Analytical Chemistry Chemical Cytometry Quantitates Superoxide

More documents

Recommendations

Info

Figure 2. LC-ESI-MS/MS traces of eicosanoids after workup from a solution in phosphate buffered saline: (A) early eluting eicosanoids and (B) latter eluting eicosanoids. the carboxyl group of lipid mediators to the AMPP amide using a well-known carbodiimide coupling reagent. Coupling conditions were optimized to give near quantitative formation of the AMPP amide. The was shown by converting the fluorescent fatty acid 10-pyrene-decanoic acid into its AMPP amide and examining the reaction mixture by fluorimetric HPLC and ESI-MS. No remaining 10-pyrenedecanoic acid was detected, and the AMPP amide was formed in >95% yield with
scanning of AMPP amides of the analytes are shown in the Supporting Information Supplementary Material Figure 6A-D. In all the spectra, some cleavage of the AMPP tag occurs giving rise to the peaks at m/z ) 169 and 183. To best ensure analytical specificity, we quantified the eicosanoids using a fragment in the analyte portion rather than in the AMPP tag. For PGD2, we used the m/z ) 307 fragment, which is likely due to cross-ring cleavage of the cyclopentanone ring and represents the most abundant non-tag fragment ion. For PGE2 and PGF2R, we used m/z ) 239 due to cleavage between C3 and C4. For AA and 6-keto-PGF1R, we used m/z ) 239, again due to C3-C4 cleavage. The major nontag fragment for TxB2 is m/z ) 337 due to cross-ring cleavage. For all HETE species, we could identify a unique fragment ion (see Table 1) to avoid cross contamination of MS/MS signals due to partially unresolved LC peaks. Finally, for LTB4, we chose m/z ) 323, which is the most abundant non-tag fragment ion but has a nonobvious origin. Next we studied a mixture of eicosanoids in phosphate buffered saline and optimized pre-ESI-MS/MS sample workup and LC-ESI- MS/MS conditions. Critical for a successful method with ultrasensitive analyte detection is high yield recovery of analytes from the sample prior to LC-ESI-MS/MS analysis. We found that solid phase extraction with a rapidly wettable matrix (Oasis HLB cartridges, Waters Inc. or Strata-X cartridges, Phenomenex) combined with analyte elution with methanol gave high yield recovery of all eicosanoids analyzed (Table 1). The data in Table 1 is based on 50 pg of each analyte submitted to recovery studies. When the same recovery studies were carried out with 5 pg of eicosanoid, results were essentially identical (data not shown). Inferior recoveries were obtained using elution of the solid phase cartridges with acidified methanol or if the sample was subjected to liquid-liquid extraction or protein precipitation using various solvents (Supporting Information). Figure 2A,B shows a typical LC-ESI-MS/MS run of an eicosanoid mixture. All of the analytes examined elute from the LC column in less than 11 min. All HETEs except 11(S)-HETE and 12(S)-HETE are baseline resolved. The partial overlap of the 11(S)- and 12(S)-HETEs is not a concern because completely selective fragment ions are being monitored. The LC elution profile and peak shape for both the derivatized and underivatized analytes (not shown) are similar. This shows that the presence of the quaternary ammonium group of the AMPP tag has no negative effect on the LC. TxB 2 gives rise to the typical broad peak shape due to interconversion between the two hemiacetal isomers. Also similar to underivatized analysis, the fragments used to monitor PGD2 and PGE2 (isobars of each other) are not completely unique to each species, but baseline LC resolution of these two eicosanoids resolves the issue. The same is true for the isobaric pair TxB2 and 6-keto-PGF1R. To ensure high yield conversion of eicosanoids to AMPP amides with minimal formation of N-acyl-ureas, we used a large excess of derivatization reagents. The AMPP tag and the EDCI coupling reagent elute in the void volume (not shown), and the HOBt elutes in the large time window between PGD2 and LTB4 (not shown). Thus removal of excess derivatization reagents is not necessary prior to LC-ESI-MS/MS. Standard curve analysis was performed starting below the limit of quantification up to 2 orders of magnitude above this limit. In Table 2. Coefficient of Variation (%) for LC-ESI-MS/MS Analysis of Eicosanoid AMPP Amides intrasample a intersample (10 µL PBS) b intersample (10 µL serum) c 6-keto-PGF1〈 4.9 16.9 not detected TxB2 2.4 3.0 3.7 PGF2 〈 8.1 6.5 6.4 PGE2 8.0 9.2 not detected PGD2 4.5 8.3 not detected LTB4 7.3 17.2 7.3 5(S)-HETE 3.3 2.8 6.4 8(S)-HETE 6.7 20.3 13.7 11(S)-HETE 5.5 12.2 8.8 12(S)-HETE 5.2 10.7 6.3 15(S)-HETE 4.0 5.8 12.4 arachidonic acid 12.7 28.2 7.9 a Coefficient of variation for the analysis of the same sample of 1.25 pg/µLofeicosanoidstandardsinjectedthreetimesontotheLC-ESI-MS/MS. b Coefficient of variation for the analysis of three independent samples of 1.25 pg/µL of eicosanoid standards (50 pg of standards spiked into phosphate buffered saline and worked up as described in the Experimental Methods section for LC-ESI-MS/MS.). c Coefficient of variation for three separate serum samples spiked with internal standards only. all cases, the mass spectrometry response was linear from the limit of quantification up to the highest amount analyzed (19 pg on-column for the Waters Quattro Micro and 5-10 pg on-column fortheWatersQuattroPremier)(SupplementalMaterialFigure2A,B