with the pI markers. Unlike the pI markers, none of the peptides studied here had any appreciable affinity for pure toluene. Moreover, the apparent inability to extract neutral, zwitterionic peptides may be due to the tendency of the oppositely charged groups in a given zwitterionic peptide to interact with one other instead of interacting with the negatively charged interiors of the reverse micelles. 5050
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The extraction selectivity and efficiency of individual peptides are regulated by the aqueous solution pH (Figure 4). In general, the normalized ion abundance of each peptide (solid line) tends to increase when the aqueous solution pH is decreased. This is consistent with most of the peptide molecules in a sample becoming positively charged at lower pH values and so being efficiently extracted by the negatively charged reverse micelles. A lower fraction of the molecules are positively charged at higher pH, and thus the extraction efficiency is lower. A notable observation from the plots in Figure 4 is that normalized ion abundances of the OVA peptide, angiotensin I, and ACTH significantly drop when the aqueous pH is lower than 6. One possible reason to explain these observations is that the carboxylate functional groups in the polymeric reverse micelle interiors become protonated when the aqueous solution pH is decreased. Therefore, the influence of aqueous solution pH on the extraction capacity of the polymeric reverse micelles was investigated. Polymeric Reverse Micelle Extraction Capacity As a Function of Aqueous Solution pH. Determining the extraction capacity of the reverse micelles as a function of pH is important for interpreting the data in Figure 4, but it also important for understanding the potential of these materials for extracting more highly complex samples, such as protein digests. The extraction capacity of the polymeric reverse micelles was determined for aqueous solution pH values ranging from 3 to 10 using bradykinin as a model peptide. For these extractions, a constant concentration of the polymer in toluene (1.0 × 10-4 M, 200 µL) was mixed with 1000 µL of bradykinin at different concentrations to determine the maximum capacity of the reverse micelles at each pH
Table 2. m/z Ratios, Residue Number, Sequences, And Calculated pI Values of Some Cytochrome c Peptides That Are Usually Observed in MALDI Spectra of the Organic Phase at Aqueous pHs Ranging from 4.0 to 12.3
Figure 5. The extraction capacity of negatively charged polymeric reverse micelles over a range of aqueous solution pH values (3-10).
(Figure 5). Figure S2 in the Supporting Information shows an example of how the capacity was determined at each pH. The extraction capacity of the polymer is at its maximum (1.5 × 10-6 M bradykinin) at pHs above 6; however, the capacity decreases by factors of 1.9-7.5 at pHs below 6. At pHs below 6, the aqueous solution pH is close to the pKa value (∼5) of typical carboxylic acid groups, which are what comprise the interiors of the reverse micelle interiors. Evidently, at lower pH values some of the carboxylate groups become protonated, thereby substantially reducing the extraction capacity of the polymeric reverse micelles. The drop in the extraction capacity at pHs below 6 corresponds well with the pH values at which the normalized ion abundances of the OVA peptide, angiotensin I,
residue number
m/z
sequence
calculated pI
1-8 9-22 23-38 26-38 26-39 28-38 28-39 39-53 39-55 74-86 88-99
860.9 1634.9 1676.9 1434.6 1562.8 1169.3 1297.5 1599.7 1842.0 1439.8 1479.7
GDVEKGKK IFVQKCAQCHTVEK GGKHKTGPNLHGLFGR HKTGPNLHGLFGR HKTGPNLHGLFGRK TGPNLHGLFGR TGPNLHGLFGRK KTGQAPGFTYTDANK KTGQAPGFTYTDANKNK YIPGTKMIFAGIK KTEREDLIAYLK
10.05 8.32 11.47 11.45 11.47 10.75 11.45 9.52 10.16 10.19 6.66
and ACTH significantly drop (Figure 4). We can now understand the lower extraction efficiencies of these individual peptides at lower pHs as being caused by the limited capacity of this polymeric material at lower pHs. Even though the extraction efficiencies drop at lower pHs, the strong correlation between the experimental results and theoretical fractional compositions at pHs above 6 for all the peptides in Figure 4 is an important finding that suggests that titration curves for peptides could be generated. Extraction of Peptide Mixtures. After the titration curves of individual peptides were generated by extractions with the reverse micelles, similar experiments were done on peptide mixtures. The first mixture contained angiotensin I, ACTH, and kinetensin and was extracted by the reverse micelles from aqueous solutions with pHs ranging from ∼5 to 11. As before,
Figure 6. The normalized ion abundances of the peptides in the organic phase after extraction (solid line) and the calculated fractional compositions of the positively charged species (dotted line) of these peptides as a function of aqueous solution pH. Analytical Chemistry, Vol. 81, No. 12, June 15, 2009
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Figure 7. The normalized ion abundances of cytochrome C peptides in the organic phase after extraction (solid line) and the calculated fractional compositions of the positively charged species (dotted line) of these peptides as a function of aqueous solution pH.
