Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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separate and identify the individual isoforms but the hitherto studies have been plagues by poor sensitivity preventing the direct analysis of biological cytosols. Indeed, most of the reports referred to the analysis of a1mg· mL -1 solution by capillary electrophoresis and 100 µg · mL -1 by HPLC. Such high MT concentrations are largely superior to those encountered in real-world biological systems and can be achieved only after a large-scale purification. Moreover, the ESI MS sensitivity is severely degraded by the coelution of other biomolecules, especially in ESI TOF MS. Consequently, the most widely applied MS technique for the screening of biological cytosols for MTs has been inductively coupled plasma mass spectrometry (ICP MS) used in combination with HPLC or capillary electrophoresis. 26,27 ICP MS is a convenient technique for the detection of the individual isoforms and the determination of the stoichiometry of the metal-complexes formed, indeed, but does not allow their identification. The few successful examples of the identification of MTs in biological cytosols by ESI MS were carried out on the basis of the molecular mass determined using quadrupole 28,29 and TOF mass spectrometers 30 with limited mass accuracy. The acquisition of sequence information by conventional bottom-up proteomics approaches is hampered by resistance of MT to tryptic digestion and frequent miscleavages in the presence of residual metals. 31 Also, as MT isoforms exhibit a significant sequence homology (70-90%), 32 many tryptic peptides are common for many isoforms thus preventing de novo identification. Indeed, examples of successful identification of MTs on the basis of MS/MS of tryptic peptides have been scarce. 30,33 The above drawbacks and the tediousness of off-line bottomup identification procedures can be alleviated by top-down MS allowing one to obtain structural information from intact proteins. 34 Although most of data have been obtained with high magnetic field strength FT ICR MS using infrared multiple photon dissociation (IRMPD) or electron capture detection (ECD), 35 the efficiency of direct fragmentation of intact protein in quadrupole collision cells is increasingly explored. 36,37 Particularly interesting is the combination of hybrid linear quadrupole ion trap with an Orbitrap mass spectrometer which offers resolution exceeding 60 000 and often sub-ppm mass accuracy. 38-42 (25) Benavente, F.; Andon, B.; Gimenez, E.; Olivieri, A. C.; Barbosa, J.; Sanz- Nebot, V. Electrophoresis 2008, 29, 4355–4367. (26) Prange, A.; Schaumloffel, D. Anal. Bioanal. Chem. 2002, 373, 441–453. (27) Szpunar, J. Analyst 2005, 130, 442–465. (28) Infante, H. G.; Cuyckens, F.; Van Campenhout, K.; Blust, R.; Claeys, M.; Van Vaeck, L.; Adams, F. C. J. Anal. At. Spectrom. 2004, 19, 159–166. (29) Polec, K.; Perez-Calvo, M.; Garcia-Arribas, O.; Szpunar, J.; Ribas-Ozonas, B.; Lobinski, R. J. Inorg. Biochem. 2002, 88, 197–206. (30) Wang, R.; Sens, D. A.; Albrecht, A.; Garrett, S.; Somji, S.; Sens, M. A.; Lu, X. Anal. Chem. 2007, 79, 4433–4441. (31) Wang, R.; Sens, D. A.; Garrett, S.; Somjii, S.; Sens, M. A.; Lu, X. Electrophoresis 2007, 28, 2942–2952. (32) Sewell, A. K.; Yokoya, F.; Yu, W.; Miyagawa, T.; Murayama, T.; Winge, D. R. J. Biol. Chem. 1995, 270, 25079–25086. (33) Feng, W.; Benz, F. W.; Cai, J.; Pierce, W. M.; Kang, Y. J. J. Biol. Chem. 2006, 281, 681–687. (34) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806–812. (35) Tolmachev, A. V.; Robinson, E. W.; Wu, S.; Pasa-Tolic, L.; Smith, R. D. Int. J. Mass Spectrom. 2009, 287, 32–38. (36) Mandal, R.; Li, X. F. Rapid Commun. Mass Spectrom. 2006, 20, 48–52. (37) Moreno-Gordaliza, E.; Canas, B.; Palacios, M. A.; Gomez-Gomez, M. M. Anal. Chem. 2009, 81, 3507–3516. 6948 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 The goal of this study was the description on the molecular level of the chemical response of a cell line exposed to the stress of CdS nanoparticles, increasingly used in diverse areas from electronics to targeted drug delivery, in view of the understanding of the mechanisms of their toxicity. As the preliminary experiments indicated MT induction at picomole levels, analytical development focused on the detection of traces of metal-complexes with individual MT isoforms and the unambiguous identification of the latter using both the accurate mass and topdown sequencing information, directly in the MT fraction, without extensive purification. EXPERIMENTAL SECTION Reagent and Standards. Analytical reagent grade chemicals purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and water (18 MΩ cm) obtained from a Milli-Q system (Millipore, Bedford, MA) were used throughout unless stated otherwise. The rabbit liver metallothionein-2 isoform standard (purity: 95%) was purchased from Enzo Life Sciences (Villeurbanne, France). It was reported by the manufacturer to be a mixture of isoforms (major: MT-2a, minor: MT-2b and MT-2c) and to contain 67% of protein and 9% of metals (Cd and Zn). The stock solution (1 mg · mL -1 ) was prepared by dissolving 1 mg of metallothionein in 1 mL of water, subdivided in 10 µL aliquots to avoid multiple thawing and freezing, and frozen at -20 °C. Working solutions were prepared daily by dilution with water at 4 °C. Instrumentation. Microbore reversed-phase HPLC (µRP HPLC) separations were carried out using an Agilent 1100 capillary HPLC system (Agilent, Tokyo, Japan) equipped with a 100 µL · min -1 splitter module. ICP MS detection was achieved using a model 7500cs instrument (Agilent) fitted with platinum cones, 1 mm i.d injector torch and a T-connector allowing the introduction of 5% O2. The µRP HPLC-ICP MS coupling was done via an Isomist interface (Glass Expansion, Melbourne, Vic, Australia) consisting of a 20 mL model Cinnabar spray chamber cooled at 2 °C and fitted with a 50 µL · min -1 Micromist nebulizer. For µRP HPLC-ESI MS experiments, the µRP HPLC system was connected to a LTQ Orbitrap Velos mass spectrometer (ThermoFisher Scientific, Bremen, Germany). The coupling was achieved via a heated electrospray ionization source (H-ESI II) (ThermoFisher Scientific). The postcolumn acidification manifold, described in detail elsewhere, 43 consisted of a zero dead volume PEEK T-piece allowing the mixing of the chromatographic effluent with a formic acid:MeOH (30/70%, v/v) solution delivered by means of a syringe pump (Pump 33 model, Harvard Apparatus, South Natick, MA) and a mixing PEEK tubing coil (250 µm i.d. × 250 mm) connected to the inlet of the electrospray ion source. Only PEEK tubing and connectors were used to avoid metal contamination. (38) Bondarenko, P. V.; Second, T. P.; Zabrouskov, V.; Makarov, A. A.; Zhang, Z. J. Am. Soc. Mass Spectrom. 2009, 20, 1415–1424. (39) Scigelova, M.; Makarov, A. Proteomics 2006, 1, 16–21. (40) Macek, B.; Waanders, L. F.; Olsen, J. V.; Mann, M. Mol. Cell. Proteomics 2006, 5, 949–958. (41) Wynne, C.; Fenselau, C.; Demirev, P. A.; Edwards, N. Anal. Chem. 2009, 81, 9633–9642. (42) Erales, J.; Gontero, B.; Whitelegge, J.; Halgand, F. Biochem. J. 2009, 419, 75–82. (43) Chassaigne, H.; ?obin?ski, R. J. Chromatogr., A 1998, 829, 127–136.
Figure 1. Schematic flowchart showing the type of information obtained in the different modes of the multimodal approach developed. The cell-line cytosol was separated by ultracentrifugation using a HimaCs 120GX model (Hitachi, Tokyo, Japan) and fractionated by size-exclusion chromatography using an Agilent 1100 HPLC system (Agilent, Wilmington, DE). The metal elution was monitored by splitting part of the effluent to an Agilent 7500ce ICP MS (Agilent, Tokyo, Japan). Procedures. The purpose of the different detection modes used in this study was schematically illustrated in Figure 1. µRP HPLC. The column was a C8 Vydac (250 mm ×1 mm i.d., 5 µm) presented in passivated 316 copper-free stainless steel housing (Alltech/Grace, Templemars, France). The injection volume was 5 µL. The elution solvents were eluent A: 5 mM ammonium acetate pH 6 and eluent B: 5 mM ammonium acetate (pH 6) in 50% (v/v) acetonitrile. Elution was carried out at 40 µL · min -1 using the following program: 0-50 min: 2-20% B; 50-52 min: 2% B; 52-60 min: 2% B. µRP HPLC-ICP MS. ICP MS was optimized daily for the maximum sensitivity by introducing directly a solution containing 1 µg · L -1 89 Y, 7 Li, 205 Tl and 140 Ce. Isotopes monitored in µRP HPLC-ICP MS were 114 Cd, 112 Cd 63 Cu, 65 Cu, 64 Zn, 66 Zn. The data were processed using Excel Microsoft software. µRP HPLC-ESI MS. Chromatographic separation conditions were identical as given above. For the postcolumn acidification experiments, the solution added was formic acid:methanol (30/ 70%, v/v) delivered at 4 µL · min -1 . Initial calibration of the mass spectrometer was performed using a mixture consisting of cafein (195.08765), MRFA peptide (524.26499) and Ultramark polymer (m/z 1221.99063). The ion source was operated in the positive ion mode at 3.2 kV. The vaporizer temperature of the source was set to 120 °C and the capillary temperature to 280 °C. Nitrogen sheath gas was set to 20, the auxiliary gas to 5 and sweep gas to 0 (arbitrary unit). The ion lenses were automatically optimized using a MT solution (1 µg · mL -1 