Anal. Chem. 2010, 82, 9122–9126
Microwave-Assisted 18O-Labeling of Proteins Catalyzed by Formic Acid Ning Liu,*,†,‡ Hanzhi Wu,‡ Hongxia Liu,§ Guonan Chen,| and Zongwei Cai*,‡ Central Laboratory, The Second Hospital of Jilin University, Changchun, China, Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR, China, The Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China, Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fuzhou University, Fuzhou 350002, Fujian, China Oxygen exchange may occur at carboxyl groups catalyzed by acid. The reaction, however, takes at least several days at room temperature. The long-time exchanging reaction often prevents its application from protein analysis. In this study, an 18O-labeling method utilizing microwaveassisted acid hydrolysis was developed. After being dissolved in 16O/18O (1:1) water containing 2.5% formic acid, protein samples were exposed to microwave irradiation. LC-MS/MS analysis of the resulted peptide mixtures indicated that oxygen in the carboxyl groups from glutamic acid, aspartic acid, and the C-terminal residues could be efficiently exchanged with 18 O within less than 15 min. The rate of back exchange was so slow that no detectable back exchange could be found during the HPLC run. Stable-isotope labeling combined with mass spectrometry analysis has been reported as a strategy to perform identification and relative quantification of proteins.1-3 Among various approaches using stable-isotope labeling, proteolytic 18O-labeling offers a universal strategy for the incorporation of 18O at the C-termini of peptides, which may be catalyzed by certain proteases during the proteolytic cleavage process.4 The simplicity of the labeling method as well as its resistance to back exchange allow the 18O-labeling to be a preferred method for isotopic labeling of peptides in the quantitative application of proteomics. A variety of proteases have been applied to the catalytic incorporation of 18O via the molecule of water (H218O) into peptide fragments during cleavage of specific peptide bonds. Some acids are known to be able to cleave peptide bonds to generate peptide fragments. Recently, methods for the acid* To whom correspondence should be addressed. Z.C.: address, Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR, China; fax, 852 34117348; e-mail,
[email protected]. N.L.: address, Central Laboratory, The Second Hospital of Jilin University, Changchun, China; fax, 86 431 88796852; e-mail,
[email protected]. † The Second Hospital of Jilin University. ‡ Hong Kong Baptist University. § Tsinghua University. | Fuzhou University. (1) Guo, K.; Li, L. Anal. Chem. 2009, 81, 3919–3932. (2) Dreger, M.; Leung, B. W.; Brownlee, G. G.; Deng, T. Protein Sci. 2009, 18, 1448–1458. (3) Ha¨gglund, P.; Bunkenborg, J.; Maeda, K.; Svensson, B. J. Proteome Res. 2008, 7, 5270–5276. (4) Fenselau, C.; Yao, X. J. Proteome Res. 2009, 8, 2140–2143.
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catalyzed cleavage of protein with microwave irradiation have been demonstrated to greatly increase the efficiency of acid hydrolysis.5-8 Among the acids that have been reported in the hydrolysis of proteins, formic acid has been found to efficiently and specifically cleave at either N-terminal or C-terminal side of aspartic acid under short-time microwave irradiation.6,8-10 Similar to the enzymatic hydrolysis of peptide bonds, acid-catalyzed hydrolysis was also achieved with the participation of 18O-water molecules. Therefore, the isotopic tag, namely 18O, can be readily introduced into the carboxyl groups of the produced peptides. In previous reports, 18O-labeling in carboxyl groups of amino acids and small peptides was achieved in mild acid solution after the incubation at room temperature for a long course (>10 days).11-13 Herein, an 18O-labeling method utilizing microwave-assisted acid hydrolysis is described. Formic acid was chosen in the experiment of acid-catalyzed 18O-labeling on proteins. The method simultaneously involved the 18O-labeling and protein digestion under the same conditions. The developed method has been successfully applied for the direct analysis of several proteins. Because the oxygen exchange with the microwave irradiation produced the stable 18O-labeled peptides with steady isotopic patterns in less than 15 min, the method could be applied as an alternative labeling strategy in quantitative proteomics. EXPERIMENTAL SECTION Dithiolthreitol (DTT), ammonium bicarbonate, Tris, phenylmethylsulfonyl fluoride (PMSF), CaCl2, bovine milk lactoglobulin B, bovine heart cytochrome C, bovine pancreas ribonuclease A, and horse heart myoglobin were purchased from Sigma(5) Zhong, H.; Zhang, Y.; Wen, Z.; Li, L. Nat. Biotechnol. 