Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6838–6846 Analysis of Inorganic Polyphosphates by Capillary Gel Electrophoresis Andrew Lee and George M. Whitesides* Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA This paper describes the development of a method that uses capillary gel electrophoresis (CGE) to analyze mixtures of inorganic polyphosphate ((Pi)n). Resolution of (Pi)n on the basis of n, the number of residues of dehydrated phosphate, is accomplished by CGE using capillaries filled with solutions of poly(N,N-dimethylacrylamide) (PDMA) and indirect detection by the UV absorbance of a chromophore, terephthalate, added to the running buffer. The method is capable of resolving peaks representing (P i)n with n up to ∼70; preparation and use of authentic standards enables the identification of peaks for (Pi)n with n ) 1-10. The main advantages of this method over previously reported methods for analyzing mixtures of (Pi)n (e.g., gel electrophoresis, CGE using polyacrylamide-filled capillaries) are its resolution, convenience, and reproducibility; gel-filled capillaries are easily regenerated by pumping in fresh, low-viscosity solutions of PDMA. The resolution is comparable to that of ion-exchange chromatography and detection of (P i)n by suppressed conductivity. The method is useful for analyzing (Pi)n generated by the dehydration of Pi at low temperature (125-140 °C) with urea, in a reaction that may have been important in prebiotic chemistry. The method should also be useful for characterizing mixtures of other anionic, oligomeric, or polymeric species without an intrinsic chromophore (e.g., sulfated polysaccharides, oligomeric phospho-diesters). This paper describes a technique for analyzing mixtures of inorganic oligo- and polyphosphates, (Pi)n, by capillary gel electrophoresis. Samples of condensed inorganic phosphate are typically mixtures of (Pi)n with different chain length, n. Mixtures of (Pi)n can have a wide range in n; (Pi)n with estimated values of n as high as 1000 have been reported. 1 The analytical method developed here characterizes samples of (Pi)n at high resolution by separating and detecting each species in a mixture. Our primary motivation to develop a new method of analysis was to explore the synthesis and reactivity of (Pi)n relevant to the chemical origins of life (i.e., the prebiotic chemistry leading to self-replicating systems in a “pre-RNA” or “RNA world”). 2-7 * To whom correspondence should be addressed. E-mail: gwhitesides@ gmwgroup.harvard.edu. (1) Clark, J. E.; Wood, H. G. Anal. Biochem. 1987, 161, 280–290. (2) Joyce, G. F. Nature 1989, 338, 217–224. 6838 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Species of (Pi)n in aqueous solution are anionic and differ from each other in the number of residues of condensed phosphate and in net negative electrostatic charge. Capillary electrophoresis (CE), in its most straightforward mode of operation (capillary zone electrophoresis (CZE), that is electrophoresis of analytes through free solution, combined with optical detection of chromophoric analytes), cannot resolve and detect (Pi)n. We developed a method that addresses the two problems of (i) separating (Pi)n in mixtures and (ii) detecting and quantifying each (Pi)n. Capillary gel electrophoresis (CGE), using capillaries filled with aqueous solutions of poly(N,Ndimethylacrylamide) (PDMA), resolved cyclic and linear (Pi)n in order of their electrophoretic mobility. Addition of the chromophoric anion, terephthalate (1,4-(CO2 - )2C6H4, abbreviated as TP 2- ), to the running buffer enabled the detection of separated (Pi)n by indirect UV absorbance. We demonstrated the resolution of mixtures of (Pi)n (with n up to ∼70) and the identification of components in mixtures with the use of authentic standards. The areas of peaks in electropherograms, determined by indirect detection, allowed us to quantify the relative concentration of each species in a mixture of (Pi)n. In addition to analyzing commercially available samples of (Pi)n, prepared by the thermal dehydration of Pi (>220 °C), we analyzed (Pi)n generated by dehydration reactions that might have occurred on the prebiotic earth and, thus, might have been involved in the chemical origins of life. 5,6,8 MOTIVATION (Pi)n and adenosine triphosphate (ATP) have an essential functional group in common: residues of dehydrated phosphate connected by phosphoanhydride bonds. (Pi)n is simpler in composition than ATP but, in principle, may provide the same chemical function (e.g., activation of -OH groups) and has led to the suggestion that (Pi)n is a molecular fossil, a species important in the origin of life, and a precursor to ATP. 9-13 We (3) Joyce, G. F. Nature 2002, 418, 214–221. (4) Gilbert, W. Nature 1986, 319, 618–618. (5) Keefe, A. D.; Miller, S. L. Origins Life Evol. Biospheres 1996, 26, 15–25. (6) Keefe, A. D.; Miller, S. L. J. Mol. Evol. 1995, 41, 693–702. (7) Pasek, M. A.; Kee, T. P.; Bryant, D. E.; Pavlov, A. A.; Lunine, J. I. Angew. Chem., Int. Ed. Engl. 2008, 47, 7918–7920. (8) Osterberg, R.; Orgel, L. E.; Lohrmann, R. J. Mol. Evol. 1973, 2, 231–234. (9) Rao, N. N.; Gomez-Garcia, M. R.; Kornberg, A. Annu. Rev. Biochem. 2009, 78, 605–647. (10) Kornberg, A. J. Bacteriol. 1995, 177, 491–496. (11) Kornberg, A.; Rao, N. N.; Ault-Riche, D. Annu. Rev. Biochem. 1999, 68, 89–125. (12) Orgel, L. E.; Lohrmann, R. Acc. Chem. Res. 1974, 7, 368–377. (13) Lohrmann, R.; Orgel, L. E. Nature 1973, 244, 418–420. 10.1021/ac1008018 © 2010 American Chemical Society Published on Web 07/27/2010