in 0.01% formic acid in 50% (v/v) methanol) introduced by infusion at 4 µL · min -1 and monitored at m/z 1225.843 (z ) 5). In all experiments, the most abundant mass of [M + 5H] 5+ ion was systematically monitored for better detection limit. Throughout this manuscript, the most abundant mass is always given for multicharged ions or uncharged molecules. In full scan mode, an m/z 1200-1500 range was scanned for the detection of apo- and metalated MTs (the resolution was set at 100 000 (m/∆m, fwhm at m/z 400)). Injection time was 400 ms. In single ion monitoring (SIM) mode, the scan mode was centered on m/z 1230 ± 50 and m/z 1377.5 ± 65 for the detection of apo- and metalated rabbit liver MTs, respectively. Pig kidney apo-MTs were measured at m/z 1250 ± 60 and then at m/z 1170 ± 70 whereas the metalated forms at m/z 1367.5 ± 87. The resolution was set at 100 000 (m/∆m, fwhm at m/z 400) and injection time was 3 s. The mass accuracy was expressed in ppm and calculated as the ratio (M theoretical - Mdetermined)/Mtheoretical multiplied by 10 6 . µRP HPLC-ESI MS/MS. The [M+5H] 5+ of apo-MT was selected for top-down sequencing experiments. High energy collision fragmentation (HCD) was carried out at 50% energy, using an isolation width of 2 in full scan mode over m/z 100-2000 mass range at a resolution of 100 000. The acquisition time (60 min) was split into 6 time segments. Segment 1: 0-30.75 min, SIM scan mode (m/z 1100-1300) at 100 000 resolution (m/∆m, fwhm at m/z 400); segment 2: 30.75-36 min, HCD at m/z 1189.4: segment 3: 36-39 min, HCD at m/z 1197.63, and m/z 1194.63; segment 4: 39-44 min, HCD at m/z 1203.24; segment 5: 44-47 min, HCD at m/z 1271.96; segment 6: 47-60 min, SIM scan mode centered at m/z 1200 ± 100 at 100 000 resolution. Data were processed using Xcalibur 2.1 software (Thermo Fisher Scientific) and masses were manually corrected using the traces of peaks of the Ultramark polymer calibrant as internal standard. Analyst Q.S. 1.1 software (Applied Biosystems MDS Sciex, Foster City, CA) was used to obtain the theoretical y ions lists of the amino acids sequence of MT subisoforms. Cell Growth and CdS Nanoparticles Exposure. Kidney pig cell line (LLC-PK1) were grown in 100 mm cell culture Petri dish with EMEM (Eagle’s minimal essential medium) media containing 1% antibiotics (penicillin, streptomycin), 2 mM Lglutamin, 1 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES), non-essential amino acids, and 5% of fetal calf serum. Petri dishes were incubated at 37 °C ina5%CO2 incubator. At subconfluence, cells were exposed during 24 h with5mLof120µM solution of 10 nm-CdS nanoparticles. Cells from 11 Petri dishes were pooled. Cells not submitted to CdS were considered as control. Recovery of the MT Fraction. After cell growth, the supernatant was discarded and cells were rinsed twice with 1 mL phosphate buffered saline (PBS). A 1-mL aliquot of 25 mM Tris/HCl buffer Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6949
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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
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Figure 5. Device to eject and mix s
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Anal. Chem. 2010, 82, 6877-6886 Imm
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NaCl, phosphate buffer saline (PBS)
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Figure 2. Product ion mass spectra
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-70 °C resolved the problem, givin
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Table 1. Intraday Precision and Acc
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Anal. Chem. 2010, 82, 6887-6894 Fer
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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
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 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: Anal. Chem. 2010, 82, 6947-6957 Ide
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
Page 233 and 234: nucleotide tails. 3-5 Thus, the amo
Page 235 and 236: allow the use of higher aptamer con
Page 237 and 238: quent ligation of the aptamers afte
Page 239 and 240: Anal. Chem. 2010, 82, 6983-6990 Imp
Page 241 and 242: Figure 2. Configuration editor wind
Page 243 and 244: Figure 4. Dependencies between volu
Page 245 and 246: Since the time for liquid expulsion
Page 247 and 248: Anal. Chem. 2010, 82, 6991-6999 Int
Page 249 and 250: Figure 1. Representation of the Car
Page 251 and 252: Table 2. Capillary Electrophoresis
Page 253 and 254: 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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