the resulting organic phase were analyzed by MALDI-MS. The normalized ion abundance of each peptide in the organic phase was then plotted as a function of aqueous pH and compared to the theoretical fractional composition of the positively charged peptides (Figure 6). The experimental titration curves of each peptide in the mixture exhibit about the same trend as they did when they were extracted individually (i.e., Figure 4). Even though the experimental data in Figure 6 do not fit the theoretical data at every pH as well as it did in Figure 4, the pHs at which the ion abundances drop and the pH range over which these ion abundances drop (i.e., the slopes) correspond very well with the calculation. Ultimately, the pH at which the ion abundance begins to drop and the slope of this drop are characteristic of each peptide, and our experimental data recapitulate these characteristics fairly well. The second peptide mixture that was studied was from a proteolytic digest of cytochrome C. This peptide mixture was extracted from aqueous solutions having pHs ranging from 4.0 to 12.3 and subsequently analyzed by MALDI-MS. The peptides that are consistently observed in MALDI spectra of the organic 5052
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phases are shown in Table 2. Additional peptides are often seen after extraction from some digests (e.g., peptides with some missed cleavages or with low m/z ratios that interfere with matrix ions), and these peptides are listed in the Table S3 of the Supporting Information. The organic phase ion abundances of some cytochrome c peptides listed in Table 2 were plotted as a function of aqueous solution pH and then compared with the fractional compositions (Figure 7). Plots of the remaining peptides in Table 2 are shown in Figure S3 of the Supporting Information. For each peptide, the best match with the experimental data is found when only positively charged species are considered in the calculation. As can be seen, all of the experimentally observed titration curves fit the theoretical data very well, except at aqueous solution pH values below 6. The poor fits at lower pH values are presumably due to the limited capacity of the polymers at lower pH values, as described above. Interestingly, the peptides for which the titration curves can be generated (Figure 7 and Figure S3 in the Supporting Information) represent 77% of the sequence of cytochrome C. The remaining 23% of the sequence is accounted for by digestion
products that are individual amino acids (e.g., K), small peptides that have m/z ratios similar to matrix ions, or peptides that have low pI values (e.g., [61-72] EETLMEYLENPK; pI ) 3.8). The failure to measure titration curves for peptides with low pI values is due to the limited capacity of the polymeric material at pHs where these peptides would be positively charged. CONCLUSION The results described in this work demonstrate that (i) the aqueous solution pH and analyte pKa values are the two main factors controlling the extraction selectivity of the polymeric reverse micelles, (ii) selective extraction of pI markers, individual peptides, and peptide mixtures over a range of aqueous solution pH values can be used to create titrationtype curves for these analytes, (iii) the experimental titrationtype curves for the peptides studied in this work correspond very well with the calculated fractional composition of their positively charged species in solution, except at the aqueous solution pH values below 6 where the extraction capacity of the polymer is limited, and (iv) titration-type curves for individual peptides from a protein digest can be simultaneously generated with our extraction protocol. The combination of peptide titration-type curves and mass information obtained from MALDI-MS data might be useful for improving protein identification processes, especially when different peptides in a complex mixture have very similar masses. Peptide pI information along with accurate mass measurements has been shown to improve protein identification in the context of peptide mass fingerprinting experiments.15,18,19 Because peptide titration curves contain even more specific information about a peptide’s acid and base content, it stands to reason that such curves should
improve protein identification beyond what is possible with a single pI value. Our extraction approach and the titration-type curves that are produced could be used in either peptide mass fingerprinting or tandem MS approaches. In use of our extraction approach for complex samples (e.g., shotgun proteomics), one issue that needs to be addressed is the limited extraction capacity of the polymers, especially at low pH values. One way that we are currently investigating this issue is by using higher polymer concentrations and polymers with a greater number of monomer groups. A very encouraging preliminary finding is that going from a 23 kDa polymer, as used in the current studies, to a 40 kDa polymer increases the extraction capacity by a factor of 5. This 5-fold increase is greater than expected based on simply considering the increase in monomeric units from ∼ 70 to ∼ 120. Further increases in polymer size may provide even further increases in extraction capacity. A second approach, which we are also currently investigating, is the use of anionic functional groups with lower inherent pKa values than carboxylates (e.g., - SO32-), which would help maintain the polymer’s capacity over a wider pH range. ACKNOWLEDGMENT This work was supported by a grant from the Office of Naval Research under Award Number N000140510501. 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 March 30, 2009. Accepted May 8, 2009. AC900661E
Analytical Chemistry, Vol. 81, No. 12, June 15, 2009
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