wished to explore the dehydration of Pi in detail and needed a method that was more convenient and reproducible than those reported so far for the resolution of mixtures of (Pi)n varying in chain length. The method we have developed should also be useful for investigating the biochemistry of (Pi)n 9-11 and for developing (Pi)n as a reagent in chemical synthesis. In addition, the method may be useful in applications for quality control: (Pi)n is a component of many commercial materials (e.g., fertilizers, food products, detergent formulations, building materials). 14 PREVIOUSLY REPORTED METHODS FOR SEPARATING MIXTURES OF (PI)N Polyacrylamide gel electrophoresis (PAGE) can resolve mixtures of (Pi)n, with n ∼ 2-450. Analysis by PAGE involves gel electrophoresis (typical runs require g3 h) and the detection of (Pi)n by staining gels with the cationic dye toluidine blue O (TBO); 1,15,16 autoradiography can detect species of (Pi)n synthesized from 32 P-ATP. 17 The disadvantages to PAGE are the difficulty of the experiments and the time required for analysis. We found it difficult to cast 20% gels that are homogeneous and provide reproducible resolution; to run a gel requires several hours (for separation, staining, and destaining). Anion-exchange chromatography is useful both for analyzing mixtures of (Pi)n and for preparing samples of purified (Pi)n. The best results for the chromatographic resolution of (Pi)n are chromatograms showing up to ∼50 peaks, in runs of less than 30 min; 18,19 this method requires HPLC instrumentation with a suppressed conductivity detector and an online KOH gradient generator (for minimizing the amount of CO2/CO3 2adsorbed from the atmosphere). Distinguishing samples containing (Pi)n with n > 45 is difficult by this method, and the resolution of species with n ∼ 100 has not been demonstrated. Other qualitative or semiquantitative methods used to analyze mixtures of (Pi)n include paper chromatography, 20,21 31 P NMR, 22-27 ESI-MS, 28 and the analysis of terminal phosphate groups with phosphoglucokinase. 20 (14) Phosphoric Acid and Phosphates. Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed.; John Wiley & Sons: New York, 1991; Vol. 18, pp 669- 718. (15) Ogawa, N.; DeRisi, J.; Brown, P. O. Mol. Biol. Cell 2000, 11, 4309–4321. (16) Robinson, N. A.; Wood, H. G. J. Biol. Chem. 1986, 261, 4481–4485. (17) Gomez-Garcia, M. R.; Kornberg, A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15876–15880. (18) Baluyot, E. S.; Hartford, C. G. J. Chromatogr., A 1996, 739, 217–222. (19) Sekiguchi, Y.; Matsunaga, A.; Yamamoto, A.; Inoue, Y. J. Chromatogr., A 2000, 881, 639–644. (20) Greenfield, S.; Clift, M. Analytical Chemistry of the Condensed Phosphates; 1 ed.; Pergamon Press: New York, 1975. (21) Osterberg, R.; Orgel, L. E. J. Mol. Evol. 1972, 1, 241–250. (22) Rao, N. N.; Roberts, M. F.; Torriani, A. J. Bacteriol. 1985, 162, 242–247. (23) Gard, D. R.; Burquin, J. C.; Gard, J. K. Anal. Chem. 1992, 64, 557–561. (24) Crutchfield, M. M.; Callis, C. F.; Irani, R. R.; Roth, G. C. Inorg. Chem. 1962, 1, 813–817. (25) Teleman, A.; Richard, P.; Toivari, M.; Penttilla, M. Anal. Biochem. 1999, 272, 71–79. (26) Moreno, B.; Urbina, J. A.; Oldfield, E.; Bailey, B. N.; Rodrigues, C. O.; Docampo, R. J. Biol. Chem. 2000, 275, 28356–28362. (27) Glonek, T.; Lunde, M.; Mudgett, M.; Myers, T. C. Arch. Biochem. Biophys. 1971, 142, 508–513. (28) Choi, B. K.; Hercules, D. M.; Houalla, M. Anal. Chem. 2000, 72, 5087– 5091. CAPILLARY ELECTROPHORESIS OF (PI)N AND DNA Capillary electrophoresis separates and resolves analytes based on differences in electrophoretic mobility. Since both the amount of negative charge and hydrodynamic drag of (Pi)n increase with n, in a way similar to that of single-stranded DNA, we expected poor resolution in the analysis of (Pi)n in free-solution capillary electrophoresis (i.e., CZE). 29 Capillaries filled with a sieving matrix, however, are capable of resolving DNA in order of chain length. 30-32 The use of replaceable solutions of entangled polymer (such as linear acrylamide or PDMA) in CGE enabled the automated and massively parallel analysis of samples of DNA and was essential to the completion of the Human Genome Project. 33,34 Previous reports of methods for analyzing (Pi)n by CGE used capillaries coated and filled with linear polyacrylamide (capillaries were prepared by filling capillaries with aqueous acrylamide and polymerizing in situ). 35,36 We, and others, found that capillaries prepared this way had short lifetimes; 37-40 these capillaries could not be reused, required time-consuming preparation of new capillaries, and limited the reproducibility of the method. Stover 36,41 and Wang 29,35 detected (Pi)n by indirect UV absorbance in CGE experiments (chromate or pyromelltic acid were the chromophores added to the running buffer). These demonstrations did not, however, identify peaks for (Pi)n beyond P3 nor did they quantify resolved (Pi)n. EXPERIMENTAL DESIGN CZE: Separation of (Pi)n in Free Solution. We used the results of CZE experiments to guide the development of a CGE technique. Although the resolution of mixtures of (Pi)n in free solution is poor, CZE experiments are easier to run than CGE experiments and allowed us to (i) test different chromophores for indirect detection and identify terephthalate (TP 2- )asan optimal choice; (ii) measure µ for analytical standards of (Pi)n in free solution and determine the influence of pH and net electrostatic charge on the mobility of (Pi)n (pH in the ranges of 6.8-7.1 and 8.4-8.7); (iii) optimize the composition of the running buffer. Table 1 gives literature values of pKa for (Pi)n. Analysis by CZE required the migration of (Pi)n to the anode and the use of capillaries with suppressed electro-osmotic flow. Capillaries covalently modified by reaction of the fused silica surface with a copolymer of N,N-dimethylacrylamide and 3-methacryloxy-propyltrimethoxysilane 42 had an electro-osmotic flow of