2004, 22, 1291– 1296. (6) Li, A.; Sowder, R. C.; Henderson, L. E.; Moore, S. P.; Garfinkel, D. J.; Fisher, R. J. Anal. Chem. 2001, 73, 5395–5402. (7) Swatkoski, S.; Gutierrez, P.; Wynne, C.; Petrov, A.; Dinman, J. D.; Edwards, N.; Fenselau, C. J. Proteome Res. 2008, 7, 579–586. (8) Hauser, N. J.; Basile, F. J. Proteome Res. 2008, 7, 1012–1026. (9) Hua, L.; Low, T. Y.; Sze, S. K. Proteomics 2006, 6, 586–591. (10) Tsugita, A.; Miyazaki, K.; Nabetani, T.; Nozawa, T.; Kamo, M.; Kawakami, T. Proteomics 2001, 1, 1082–1091. (11) Murphy, R. C.; Clay, K. L. Biomed. Mass Spectrom. 1979, 6, 309–314. (12) Ponnusamy, E.; Jones, C. C.; Fiat, D. J. Labelled Compd. Radiopharm. 1987, 24, 773–778. (13) Niles, R.; Witkowska, H. E.; Allen, S.; Hall, S. C.; Fisher, S. J.; Hardt, M. Anal. Chem. 2009, 81, 2804–2809. 10.1021/ac101888f 2010 American Chemical Society Published on Web 10/12/2010
Aldrich (St. Louis, MO, USA). HPLC grade formic acid was from Panreac (Barcelona, Spain). HPLC-grade acetonitrile was obtained from Tedia (Fairfield, OH, USA). 18O water (95%) was purchased from ISOTEC (Miamisburg, OH, USA). Water was purified from a Milli-Q Ultrapure water system (Millipore, Billerica, MA, USA). Chicken eggs were broken, and egg whites were carefully separated from the yolk. After being homogenized, the isolated egg whites were lyophilized. Microwave-Assisted Acid-Catalyzed 16O/18O Exchange. The stock solutions of each protein were prepared by dissolving 400 nmol of each protein in 1000 µL of 5% formic acid in a 1.5 mL vial. Twenty-five microliters of each of the protein stock solutions were mixed with an equal volume of 18O water in 1.5 mL polypropylene centrifuge vials. For the egg white sample, 1 mg of the lyophilized egg whites was dissolved in 200 µL of 5% formic acid in a 1.5 mL vial and vortexed for 5 min. Twentyfive microliters of the egg white sample was mixed with an equal volume of 18O-water in 1.5 mL polypropylene centrifuge vials. Another 25 µL of the egg white sample was mixed with equal volume of natural water to serve as a control reference. The sample vials were then exposed to microwave irradiation in a domestic 1100 W (2450 MHz) microwave oven for 10-15 min. During the microwave irradiation, 200 mL of water was placed inside the microwave oven to absorb extra microwave energy. After the microwave treatment, the sample solutions were cooled to room temperature, centrifuged, and then subject to LC-MS/MS analysis or stored in -20 °C for future use. For in-gel 18O-labeling, samples were boiled for 5 min in sample loading buffer containing DTT or β-mercaptoethanol and then separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) gel. After Coomassie brilliant blue (CBB) staining, the protein bands were excised, destained, and then dehydrated, as described previously.14 The dried gel pieces were subsequently rehydrated in 200 µL of 16 O/18O (1:1) water containing 2.5% formic acid and 20 mM DTT for 20 min, followed by exposure of microwave irriadiation for 15 min. After the samples were cooled, the supernatants were collected in another Eppendorf vial, followed by lyophilization. The freeze-dried samples were redissolved in 10 µL of 0.1% trifluoroacetic acid (TFA) in 5% acetonitrile (ACN) right before LC-MS/MS analysis. Capillary LC-MS/MS Analysis. Capillary LC-MS/MS experiments were conducted on an ABI QSTAR spectrometer coupled with an Agilent 1100 micropump system. Eight microliters of the sample solution was injected directly onto a 0.3 mm × 25 cm column packed with C18 material (LC Packings C18 PepMap 100, 5 µm, 100 A; LC Packings, Amsterdam, The Netherlands). Peptides were eluted at a flow rate of 10 µL/min with mobile phase A (100% water, 0.1% formic acid), to which mobile phase B (100% acetonitrile, 0.1% formic acid) was added by a linear gradient (initially, 5% B for 5 min, and then increased to 50% B at 100 min, to 95% B at 110 min, and then kept isocratic for 10 min). Mass spectrometric analysis was performed on an ABI QSTAR spectrometer using information dependent acquisition mode (IDA; Analyst QS, Applied Biosystems) by selecting the four most intense ions for MS/MS analysis. A survey scan of 300-2000 Da (14) Liu, N.; Song, W.; Lee, K. C.; Wang, P.; Chen, H.; Cai, Z. J. Am. Soc. Mass Spectrom. 2009, 20, 312–320.
