Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6926–6932 Study of Highly Selective and Efficient Thiol Derivatization Using Selenium Reagents by Mass Spectrometry Kehua Xu, †,‡ Yun Zhang, † Bo Tang,* ,‡ Julia Laskin, § Patrick J. Roach, § and Hao Chen* ,† Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701, Key Laboratory of Molecular and Nano Probes, Ministry of Education, College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, China, 250014, and Chemical and Materials Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, MSIN K8-88, Richland, Washington 99352 This paper reports a systemic mass spectrometry (MS) investigation of a novel strategy for labeling biological thiols, involving the cleavage of the Se-N bond by thiol to form a new Se-S bond. Our data show that the reaction is highly selective, rapid, reversible, and efficient. Among 20 amino acids, only cysteine is reactive toward Se-N containing reagents and the reaction occurs in seconds. With the addition of dithiothreitol, peptides derivatized by selenium reagents can be recovered. The high reaction selectivity and reversibility provide potential in both selective identification and isolation of thiols from mixtures. Also, with dependence on the selenium reagent used, derivatized peptide ions exhibit tunable dissociation behaviors (either facile cleavage or preservation of the formed Se-S bond upon collision-induced dissociation), a feature that is useful in proteomics studies. Equally importantly, the thiol derivatization yield is striking, as reflected by 100% conversion of protein �-lactoglobulin A using ebselen within 30 s. In addition, preliminary applications such as rapid screening of thiol peptides from mixtures and identification of the number of protein free and bound thiols have been demonstrated. The unique selenium chemistry uncovered in this study would be valuable in the MS analysis of thiols and disulfide bonds of proteins/peptides. Biological thiols, such as glutathione (GSH) and thiol proteins, are critical physiological components found in animal tissues and fluids and involved in a plethora of important cellular functions. 1,2 In the past decades chemical characterization of biological thiols has attracted significant attention. Various measurement methods have been developed including UV, 3 fluorescence (FL) * To whom correspondence should be addressed. Hao Chen: phone, 740- 593-0719; fax, 740-597-3157; e-mail, chenh2@ohio.edu. Bo Tang: phone, (86)531- 86180010; e-mail, tangb@sdnu.edu.cn. † Ohio University. ‡ Shandong Normal University. § Pacific Northwest National Laboratory. (1) Basford, R. E.; Huennekens, F. M. J. Am. Chem. Soc. 1955, 77, 3873– 3877. (2) Rahman, I.; MacNee, W. Free Radical Biol. Med. 2000, 28, 1405–1420. (3) Kusmierek, K.; Chwatko, G.; Glowacki, R.; Bald, E. J. Chromatogr., B 2009, 877, 3300–3308. 6926 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 spectroscopy, 4-6 and mass spectrometry (MS), 7-12 in which the derivatization of thiol groups with a suitable chemical reagent is necessary for increasing thiol stability and improving detection selectivity. While spectroscopic methods such as FL are very useful for detection and imaging of thiol compounds, MS can provide molecular weight and structural information. The inherent sensitivity and chemical specificity offered by MS 13-16 is essential in further identifying biological thiols and investigating their physiological functions on a molecular level. Also, a number of excellent MS studies of thiols and disulfides of proteins/peptides based on the novel ion chemistry have been reported. 10,11,17-20 At present, MS labeling strategies are mainly based on three types of chemical reactions. Nucleophilic substitution such as using heptafluorobutyl chloroformate 21 and iodoacetamide is commonly used for the analysis of protein tryptic digest; 22 however, these reagents are not specific for thiol compounds, that is, they can also couple with amino/hydroxyl groups. The second approach involves Michael- (4) Lee, J. H.; Lim, C. S.; Tian, Y. S.; Han, J. H.; Cho, B. R. J. Am. Chem. Soc. 2010, 132, 1216–1217. (5) Pullela, P. K.; Chiku, T.; Carvan, M. J.; Sem, D. S. Anal. Biochem. 2006, 352, 265–273. (6) Tang, B.; Yin, L.; Wang, X.; Chen, Z.; Tong, L.; Xu, K. Chem. Commun. 2009, 5293–5295. (7) Gorman, J. J.; Wallis, T. P.; Pitt, J. J. Mass Spectrom. Rev. 2002, 21, 183– 216. (8) Bilusich, D.; Bowie, J. H. Mass Spectrom. Rev. 2009, 28, 20–34. (9) Chrisman, P. A.; Pitteri, S. J.; Hogan, J. M.