Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 1. ESI-MS spectrum showing the selective reaction of ebselen with cysteine in the presence of methionine, tryptophan, and valine. The insets show the agreement of the simulated product peak isotopic distribution (top) with the recorded reaction product ion of m/z 397 (bottom). some cases) at room temperature. On the basis of these revealed features of the reaction, associated analytical applications were explored, such as the selective identification of thiol-containing peptides from mixtures and measurement of the number of cysteine residues of proteins. In addition, the dissociation behaviors of the derivatized protein/peptide ions were also examined. EXPERIMENTAL SECTION Electrospray ionization (ESI) of samples was performed using a Thermo Finnigan LCQ DECA Mass Spectrometer (San Jose, CA) or a hybrid triple-quadrupole-linear ion trap mass spectrometer (Q-trap 2000; Applied Biosystems/MDS SCIEX, Concord, Canada). The sample injection flow rate was 10 µL/min. A high voltage of +5 kV was applied to the spray probe for the positive ion mode. For the Thermo Finnigan LCQ DECA mass spectrometer, the optimized heated transfer capillary tube temperature was 150 °C. Collision-induced dissociation (CID) was used for further structural confirmation of the product ions. Data acquisition was performed using Xcalibur (rev. 2.0.7, Thermo Scientific, San Jose, CA). For the Q-trap 2000 mass spectrometer, the mass spectrometer curtain gas (N 2) was kept as 20 (manufacturer’s units) and the declustering potential was set at 10 V. Collision induced dissociation (CID) was carried out to provide ion structural information using enhanced product ion scan mode. Precursor ion scanning (PIS) was used to select reaction products by monitoring the characteristic fragment ions, and N2 was used as the collision gas. Data acquisition was performed using the Analyst software (version 1.4.2, Applied Biosystems/MDS SCIEX, Concord, Canada). Deconvolution of mass spectra was carried out using Mag-Tran 1.03b2 software (Amgen Inc., Thousand Oaks, CA) written based on the ZScore algorithm. 40 High-Resolution MS. High-resolution LTQ-Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) with a (40) Zhang, Z.; Marshall, A. J. Am. Soc. Mass Spectrom. 1998, 9, 225–233. 6928 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 modified nanoelectrospray ionization (nano-ESI) was used for collecting high-resolution data. The system was operated in the positive ion mode with a resolving power of 60 000 at m/z 400. The Molecular Weight Calculator (http://ncrr.pnl.gov/software/) was used to simulate the isotope distribution for thiol-derivatized products. Chemicals. The 20 amino acids, angiotensin II human (FW 1046 Da), bradykinin acetate (FW 1060 Da), MRFA (Met-Arg- Phe-Ala) acetate salt (FW 523 Da), �-lactoglobulin A from bovine milk, tris(2-carboxyethyl)phos-phine hydrochloride (TCEP), DLdithiothreitol (DTT), TPCK-treated trypsin from bovine pancreas (MW ∼23.8 KDa), ammonium bicarbonate, 1,4-benzoquinone, and N-(phenylseleno)phthalimide were purchased from Sigma-Aldrich (St. Louis, MO). Glutathione (GSH) (reduced form, MW 307 Da) and ebselen were obtained from TCI America (Tokyo, Japan) and Calbiochem (Cincinnati, OH), respectively. HPLC-grade methanol and acetonitrile from GFS Chemicals (Columbus, OH) and Sigma- Aldrich (St. Louis, MO) were used, and acetic acid was purchased from Fisher Scientific (Pittsburgh, PA). The deionized water used for sample preparation was obtained using a Nanopure Diamond Barnstead purification system (Barnstead International, Dubuque, IA). Protein Reduction. A volume of 175 µL of 0.1 mM �-lactoglobulin A in methanol/water (1:1 by volume) containing 2% acetic acid and 17.5 µL of 50 mM TCEP in 20 mM ammonium bicarbonate aqueous solution were mixed resulting in the molar ratio of 1:50 (protein/TCEP). The protein was reduced by TCEP for 3.5 h at room temperature. Then Millipore-ZipTip Pipette Tips were used to remove TCEP and ammonium bicarbonate via desalting. RESULTS AND DISCUSSION Reaction Selectivity. In this experiment, the selectivity of the derivatization reaction was examined first using amino acids as
Figure 2. (a) ESI-MS spectrum showing the reaction of GSH with ebselen; (b) CID MS 2 spectrum of ebselen derivatized GSH product ion (m/z 583); (c) ESI-MS spectrum showing the reaction of GSH with reagent 2; (d) CID MS 2 spectrum of the product ion of GSH derivatized by reagent 2 (m/z 464); (e) ESI-MS spectrum showing the selective reaction of ebselen with GSH in the presence of angiotensin II, bradykinin, and MRFA; (f) precursor ion scanning based on the monitoring of the characteristic fragment ion of m/z 276 showing the selective detection of GSH. reaction substrates and using ebselen as a reagent. We found that ebselen reacts exclusively with side chains of cysteine residues that contain a free thiol. As illustrated in Figure 1, the ESI-MS spectrum displays the reaction of ebselen (10 µM) with cysteine (5 µM) in the presence of methionine, tryptophan, and valine (5 µM for each) in methanol/water (1:1 by volume) containing 0.5% acetic acid. A dominant peak at m/z 397 corresponds to the reaction product between ebselen and cysteine. The inset shows the zoom-in area for the reaction product ion C 16H17O3N2SSe (m/z 397) (bottom) in comparison with the calculated product isotope distribution (top). Exact match is observed, confirming the peak assignment. The characteristic isotope distribution helps to identify products of the derivatization reaction. Upon CID, the ion at m/z 397 fragments into m/z 379, 353, and 276 by losses of one water molecule, one carbon dioxide, and one cysteine, respectively (data not shown), confirming the product ion structure and the covalent nature of the newly formed bond. In addition, peaks at m/z 118, 122, 150, 205, and 276 are seen in the Figure 1, corresponding to the protonated valine, cysteine, methionine, tryptophan, and ebselen, respectively. We also tested the reaction of ebselen with cysteine in the presence of 19 other natural amino acids (Figure 1-S in the Supporting Information). Again, only the reaction product of ebselen and cysteine (m/z 397) were observed, demonstrating that ebselen has exclusive specificity toward cysteine derivatization. Similarly, it was also found that another selenium reagent 2 has similar selectivity for cysteine in the presence of other amino acids. Furthermore, in our observation, the derivatization reaction takes place rapidly and Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6929
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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
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 and 182: the NB signal in a much better foot
Page 183: Scheme 1. Reactions of Selenium Rea
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
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
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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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