was collected for 3 s followed by 5 s MS/MS scans of 40-1500 Da using the standard rolling collision energy settings. RESULTS AND DISSCUSION Microwave-Assisted 18O-Labeling Catalyzed by Dilute Formic Acid. Microwave technology has been widely used in hydrolysis of proteins by acids, in which nonspecific cleavages could be observed. Among the acids typically used in hydrolysis of proteins,5,15 mild formic acid has been proved to be more specific than other acids and, thus, commonly used for chemical cleavage of proteins.6-10 It has been well-known that oxygen on the carboxyl groups of two amino acid residues, namely, the aspartic acid (D, Asp) and glutamic acid (E, Glu), as well as the C-terminal residues, can be exchanged with that in solvent water under acidic conditions. However, the speed of individual oxygen exchange was found to be peptide and site specific,11 which often took a long time to complete at room temperature. The application of microwave irradiation on the formic acid-catalyzed 18O-labeling was investigated directly on proteins in order to increase the exchange speed. Protein samples were dissolved in 16O/18O (1:1) water containing 2.5% formic acid, followed by exposure to microwave irradiation. The produced peptide mixtures were subject to LC-MS/MS analyses. Specific cleavages were observed to take place at either the N-terminal or C-terminal side of aspartic acid residues. Meanwhile, the oxygen exchange between solvent water and most of carboxyl groups of peptides was completed in less than 15 min. Up to 2(ND + NE + 1) of oxygen atoms per peptide were labeled with 18O, where ND and NE refer to numbers of Asp (D) and Glu (E) residues in the peptide, respectively, and 1 refers to the C-terminal carboxyl group. Figure 1A-D showed the representative mass spectra of several peptide fragments obtained from the analysis of myoglobin. The ESI-MS spectrum of the fragment peptide 110AIIHVLHSKHPG121 that contains only two oxygen atoms on the C-terminal carboxyl group was presented in Figure 1A. The triply charged ion peak at m/z 436.97 represented the ion of molecule containing two 16O atoms in the C-terminal carboxyl group, while the triply charged ions at m/z 438.26 and 437.60 represented molecules containing two 18O atoms and one 16O atom plus one 18O atom, respectively. For the doubly charged ion cluster of peptide 142 IAAKYKELGFQG153 (Figure 1B), there were four oxygen atoms (two at the side chain of E148 and two at the C-terminal carboxyl group of G153) that were liable to the acid-catalyzed oxygen exchange. Similarly, six and eight 18O atoms were introduced into the peptides 5GEWQQVLNVWGKVEA19 (Figure 1C) and 21IAGHGQEVLIRLFTGHPETLEKF43 (Figure 1D), respectively, although the oxygen exchange catalyzed by formic acid with the aid of microwave irradiation occurred within a short time. The identified sequences of above peptides from myoglobin were summarized in Table S1 in the Supplemental Information. The mechanism of exchanging oxygen in carboxyl groups of peptides with that in water at low pH was reported previously by Myron L. Bende and his co-workers.16 The exchange reaction was thought to proceed through the addition of water to the carboxylic acid to form an intermediate that broke down to give the (15) Marcus, H. S. L.; Li, L. J. Am. Soc. Mass Spectrom. 2005, 16, 471–481. (16) Bender, M. L.; Stone, R. R.; Dewey, R. S. J. Am. Chem. Soc. 1956, 78, 319–321.