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Anal. Chem. 2010, 82, 6745-6750 Let
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purchased from Invitrogen-Molecular
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Figure 3. Electropherograms of TPP-
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Anal. Chem. 2010, 82, 6751-6755 Res
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(pH 8.0) with cysteine and cystamin
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Figure 4. Ion mobility mass spectra
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GOx glucose + O298 gluconic acid +
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coater at 2000 rpm for 20 s, and th
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Figure 5. Comparison of cyclic volt
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in channels with either no grooves
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indicators of atmospheric processin
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Figure 1. GC/MS total ion chromatog
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Table 2. Concentrations and Stable
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on a substrate are preferred. 20-24
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Figure 4. SERS analysis of NAADP co
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Anal. Chem. 2010, 82, 6775-6781 Hig
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tion, 2 µL of proprionaldehyde wer
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Figure 4. Analysis of 2a by HPLC-MS
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eaction of 1a with PBH can be condu
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Numerous references had demonstrate
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Figure 1. TEM images of the prepare
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Page 45 and 46: esult in a big SPR signal change wi
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Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
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Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
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Page 65 and 66: Polymerase (1 U per sample). Reacti
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Page 75 and 76: Table 2. Concentrations and Ratios
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Page 79 and 80: mg/mL protein, followed by separati
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Page 83 and 84: Figure 4. High-resolution MS/MS spe
Page 85 and 86: Figure 6. Characterization of PSMs
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Page 91 and 92: Figure 3. (A) The value of Ex650 nm
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Page 99 and 100: Figure 2. Standards of (Pi)n, n ) 1
Page 101 and 102: Figure 5. Quantitative calibration
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Page 105 and 106: Figure 1. 226 Ra spectrum by liquid
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Page 117 and 118: data as well. The 41 genes in Table
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Page 123 and 124: Table 1. Absolute Quantification Re
Page 125 and 126: ment. Therefore, the long-time drea
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Page 129 and 130: Figure 2. Influence of a surfactant
Page 131 and 132: Figure 5. Device to eject and mix s
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Page 135 and 136: NaCl, phosphate buffer saline (PBS)
Page 137 and 138: Figure 2. Product ion mass spectra
Page 139 and 140: -70 °C resolved the problem, givin
Page 141 and 142: Table 1. Intraday Precision and Acc
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Figure 1. Infrared spectra of (A) u
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Figure 3. Cyclic voltammograms obta
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Figure 6. Calculated charge from ch
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Anal. Chem. 2010, 82, 6895-6903 Ele
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Table 1. Chemical Structure, pKa Va
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pH with a tilted baseline (Figure 1
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Table 2. Linearity and Detection Li
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ascorbic acid (AA), uric acid (UA),
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the multielement capabilities, the
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RESULTS AND DISCUSSION Sulfur Detec
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Table 2. Molecular Properties and C
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Anal. Chem. 2010, 82, 6911-6918 Dir