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2005, 16, 1020–1030. (10) Diedrich, J. K.; Julian, R. R. J. Am. Chem. Soc. 2008, 130, 12212–12213. (11) Gunawardena, H. P.; O’Hair, R. A. J.; McLuckey, S. A. J. Proteome Res. 2006, 5, 2087–2092. (12) Seiwert, B.; Karst, U. Anal. Chem. 2007, 79, 7131–7138. (13) Wang, Y.; Vivekananda, S.; Zhang, K. Anal. Chem. 2002, 74, 4505–4512. (14) Pingitore, F.; Wesdemiotis, C. Anal. Chem. 2005, 77, 1796–1806. (15) Blanksby, S. J.; Bierbaum, V. M.; Ellison, B. G.; Kato, S. Angew. Chem., Int. Ed. 2007, 46, 4948–4950. (16) Gronert, S. Chem. Rev. 2001, 101, 329–360. (17) Kim, H. I.; Beauchamp, J. L. J. Am. Soc. Mass Spectrom. 2009, 20, 157– 166. (18) Chrisman, P. A.; McLuckey, S. A. J. Proteome Res. 2002, 1, 549–557. (19) Li, J.; Shefcheck, K.; Callahan, J.; Fenselau, C. Int. J. Mass Spectrom. 2008, 278, 109–133. (20) Qiao, L.; Bi, H.; Busnel, J.-M.; Liu, B.; Girault, H. H. Chem. Commun. 2008, 47, 6357–6359. (21) Simek, P.; Husek, P.; Zahradnickova, H. Anal. Chem. 2008, 80, 5776– 5782. (22) Williams, D. K., Jr.; Meadows, C. W.; Bori, I. D.; Hawkridge, A. M.; Comins, D. L.; Muddiman, D. C. J. Am. Chem. Soc. 2008, 130, 2122–2123. 10.1021/ac1011602 © 2010 American Chemical Society Published on Web 07/16/2010
Scheme 1. Reactions of Selenium Reagents 1 and 2 with Thiols of Proteins/Peptides addition of thiols onto unsaturated CdC bonds such as using acrylate 23 or maleimide derivatives as common labeling agents. 24 These reagents have selectivity toward thiols and have been often used in practical thiol analysis. However, the Michael-addition product is irreversible, which cannot allow enriching and purifying analyte compounds from complex matrices. 25 The third approach involves thiol exchange reaction such as using 5,5′-dithiobis(2nitrobenzoic acid) known as Ellman’s reagent; 26,27 a large excess amount of the disulfide reagent is necessary to ensure complete derivatization of all thiols in the sample. As a result, the newly formed disulfides in the reaction can possibly further react with residual thiols of the target molecule to form undesirable disulfides. For example, Udgaonkar et al. used Ellman’s reagent in a 100-fold molar excess to label the thiol protein based on the exchange reaction in the study of the cooperativity of a fast protein folding reaction by MS. 27 Besides the problems mentioned above, these derivatization reactions have other limitations, including a long reaction time, low conversion yield, or more than one possible site for tagging. Therefore, new derivatization chemistry suitable for MS detection of thiol, particularly with high thiol selectivity, fast reaction speed, good reaction reversibility, and high conversion yield, is still needed for the structural analysis of peptides and proteins in biological samples. Because selenium is an essential element in vivo 28 and a key component of selenoproteins known as antioxidant enzymes, selenium chemistry has recently attracted increasing attention. Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (reagent 1, Scheme 1), has been considered as a mimetic of glutathione peroxidase (GPx) 29-32 and an anti-inflammatory drug since being found. 33,34 The mechanism for ebselen-catalyzed thiol oxidation (23) Wang, G.; Hsieh, Y.; Wang, L.; Prelusky, D.; Korfmacher, W. A.; Morrison, R. Anal. Chim. Acta 2003, 492, 215–221. (24) Seiwert, B.; Hayen, H.; Karsta, U. J. Am. Soc. Mass Spectrom. 2008, 19, 1–7. (25) Jalili, P. R.; Ball, H. L. J. Am. Soc. Mass Spectrom. 2008, 19, 741–750. (26) Sevcikova, P.; Glatz, Z.; Tomandl, J. J. Chromatogr., A 2003, 990, 197– 204. (27) Kumar Jha, S.; Udgaonkar, J. B. J. Biol. Chem. 2007, 282, 37479–37491. (28) Yoneda, S. J.; Kazuo, T. S. Toxicol. Appl. Pharmacol. 1997, 143, 274–280. (29) Forstrom, J. W.; Zakowski, J. J.; Tappel, A. L. Biochemistry 1978, 17, 2639– 2644. (30) Birringer, M.; Pilawa, S.; Flohé, L. Nat. Prod. Rep. 2002, 19, 693–718. (31) Jacob, C.; Giles, G. I.; Giles, N. M.; Sies, H. Angew. Chem, Int. Ed. 2003, 42, 4742–4758. (32) Haenen, G. R.; De Rooij, B. M.; Vermeulen, N. P.; Bast, A. Am. Soc. Pharmacol. Exp. Therapeut. 