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Figure 1. Mass spectra of the 18O-labeled fragment peptides from myoglobin. The four peptides (A) AIIHVLHSKHPG, (B) IAAKYKELGFQG, (C) GEWQQVLNVWGKVEA, and (D) IAGHGQEVLIRLFTGHPETLEKF were labeled with 16O/18O (1:1) water containing 2.5% formic acid under the microwave irradiation for 15 min.
exchanged product. The method has been well established, and the exchange reaction tended to proceed better at elevated temperature (70-80 °C). There might be two reasons why the microwave could accelerate the rate of the exchange reaction. One was the effect of rapid heating by microwave irradiation, by which the collision frequency of the reactant molecules became greater at high temperature. The other one, more importantly, was that the proportion of reactant molecules with sufficient energy to react was significantly higher after microwave irradiation. The stability of the isotopic labeling was examined for the procedures of sample storage and the HPLC experiment. The rate of the back exchange was so slow that no detectable back exchange was found during the sample storage and HPLC separation of the 18O-labeled peptides that were eluted with acidic mobile phase containing a large amount of unlabeled water (data not shown). To further investigate the back exchange during the storage at room temperature, the 18Olabeled samples were freeze-dried and then redissolved in 2.5% formic acid aqueous solution containing no 18O-labeled water at room temperature. Aliquots (10 µL of each) at different time courses were transferred into new vials and subject to the LCMS/MS analysis. Figure 2 showed mass spectra of the quadruply charged ion for one of the fragment peptides from myoglobin (45KFKHLKTEAEMKASE59) at different times. Although up to eight oxygen atoms in the peptide could be back exchanged, 9124
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Figure 2. Back exchange of 18O to 16O for peptide 45KFKHLKTEAEMKASE59 in water at room temperature for (A) 0 day, (B) 4 days, and (C) 11 days after the 18O-labeling.
it was found that the back exchange was very slow at room temperature. The obtained data over the long-term experiment indicated that the labeled 18O atoms were exchanged back to 16 O after placing the aqueous peptide sample for 11 days at room temperature. A similar back exchange rate was observed in other fragment peptides. Furthermore, the back exchange was not detected in the peptide samples dissolved in water containing 2.5% formic acid and stored at -80 °C for 2 months. Compared with the widely used trypsin-catalyzed 18O-labeling method,17-19 the developed acid-catalyzed 18O-labeling method did not utilize any enzyme and, thus, eliminated the problem of residual enzymatic activity left after the labeling reaction.20-22 Moreover, the completion of acid-catalyzed reaction accelerated by microwave irradiation only needed 15 min, whereas the trypsincatalyzed labeling method usually required a long incubation. The method applicability of microwave-assisted 18O-labeling by formic acid was further examined with a protein complex sample isolated from egg whites. The isolated protein sample was dissolved in 16O/18O (1:1) water containing 2.5% formic acid, followed by microwave irradiation. For the purpose of comparison, an aliquot of the protein complex sample was treated in the same way except that the sample was dissolved in natural water, which could be served as a reference to facilitate peptide identification. The resulted samples were subject to LC-MS/MS analysis. For the peptides identified as fragments from the major proteins in egg whites, each peptide was found to be labeled with up to N 18O atoms, where N referred to the total number of the exchangeable oxygen atoms in the individual peptide. The population of the isotopomers of each peptide was approximate to binominal distribution, which was an indication for exchange equilibrium. A summary for some identified peptides as well as the relevant data of 18O incorporation was provided in Table S2, Supporting Information. Because individual oxygen exchange was supposed to be independent and the rate of 18O incorporation was reported to be sequence and residue dependent, the isotopic pattern of the sample that was labeled in 16O/18O (1:1) water might be complicated and different from that of the pooled mixture (1: 1) of the two samples labeled individually in 18O and 16O water. Therefore, it was reasonable to calculate the “final” isotopic pattern of the reaction mixture when the exchange reached kinetic equilibrium but was not at transient state. An algorithm similar to that described by Niles13 was used to calculate the relative quantities of the mixed population of isotopomers at kinetic equilibrium of exchange reaction, of which the spectrum (I) might be represented as below: (17) Ang, C. S.; Veith, P. D.; Dashper, S. G.; Reynolds, E. C. Proteomics 2008, 8, 1645–1660. (18) Liu, H.; Zhang, Y.; Meng, L.; Qin, P.; Wei, J.; Jia, W.; Li, X.; Cai, Y.; Qian, X. Anal. Chem. 2007, 79, 7700–7707. (19) Carreira, R. J.; Rial-Otero, R.; Lo´pez-Ferrer, D.; Lodeiro, C.; Capelo, J. L. Talanta 2008, 76, 400–406. (20) Sevinsky, J. R.; Brown, K. J.; Cargile, B. J.; Bundy, J. L.; Stephenson, J. L., Jr. Anal. Chem. 2007, 79, 2158–2162. (21) Petritis, B. O.; Qian, W. J.; Camp, D. G., 2nd; Smith, R. D. J. Proteome Res. 2009, 8, 2157–2163. (22) Storms, H. F.; van der Heijden, R.; Tjaden, U. R.; van der Greef, J. Rapid Commun. Mass Spectrom. 2006, 20, 3491–3497.
