Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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(pH 7.2) containing 1 mM dithiothreitol (DTT) and 0.1 mM phenylmethyl-sulfonylfluoride (PMSF) was added. Cells were scrapping off and recovered into an eppendorf tube. Another 500 µL of buffer was added to recover the maximum number of cells. Cellular lysis was carried out by a successive freeze-thaw procedure by immersing the tube into liquid nitrogen for 3 min followed by 3 min in a water bath at 37 °C (all steps repeated three times). An aliquot of 50 µL was kept aside to quantify proteins amount by the Bradford protein assay. A 1.45 µL PMSF aliquot was added to the remaining 1.45 mL sample to reach a final concentration of 0.2 mM. Then, the sample was ultracentrifuged (4 °C, 120 000 g, 20 min). The recovered supernatant was heated at 95 °C for 5 min and ultracentrifuged (4 °C, 120 000 g, 20 min). A 100 µL aliquot of the supernatant was fractionated using a Superdex peptide HR 10/30 (GE Healthcare, Uppsala, Sweden) size exclusion column (10 × 300 mm × 5 µm) and isocratic elution at 0.7 mL · min -1 50 mM TRIS HCl pH 7.4. The SEC column was cleaned beforehand by flushing with mobile phase containing5mM�-mercaptoethanol and 2 mM EDTA in order to remove adsorbed metal ions. The main Cd-containing fraction (8.75 - 10.73 mL) was collected from nine injections, freezedried, resolubilized in 900 µL of water and desalted using a 5 mL HiTrap column (GE Healthcare, Uppsala, Sweden) by elution with NH3aq (pH 8.0) at 1.5 mL · min -1 . The Cd-containing fraction was heartcut, freeze-dried, and resolubilized in 25 µL prior to µHPLC analyses. No special precautions, for example, by Ar or N2 saturation during the sample preparation, were taken to reduce oxidation of MTs. RESULTS AND DISCUSSION ESI MS Analysis of Rabbit Liver MT-2 Standard. As poor detection limits are the principal drawback of the existing methods for MT detection, effort has been focus on the optimization of the signal-to-noise ratio. The choice of the scan mode between full scan and single ion monitoring (SIM) was first investigated. In the infusion conditions proper for the ionization of the Cd7- MT form (5 mM AcNH4, pH6in50%(v/v) MeOH) the S/N ratio of the most intense peak at m/z 1380.691 (δ 0.3 ppm, [M+H + ] 5+ ) was 10 times higher in the SIM mode (centered at m/z 1367.5 ± 85) than in the full scan (m/z 1325-1410) mode. Similar results were observed for the corresponding apo- MT signal (m/z 1225.848 (δ 0.3 ppm) in 0.01% FA in 50% (v/v) MeOH, pH 1.6) for which the S/N ratio was 7 times higher in the SIM mode (centered at m/z 1230 ± 13) than in the full scan mode (m/z 1215-1250). Note that in full scan the injection time was decreased from 3000 to 400 ms in order to limit the number of concomitant ions entering the ion trap but the MT peak S/N ratio was not affected. In order to evaluate the detection limits in the chromatographic mode taking into account the abundance of the individual isoforms, the purity of the MT standard available was investigated by µHPLC-ICP MS. The chromatogram (Figure 2a) obtained in the conditions of full stoichiometric metalation (seven Cd atoms) shows two major peaks ( 114 Cd). The contribution of Zn and Cu is insignificant (
difference, ppm compound Table 1. Identification of Rabbit Liver MT2 Subisoforms apo MTs complexed MTs form time, m/z, z ) 5, most abundant most abundant difference, amino acid m/z, z ) 5, most abundant most abundant N° min measured mass, measured mass, theoretical ppm composition compound compound measured mass, measured mass, theoretical 1 34.3 1230.049 6145.209 6145.212 0.5 Ac-M1D3P2N1C20S9A6T4G4K8E1Q1I1 N Ac-MT-2b 1384.894 6919.435 6919.427 -1.2 Cd7 N Ac-MT2b 1375.300 6871.464 6871.450 -2.0 Cd6Zn N Ac-MT2b 1375.100 6870.464 6870.452 -1.7 Cd6Cu N Ac-MT2b 1365.506 6822.494 6822.476 -2.6 Cd5ZnCu N Ac-MT2b 1365.906 6824.494 6824.474 -2.9 Cd5Zn2 N Ac-MT2b 1365.306 6821.494 6821.478 -2.3 Cd5Cu2 N Ac-MT2b 2 36.45 1217.4455 6082.1911 6082.1909 -0.04 M1D3P3N2C20S8A9T3G4K7Q1I1 MT-2a 1372.290 6856.414 6856.406 -1.1 Cd 7 -MT2a 1362.697 6808.449 6808.429 -2.9 Cd6Zn MT2a 3 36.7 1223.446 6112.194 6112.201 1.2 M1D3P3N2C20S8A8T4G4K7Q1I1 MT-2c / / / / / 4 38.5 1243.862 6214.274 6214.281 1.2 Ac-M1D3P2N1C20S9A7T3G3K8E1Q1I1R1 N Ac-MT-2d 1398.707 6988.499 6988.486 -1.8 Cd7 N Ac-MT2d 1389.113 6940.529 6940.520 -1.2 Cd6Zn N Ac-MT2d 5 38.9 1231.849 6154.209 6154.212 0.5 Ac-M1D3P3N2C20S8A8T4G4K7Q1I1 N Ac-MT-2c 1386.694 6928.434 6928.427 -1.0 Cd7 N Ac-MT2c 1376.900 6879.464 6879.452 -1.7 Cd6Cu N Ac-MT2c 1377.101 6880.469 6880.451 -2.6 Cd6Zn N Ac-MT2c 6 39.3 1225.848 6124.204 6124.201 -0.4 Ac-M1D3P3N2C20S8A9T3G4K7Q1I1 N Ac-MT-2a 1380.693 6898.429 6898.416 -1.8 Cd 7 N Ac-MT2a 1371.299 6851.459 6851.440 -2.7 Cd6Zn N Ac-MT2a 1361.305 6801.489 6801.466 -3.3 Cd5ZnCu N Ac-MT2a 1361.705 6803.489 6803.464 -3.6 Cd5Zn2 N Ac-MT2a 1361.104 6800.484 6800.467 -2.4 Cd5Cu2 N Ac-MT2a 7 42.3 1249.072 6240.324 6240.333 1.5 Ac-M 1D 3P 2N 1C 20S 8A 7T 3G 3K 8Q 1I 1E 1R 1L 1 N Ac-MT-2e 1403.917 7014.549 7014.548 -0.1 Cd 7 N Ac-MT2e <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010 6951
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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
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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 and 204: Anal. Chem. 2010, 82, 6947-6957 Ide
Page 205: Figure 1. Schematic flowchart showi
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
Page 255 and 256: 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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