1990, 37, 412–422. has been proposed: 35,36 the Se-N bond of ebselen is cleaved by thiol RSH to produce the corresponding selenenyl sulfide Se-S, which further reacts with excess thiol RSH to produce selenol and disulfide compound RSSR. However, Mugesh et al. recently reported that ebselen as a GPx-like redox catalyst is indeed inactive, and the reaction of ebselen with PhSH only produces the Se-S product, not disulfide PhSSPh, even with an excess amount of PhSH. 36 Very recently, based on this novel selenium chemistry, we synthesized fluorescent probes carrying Se-N bonds and used them for detecting and imaging thiols in living cells 6,37 by FL. It was shown that the FL probes capture cellular thiols selectively in the presence of diverse species such as inorganic metal ions, dopamine, histamine, L-adrenaline, etc. Following that, Zhang et al. used piazselenole containing Se-N bond to probe physiological thiols based on electrochemical reactions. 38 Highly specific derivatization of thiols using compounds containing Se-N bonds makes them promising candidates to derivatize peptides and proteins for subsequent MS characterization. Although the analysis of protein thiols and disulfide bonds is important in proteomics applications, the selenium chemistry received surprisingly limited attention. 39 Here we present a systematic study of derivatization of amino acids, peptides, and proteins using Se-N containing reagents and examine the utility of this chemistry for analytical applications involving mass spectrometry. In this study, we carried out a series of reactions of two Se-N containing reagents, ebselen and N-(phenylseleno)phthalimide (reagent 2, Scheme 1), with various biological thiol compounds such as cysteine, reduced peptide glutathione GSH, and �-lactoglobulin A protein. As shown in Scheme 1, the Se-N bonds in reagents 1 and 2 are cleaved by thiols to form new Se-S bonds as shown in products 3 and 4, respectively (Scheme 1). Our experimental results showed that this thiol derivatization reaction is highly selective, rapid, reversible, and efficient (quantitative in (33) Müller, A.; Cadenas, E.; Graf, P.; Sies, H. Biochem. Pharmacol. 1984, 33, 3235–3239. (34) Sies, H.; Masumoto, H. Adv. Pharmacol. 1997, 38, 229–246. (35) Mugesh, G.; du Mont, W.-W.; Sies, H. Chem. Rev. 2001, 101, 2125–2179. (36) Sarma, B. K.; Mugesh, G. J. Am. Chem. Soc. 2005, 127, 11477–11485. (37) Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. J. Am. Chem. Soc. 2007, 129, 11666–11667. (38) Wang, W.; Li, L.; Liu, S.; Ma, C.; Zhang, S. J. Am. Chem. Soc. 2008, 130, 10846–10847. (39) Sakurai, T.; Kanayama, M.; Shibata, T.; Itoh, K.; Kobayashi, A.; Yamamoto, M.; Uchida, K. Chem. Res. Toxicol. 2006, 19, 1196–1204. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6927
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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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Figure 4. Schematic representation
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esult in a big SPR signal change wi
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also reduces chemical noise, which
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Table 1. Extraction Yields, Liquid
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scanning of AMPP amides of the anal
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Anal. Chem. 2010, 82, 6797-6806 δ
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Figure 1. Schematic view of the pre
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from the specimen and enclosed in a
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Figure 4. (A) IRMS mass-44 chromato
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Table 3. Ambient Measurement Result
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Anal. Chem. 2010, 82, 6807-6813 Dir
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Polymerase (1 U per sample). Reacti
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of which was constant for all ampli
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analytically useful signals at less
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carbon black and RP-C18 for the ext
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solution in an equal volume, and 1
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Table 2. Concentrations and Ratios
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Anal. Chem. 2010, 82, 6821-6829 Mac
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mg/mL protein, followed by separati
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Figure 2. Productivity of SEQUST an
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Figure 4. High-resolution MS/MS spe
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Figure 6. Characterization of PSMs