1 2 1 · S + · S(+2) + · S(+4) 4 (0) 4 4 1 4 6 4 1 ·S + ·S ·S ·S ·S ) + + + 16 (0) 16 (+2) 16 (+4) 16 (+6) 16 (+8) 1 6 15 20 ·S + ·S ·S ·S ) + + + 64 (0) 64 (+2) 64 (+4) 64 (+6) 6 1 15 ·S ·S ·S + + 64 (+8) 64 (+10) 64 (+12)
I(2) ) I(4) I(6)
···
N
I(N) )
n CN
∑2
N
· S(+2n)
n)0
where N (0, 2, 4, ...) corresponds to the total number of oxygen atoms in the peptide available for exchange with those in the solvent water; S(+2n) represents the spectrum of the unlabeled peptide species shifted by 2n Da. Specially, I(N) corresponds to the unlabeled peptide when N is 0. It should be noted that the above expression represented the data of peptides with single charge in the mass spectra, which was usually recorded in MALDI-TOFMS analysis. For the spectra obtained from mass spectrometers equipped with an electrospray ionization (ESI) ion source, peptide peaks with multiple charges were often observed. Taking the charge state (z) of the peptide peak into account, the above expression of spectrum (I) should be modified as: N
I(N) )
n CN
∑2
N
n)0
( )
·S
+
2n z
where S(+2n/z) represents the spectrum of the unlabeled peptide species (S) shifted by 2n/z Da, which is multiply charged with z protons. Therefore, the relative quantities of the population of peptide isotopomers generated in 16O/18O (1: 1) could be estimated using the above expression. It should be noted that because the unlabeled peptide had original isotopic peaks due to the 13C contribution, the actual distribution of the isotopomers at kinetic equilibrium was not a strict binomial distribution. In-Gel 18O-Labeling Catalyzed by Diluted Formic Acid with Microwave Irradiation. In addition to the in-solution labeling, applicability of the developed method for the in-gel 18Olabeling of proteins separated from bands of SDS-PAGE was also demonstrated. A mixture of myoglobin and lactoglobulin B was separated on a 12% SDS PAGE gel. The gel pieces containing the proteins were excised, destained, and dehydrated. The dried gel pieces were subsequently rehydrated in 16 O/18O (1:1) water containing 2.5% formic acid and 20 mM DTT for 30 min, followed by the exposure of microwave irradiation. The resulted fragment peptides were collected and then lyophilized. The freeze-dried sample was redissolved in 10 µL of 0.1% TFA in 5% ACN prior to the LC-MS/MS analysis. Most of the fragment peptides from the in-gel 18O-labeling of myoglobin were found to be labeled with 18O. The carboxyl oxygen exchange was greatly accelerated by microwave irradiation. Similar results were also obtained from the in-gel 18 O-labeling of lactoglobulin B, which were summarized in Table 1. Deamidated peptide was detected after the labeling reaction of some peptides. A chromatographic peak of the deamidated Analytical Chemistry, Vol. 82, No. 21, November 1, 2010
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Table 1. Summary of Fragment Peptides of Lactoglobulin B Resulted from the Microwave-Assisted Formic Acid-Catalyzed In-Gel 18O Exchange peptide sequencea
ion charge status
calculated m/z (monoisotopic)
measured m/zb (monoisotopic)
residues
LIVTQTMKGL
2
552.33 (0)
17-26
LIVTQTMKGLD
2
609.84 (0)
IQKVAGTWYSLAMAAS
2
848.94 (0)
IQKVAGTWYSLAMAASD
2
906.45 (0)
ISLL
1
445.30 (0)
AQSAPLRVYVEELKPTPEG
2
1042.55 (0)
3
695.37 (0)
2
1100.07 (0)
3
733.71 (0)
ALNENKVLVL
2
556.84 (0)
ALNENKVLVLD
2
614.35 (0)
EALEKF
2
368.70 (0)
EALEKFD
2
426.21 (0)
