Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6904–6910 Human Plasma Copper Proteins Speciation by Size Exclusion Chromatography Coupled to Inductively Coupled Plasma Mass Spectrometry. Solutions for Columns Calibration by Sulfur Detection Souleiman El Balkhi, †,§,| Joël Poupon,* ,† Jean-Marc Trocello, ‡,§ France Massicot, | France Woimant, ‡,§ and Olivier Laprévote †,| Laboratoire de toxicologie biologique, Service de Neurologie, Centre de Référence Pour la Maladie De Wilson, AP-HP, Hôpital Lariboisière, 2, rue Ambroise Paré, 75475 Paris cedex 10, France, and ChimiesToxicologie Analytique et Celullaire, EA 4463, Faculté de Pharmacie, Université Paris Descartes, 4, avenue de l’Observatoire, 75006 Paris, France Among the hyphenated techniques used to probe and identify metalloproteins, size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS) has shown to have a central place. However, the calibration of SEC columns reveals to be tedious and always involves UV detection prior to ICP-MS. The presence of sulfur in 98% of proteins allows their detection by quadrupole ICP-MS, despite the isobaric interference ( 16 O 16 O) on S, by monitoring 32 S 16 O at mass to charge ratio (m/z) 48. The formation of SO occurs spontaneously in the argon plasma but can be optimized by the introduction of oxygen gas into a reaction cell (RC) to achieve nM levels. In this article, sulfur detection was discussed upon instrumental conditions and S detection was then optimized by applying O 2 as a reaction gas. SO formation was used to calibrate SEC columns without UV detection. This simple SEC-ICP- MS method was used for plasma copper proteins in plasma healthy subjects (HS) and an untreated Wilson disease (WD) patient. Copper proteins identified in healthy subjects were transcuprein, ceruloplasmin (Cp) and albumin. The method led to results in good agreement with other methods of determination. Copper bound to Cp in the WD patient was lowered with regard to the HS, and the exchangeable Cu was highly increased. In the postgenomics era, proteomics has become a central branch in life sciences. Information from proteomics (and peptidomics) studies may reveal alterations in gene expression due to a disease and facilitate the understanding of the metabolic pathways such as copper metabolism in Wilson and Menkes diseases. 1 Since one-third * Author for correspondence: Dr Joël Poupon, Laboratoire de Toxicologie biologique, Hôpital Lariboisière, 2 rue Ambroise Paré, 75475 Paris cedex 10, France, E-Mail: joel.poupon@lrb.aphp.fr, Phone: 33 1 49 95 66 00, Fax: 33 1 49 95 65 71. † Laboratoire de toxicologie biologique. ‡ Service de Neurologie. § Centre de Référence Pour la Maladie De Wilson. | Université Paris Descartes. 6904 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 of all proteins are metalloproteins, a new field recently defined by Haraguchi, namely “metallomics”, has been introduced. This topic attracted a fast growing interest during the past decade. It integrates different analytical approaches that focus on metalloproteins and metal-containing biomolecules. Metal ions in metalloproteins have a regulatory and structural role allowing them to fulfill their different functions. The biocatalysis of some specific enzymatic reactions including gene DNA synthesis, metabolism, antioxidation, and detoxification are examples of these functions. 2,3 Since the separation, identification, and quantification of metalloproteins are mandatory in our understanding of their complex role in biological systems, a growing number of studies are covering these fields. Usually, authors employ hyphenated techniques combining a separation method followed by mass spectrometry analysis. Matrix-assisted laser desorption/ionization (MALDI) MS, electrospray ionization (ESI) MS, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) laser ablation (LA) ICP-MS and high performance liquid chromatography (HPLC) ICP-MS with different modes of separation have been proposed. More recently, capillary electrophoresis (CE) ICP-MS and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR- MS) have been used for this purpose. 3,4 The overwhelming majority of recent applications concerning probing and quantification of metalloproteins were developed using ICP-MS coupled to size-exclusion chromatography (SEC). 5-9 ICP-MS is the most widely used mode of detection because of its extremely low limits of detection (LOD), a wide dynamic range, (1) Wang, M.; Feng, W. Y.; Zhao, Y. L.; Chai, Z. F. Mass Spectrom. Rev. 2009, 29, 326–348. (2) Haraguchi, H. J. Anal. At. Spectrom. 2004, 19, 5–14. (3) Shi, W.; Chance, M. R. Cell. Mol. Life Sci. 2008, 65, 3040–3048. (4) Szpunar, J. Analyst 2005, 130, 442–465. (5) Cabrera, A.; Alonzo, E.; Sauble, E.; Chu, Y. L.; Nguyen, D.; Linder, M. C.; Sato, D. S.; Mason, A. Z. Biometals 2008, 21, 525–543. (6) Franca Maltez, H.; Villanueva Tagle, M.; Fernandez De La Campa Maria Del, R.; Sanz Medel, A. Anal. Chim. Acta 2009, 650, 234–240. (7) Gonzalez-Fernandez, M.; Garcia-Barrera, T.; Arias-Borrego, A.; Bonilla- Valverde, D.; Lopez-Barea, J.; Pueyo, C.; Gomez-Ariza, J. L. Anal. Bioanal. Chem. 2008, 390, 17–28. (8) Lopez-Avila, V.; Sharpe, O.; Robinson, W. H. Anal. Bioanal. Chem. 2006, 386, 180–187. (9) Sviridov, D.; Meilinger, B.; Drake, S. K.; Hoehn, G. T.; Hortin, G. L. Clin. Chem. 2006, 52, 389–397. 10.1021/ac101128x © 2010 American Chemical Society Published on Web 07/19/2010