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dilution and hybridization buffer.
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solution under appropriate incubati
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Figure 4. Standardization curve for
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Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
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Figure 2. With a suitable removal o
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the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
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trode 28 by a finite element using
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Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
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Figure 2. Normalized product ion ab
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Figure 4. EID (a) and IRMPD (b) of
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Anal. Chem. 2010, 82, 6947-6957 Ide
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Figure 1. Schematic flowchart showi
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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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Figure 5. Extracted ion current ESI
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m/z 1172.935 was observed for Ser14
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linked products via affinity tags.
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Scheme 2. Fragmentation Mechanism o
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Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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Figure 5. (A) MALDI-TOF/TOF product
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Anal. Chem. 2010, 82, 6969-6975 Ana
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Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
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for the fill time, and we find that
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nucleotide tails. 3-5 Thus, the amo
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allow the use of higher aptamer con
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quent ligation of the aptamers afte
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Anal. Chem. 2010, 82, 6983-6990 Imp
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Figure 2. Configuration editor wind
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Figure 4. Dependencies between volu
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Since the time for liquid expulsion
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Anal. Chem. 2010, 82, 6991-6999 Int
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Figure 1. Representation of the Car
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Table 2. Capillary Electrophoresis
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Figure 4. (A) Typical raw electroph
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field. DNA profiles were delivered
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Another approach to handle this lim
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(principal components, PCs) in whic
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Figure 5. Relevant score plots and
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CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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Figure 2. Qdot-based FLFTS response
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Figure 6. Fluorescence imaging of Q
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Anal. Chem. 2010, 82, 7015-7020 Sel
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Table 1. Effect of 1 D-LC Condition
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Figure 3. Peak-production rate vers
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Anal. Chem. 2010, 82, 7021-7026 Cel
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luciferase (T7 control vector) was
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Figure 3. Inhibitory effects of luc
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Anal. Chem. 2010, 82, 7027-7034 Pat
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Electrochemistry. Electrochemical m
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Figure 2. SEM images of Au substrat
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Table 3. Film Thickness Measurement
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Anal. Chem. 2010, 82, 7035-7043 Qua
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Figure 1. (A) FL spectra of BSPOTPE
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Figure 4. (A) Variation in the FL i
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Figure 6. (A) FL spectra of BSPOTPE
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Scheme 1. Proposed Mechanism for Fl
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one or more drawbacks including poo
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Figure 2. Free fluorophores do not
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Anal. Chem. 2010, 82, 7049-7052 Dev
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Figure 2. Comparative analysis of d
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