552.33 (0) 554.34 (2) 609.85 (0) 613.84 (4) 848.96 (0) 850.95 (2) 906.47 (0) 910.47 (4) 445.30 (0) 449.30 (2) 1042.57 (0) 1050.57 (8) 695.38 (0) 700.71 (8) 1100.08 (0) 1110.08 (10) 733.71 (0) 740.38 (10) 556.84 (0) 560.85 (4) 614.36 (0) 620.37 (6) 368.70 (0) 374.72 (6) 426.21 (0) 434.22 (8)
AQSAPLRVYVEELKPTPEGD
17-27 28-43 28-44 45-48 50-68 50-68 50-69 50-69 102-111 102-112 146-151 146-152
a The acidic amino acid residues (D and E) as well as the C-terminal residues are highlighted in bold. b The number in brackets (N ) 0, 2, 4, 6, 8, 10) represents the number of 18O atoms incorporated into an individual peptide. In addition to the m/z value of peptides without the 18Olabeling (N ) 0), the peptide ion with the maximal 18O-labeling was listed.
peptide was observed right after that of the corresponding undeamidated peptide. Although the MS/MS spectra of the deamidated peptide and undeamidated peptide were very similar, interpretation of the spectrum confirmed the detection of deamidated species. For example, a peak cluster (m/z 552.3-555.3) eluted at 30.45 min was identified as a fragment (17LIVTQTMKGL26) from lactoglobulin B, as illustrated in Figure S2A, Supporting Information. The peak cluster (m/z 553.8-557.8) identified as the deamidated species was eluted at 31.27 min (Figure S2B, Supporting Information). The deamidated peptide peak was broader probably because it has more oxygen atoms available for exchange. A similar phenomenon of deamidation was observed in some other fragment peptides. The obtained data showed that the rate of deamidation reaction was much slower than that of the oxygen exchange. Therefore, the generated amount of deamidated species, if any, was usually very limited under the current experimental conditions. Furthermore, the deamidated peptides could be separated from the corresponding undeamidated species under the ordinary liquid chromatography conditions. Therefore, the effect of deamidation reaction on the 18 O-labeling method might be eliminated when LC-MS/MS was used for the peptide analysis. CONCLUSIONS A simple, fast, and efficient method to introduce 18O into the carboxyl groups in proteins catalyzed by formic acid with the aid of microwave irradiation was developed. The application of microwave irradiation on the acid-catalyzed 18O-labeling
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provided the exchange reaction within 15 min. The back exchange 18O to 16O during the HPLC run and long-term storage at -80 °C could be neglected. The method simultaneously involved two different reactions, namely, the 18Olabeling and protein digestion under the same conditions. The produced peptides labeled with 18O were stable and suitable for the HPLC-MS/MS analysis, allowing the selection of one or more 18O-labeled compounds as isotopic reference for quantitative or semiquantitative analysis. Therefore, the method could be used as an alternative labeling strategy in quantitative proteomics. Moreover, because the 18O exchange could be incorporated to other molecules containing carboxyl groups, the method application might be extended to a wide range of bioanalysis. ACKNOWLEDGMENT The authors would like to thank the National Nature Sciences Foundation of China (20928005) and Faculty Research Grant of Hong Kong Baptist University for the financial support. 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 15, 2010. Accepted September 29, 2010. AC101888F