the multielement capabilities, the continuous spectra signals survey and a high sample throughput. 10 In addition, SEC has the advantage, in theory, to preserve metalloproteins from solid phase/analytes interactions. Maintaining the integrity of the probed proteins is actually of a critical importance when metal ions simply coordinate to organic ligands. Nevertheless, the calibration of SEC columns presents some drawbacks rending SEC-ICP coupling laborious because of (i) a tedious installation of a UV detector in parallel to the ICP-MS, (ii) the use of an additional software for the integration of chromatographic peaks, and (iii) the increase of dead volume. This way of SEC calibration involves the injection of high concentrations of proteins. These are expensive and can irreversibly bound to columns leading to columns clogging. It is noteworthy that other detection techniques have been proposed to calibrate SEC columns such as IR, NMR, MS, evaporative light scattering, and viscometers 11 but their coupling to ICP has not yet been reported. The capability of ICP-MS to detect in a single run the metal ions of interest and hetero nonmetallic elements present in proteins such as S and P offers great opportunities. It allows a specific detection, molecular characterization, and even quantification of some metalloproteins. For instance, when the amino acid (AA) sequence is known for a given protein, and hence the number of AA containing S, the determination of S provides an accurate value of molar protein concentration. 12,13 Unfortunately, these heteroelements (i.e., S and P) have high ionization energies resulting in low sensitivities in ICP-MS. Moreover, both S and P are extensively interfered by isobaric polyatomic ions generated in the atmospheric pressure argon plasma ( 16 O 16 O for 32 S and 15 N 16 O for 31 P). The use of high resolution mass spectrometry systems such as sector field ICP- MS with a resolution higher than 1 800 m/∆m 14-17 allows distinguishing of these isobaric species. Alternatively, the detection of S remains feasible with lower resolution (m/∆m 400) quadrupole ICP-MS by using a reaction cell. This device is a quadrupole filled with a reaction gas (oxygen or xenon) and placed between the ion lenses and the quadrupole mass filter of the ICP-MS instrument. Oxygen used as reacting gas in the reaction cell converts S ions into 32 S 16 O. Sulfur is then measured as SO at m/z 48 which shows less interferences. This technique was previously used for the determination of some sulfured amino acids. 18,19 In the same way, the measurement of phosphorylation degree of some peptides 20 and the determination of metal-sulfur ratios in some metalloproteins 12 were obtained by detecting S and (10) Lobinski, R.; Moulin, C.; Ortega, R. Biochimie 2006, 88, 1591–1604. (11) Striegel, A. M. Anal. Chem. 2005, 77, 104 A113 A. (12) Hann, S.; Koellensperger, G.; Obinger, C.; Furtmuller, P. G.; Stingeder, G. J. Anal. At. Spectrom. 2004, 19, 74–79. (13) Profrock, D.; Leonhard, P.; Prange, A. Anal. Bioanal. Chem. 2003, 377, 132–139. (14) Bettmer, J.; Montes Bayon, M.; Encinar, J. R.; Fernandez Sanchez, M. L.; Fernandez de la Campa Mdel, R.; Sanz Medel, A. J. Proteomics 2009, 72, 989–1005. (15) Rappel, C.; Schaumloffel, D. Anal. Bioanal. Chem. 2008, 390, 605–615. (16) Wind, M.; Wesch, H.; Lehmann, W. D. Anal. Chem. 2001, 73, 3006–3010. (17) Zinn, N.; Kruger, R.; Leonhard, P.; Bettmer, J. Anal Bioanal. Chem. 2008. (18) Schaumloffel, D.; Giusti, P.; Preud’Homme, H.; Szpunar, J.; Lobinski, R. Anal. Chem. 2007, 79, 2859–2868. (19) Yeh, C. F.; Jiang, S. J.; Hsi, T. S. Anal. Chim. Acta 2004, 502, 57–63. (20) Bandura, D. R.; Baranov, V. I.; Tanner, S. D. Anal. Chem. 2002, 74, 1497– 1502. Pas 32 S 16 O + and 33 P 16 O + species (m/z 48 and 49, respectively) together with metals of interest. More recently, Wang used SEC-ICP-MS for a quantitative analysis of proteins via S determination. 21 Interestingly, by using argon plasma, under atmospheric condition, oxides are formed spontaneously at percentages corresponding to ≈3% of measured elements. 