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containing T-T mismatches. 23 Based
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Figure 1. Extinction spectra of sol
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Figure 3. (A) The value of Ex650 nm
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Figure 5. Extinction spectra and co
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wished to explore the dehydration o
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constant medium for separation; we
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Figure 2. Standards of (Pi)n, n ) 1
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Figure 5. Quantitative calibration
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Anal. Chem. 2010, 82, 6847-6853 Met
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Figure 1. 226 Ra spectrum by liquid
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Table 1. Counting Properties and De
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Table 3. Analysis of 226 Ra in Sedi
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mercial microarray scanner and fabr
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Figure 2. Optical transmission meas
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Figure 5. Volcano plots detailing t
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data as well. The 41 genes in Table
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corresponding compound if its chemi
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Figure 1. Schematic illustration of
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Table 1. Absolute Quantification Re
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ment. Therefore, the long-time drea
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Figure 1. Chemically actuated micro
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Figure 2. Influence of a surfactant
Page 131 and 132: Figure 5. Device to eject and mix s
Page 133 and 134: Anal. Chem. 2010, 82, 6877-6886 Imm
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
Page 143 and 144: Anal. Chem. 2010, 82, 6887-6894 Fer
Page 145 and 146: Figure 1. Infrared spectra of (A) u
Page 147 and 148: Figure 3. Cyclic voltammograms obta
Page 149 and 150: Figure 6. Calculated charge from ch
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Page 153 and 154: Table 1. Chemical Structure, pKa Va
Page 155 and 156: pH with a tilted baseline (Figure 1
Page 157 and 158: Table 2. Linearity and Detection Li
Page 159 and 160: ascorbic acid (AA), uric acid (UA),
Page 161 and 162: the multielement capabilities, the
Page 163 and 164: RESULTS AND DISCUSSION Sulfur Detec
Page 165 and 166: Table 2. Molecular Properties and C
Page 167 and 168: Anal. Chem. 2010, 82, 6911-6918 Dir
Page 169 and 170: dilution and hybridization buffer.
Page 171 and 172: solution under appropriate incubati
Page 173 and 174: Figure 4. Standardization curve for
Page 175 and 176: Anal. Chem. 2010, 82, 6919-6925 Ele
Page 177 and 178: the ×10 objective, to have a large
Page 179 and 180: Figure 2. With a suitable removal o
Page 181: the NB signal in a much better foot
Page 185 and 186: Figure 2. (a) ESI-MS spectrum showi
Page 187 and 188: Figure 4. (a) ESI-MS spectrum showi
Page 189 and 190: Anal. Chem. 2010, 82, 6933-6939 Dif
Page 191 and 192: trode 28 by a finite element using
Page 193 and 194: Figure 4. Comparison between simula
Page 195 and 196: Figure 6. Comparison between simula
Page 197 and 198: educed in the vicinity of double bo
Page 199 and 200: Figure 2. Normalized product ion ab
Page 201 and 202: Figure 4. EID (a) and IRMPD (b) of
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Page 205 and 206: Figure 1. Schematic flowchart showi
Page 207 and 208: difference, ppm compound Table 1. I
Page 209 and 210: isoforms, its successful use, in th
Page 211 and 212: Figure 5. Extracted ion current ESI
Page 213 and 214: m/z 1172.935 was observed for Ser14
Page 215 and 216: linked products via affinity tags.
Page 217 and 218: Scheme 2. Fragmentation Mechanism o
Page 219 and 220: Figure 1. (A) ESI-LTQ-CID-MS 2 prod
Page 221 and 222: Figure 3. (A) ESI-LTQ-CID-MS 2 prod
Page 223 and 224: Figure 5. (A) MALDI-TOF/TOF product
Page 225 and 226: Anal. Chem. 2010, 82, 6969-6975 Ana
Page 227 and 228: Figure 3. Equilibrium response as a
Page 229 and 230: Figure 6. The average measured resp
Page 231 and 232: 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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