22,23 These percentages are influenced by instrumental parameters of ICP-MS. 24,25 Consequently, in applications involving high concentrations of proteins, such as undiluted or weakly diluted plasma, the amount of produced SO could be sufficient for its detection. Moreover, recommended concentrations of proteins in SEC column calibration kits are around 1-10 g · L -1 (i.e., S concentration higher than 50 µM) which is enough to monitor SO without any addition of O2. In other words, the calibration of SEC columns should be possible by using SEC-ICP-MS in standard mode and without any prior UV detection. In this study, we present a simple SEC-ICP-MS method for plasma copper proteins speciation. This method was applied to healthy subjects (HS) and an untreated Wilson disease (WD) patient. The distribution of copper in the blood is still an important subject of research. The knowledge of copper distribution contributes to better understand copper metabolism and to better diagnose and monitor related diseases. In WD, a mutation in the gene ATP7B leads to a dysfunction of ceruloplasmin (Cp) which is the major protein binding Cu. This binding is mediated by the protein ATP7B that lacks in WD. Clinically, serum Cp concentration diminishes and the so-called “free Cu” increases and becomes toxic due to Cu deposits in target organs (liver, brain, kidney, and eyes). If not treated, irreversible damages can occur. 26-28 For the speciation purpose, we demonstrate that SEC column calibration can be conducted with a simple SEC-ICP-MS at moderate m/z resolution and without any UV detection. ICP-MS operational parameters influence on oxides formations in standard mode (without O2) was studied. The method was then optimized by the application of O2 as reaction gas in the dynamic reaction cell (DRC). Excellent limits of detection (LODs) for S and Cu were obtained after optimization. Copper proteins identification, stability and quantification are explored. EXPERIMENTAL SECTION Reagents and Chemicals. Mobile phase (NH4NO3 200 mM) was prepared daily by dissolving 16 g of NH4NO3 (Sigma Chemical Co, St-Quentin Fallavier, France) in 1Lofultrapure water Milli-Q (Millipore, Molsheim, France) and degassed by vacuum filtration on a 0.22 µm Millipore filters. For SEC column calibration, two SEC marker kits were used. Kit 1 (obtained from Sigma) included: Blue dextran (2000 kDa), thyroglobulin (670 kDa), apoferritin (443 kDa), �-amylase (200 (21) Wang, M.; Feng, W.; Lu, W.; Li, B.; Wang, B.; Zhu, M.; Wang, Y.; Yuan, H.; Zhao, Y.; Chai, Z. Anal. Chem. 2007, 79, 9128–9134. (22) Divjak, B.; Goessler, W. J. Chromatogr., A 1999, 844, 161–169. (23) Du, Z.; Houk, R. S. J. Anal. At. Spectrom. 2000, 15, 383–388. (24) Gray, A. L.; Williams, J. G. J. Anal. At. Spectrom. 1987, 2, 599–606. (25) Vaughan, M. A.; Horlick, G. Appl. Spectrosc. 1986, 40, 434–445. (26) Das, S. K.; Ray, K. Nat. Clin. Pract. Neurol. 2006, 2, 482–493. (27) El Balkhi, S.; Poupon, J.; Trocello, J. M.; Leyendecker, A.; Massicot, F.; Galliot-Guilley, M.; Woimant, F. Anal. Bioanal. Chem. 2009, 394, 1477– 1484. (28) Linder, M. C.; Hazegh-Azam, M. Am. J. Clin. Nutr. 1996, 63, 797S–811S. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6905
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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
Page 109 and 110: Table 3. Analysis of 226 Ra in Sedi
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Page 113 and 114: Figure 2. Optical transmission meas
Page 115 and 116: Figure 5. Volcano plots detailing t
Page 117 and 118: data as well. The 41 genes in Table
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Page 121 and 122: Figure 1. Schematic illustration of
Page 123 and 124: Table 1. Absolute Quantification Re
Page 125 and 126: ment. Therefore, the long-time drea
Page 127 and 128: Figure 1. Chemically actuated micro
Page 129 and 130: 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
Page 151 and 152: Anal. Chem. 2010, 82, 6895-6903 Ele
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: ascorbic acid (AA), uric acid (UA),
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 and 182: the NB signal in a much better foot
Page 183 and 184: Scheme 1. Reactions of Selenium Rea
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
Page 203 and 204: Anal. Chem. 2010, 82, 6947-6957 Ide
Page 205 and 206: Figure 1. Schematic flowchart showi
Page 207 and 208: difference, ppm compound Table 1. I
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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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Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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