in the Supporting Information). We define limit of quantification as the amount of eicosanoid AMPP amide needed to give an ESI- MS/MS signal that is 10-fold the noise (as determined using the Waters MassLynx software). The limits of quantification for all eicosanoid AMPP amides are listed in Table 1. The values using the Waters Quattro Micro are 5 pg for all eicosanoids except 12(S)- HETE (10 pg) and AA (10 pg). With the Waters Quattro Premier, the values are 0.2-0.5 pg for all eicosanoids except 12(S)-HETE (0.9 pg) and AA (1 pg). We also determined limit of quantification of underivatized eicosanoids analyzed by LC-ESI-MS/MS in negative ion mode. The values are shown in Table 1 for the Waters Quattro Micro and the Waters Quattro Premier. The new method using AMPP amides is found to be 10- to 20-fold more sensitive on both instruments. This constitutes a significant improvement in detection sensitivity of eicosanoids. To validate the above stated estimates of the limit of quantification, we injected eicosanoids standard mixtures six times on the Waters Quattro Premier, where each analyte was injected at roughly twice the estimated limit of quantification. Values of CV for analyte to internal standard peak ratios for each analyte are listed in Table 1. These CVs validate our estimates for the limits of quantification by demonstrating that eicosanoid analysis can be performed with reasonable reproducibility proximal to these stated values. Next, we more thoroughly investigated the reproducibility of the AMPP derivatization method for eicosanoid analysis. The results are summarized in Table 2. Three repetitive LC-ESI-MS/ MS analyses of an identical sample (19 pg on-column levels of each eicosanoid) gave coefficients of variation in the range of the 2.4-12.7% range. We also prepared three independent mixtures of eicosanoids (50 pg each) in phosphate buffered saline, submitted each to sample workup, and analyzed each by LC-ESI-MS/ <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010 6795
Page 1 and 2: Anal. Chem. 2010, 82, 6745-6750 Let
Page 3 and 4: purchased from Invitrogen-Molecular
Page 5 and 6: Figure 3. Electropherograms of TPP-
Page 7 and 8: Anal. Chem. 2010, 82, 6751-6755 Res
Page 9 and 10: (pH 8.0) with cysteine and cystamin
Page 11 and 12: Figure 4. Ion mobility mass spectra
Page 13 and 14: GOx glucose + O298 gluconic acid +
Page 15 and 16: coater at 2000 rpm for 20 s, and th
Page 17 and 18: Figure 5. Comparison of cyclic volt
Page 19 and 20: in channels with either no grooves
Page 21 and 22: indicators of atmospheric processin
Page 23 and 24: Figure 1. GC/MS total ion chromatog
Page 25 and 26: Table 2. Concentrations and Stable
Page 27 and 28: on a substrate are preferred. 20-24
Page 29 and 30: Figure 4. SERS analysis of NAADP co
Page 31 and 32: Anal. Chem. 2010, 82, 6775-6781 Hig
Page 33 and 34: tion, 2 µL of proprionaldehyde wer
Page 35 and 36: Figure 4. Analysis of 2a by HPLC-MS
Page 37 and 38: eaction of 1a with PBH can be condu
Page 39 and 40: Numerous references had demonstrate
Page 41 and 42: Figure 1. TEM images of the prepare
Page 43 and 44: Figure 4. Schematic representation
Page 45 and 46: esult in a big SPR signal change wi
Page 47 and 48: also reduces chemical noise, which
Page 49: Table 1. Extraction Yields, Liquid
Page 53 and 54: Anal. Chem. 2010, 82, 6797-6806 δ
Page 55 and 56: Figure 1. Schematic view of the pre
Page 57 and 58: from the specimen and enclosed in a
Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
Page 61 and 62: Table 3. Ambient Measurement Result
Page 63 and 64: Anal. Chem. 2010, 82, 6807-6813 Dir
Page 65 and 66: Polymerase (1 U per sample). Reacti
Page 67 and 68: of which was constant for all ampli
Page 69 and 70: analytically useful signals at less
Page 71 and 72: carbon black and RP-C18 for the ext
Page 73 and 74: solution in an equal volume, and 1
Page 75 and 76: Table 2. Concentrations and Ratios
Page 77 and 78: Anal. Chem. 2010, 82, 6821-6829 Mac
Page 79 and 80: mg/mL protein, followed by separati
Page 81 and 82: Figure 2. Productivity of SEQUST an
Page 83 and 84: Figure 4. High-resolution MS/MS spe
Page 85 and 86: Figure 6. Characterization of PSMs
Page 87 and 88: containing T-T mismatches. 23 Based
Page 89 and 90: Figure 1. Extinction spectra of sol
Page 91 and 92: Figure 3. (A) The value of Ex650 nm
Page 93 and 94: Figure 5. Extinction spectra and co
Page 95 and 96: wished to explore the dehydration o
Page 97 and 98: constant medium for separation; we
Page 99 and 100: Figure 2. Standards of (Pi)n, n ) 1
Page 101 and 102:
Figure 5. Quantitative calibration
Page 103 and 104:
Anal. Chem. 2010, 82, 6847-6853 Met
Page 105 and 106:
Figure 1. 226 Ra spectrum by liquid
Page 107 and 108:
Table 1. Counting Properties and De
Page 109 and 110:
Table 3. Analysis of 226 Ra in Sedi
Page 111 and 112:
mercial microarray scanner and fabr
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
Page 119 and 120:
corresponding compound if its chemi
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 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 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
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
Page 257 and 258:
Another approach to handle this lim
Page 259 and 260:
(principal components, PCs) in whic
Page 261 and 262:
Figure 5. Relevant score plots and
Page 263 and 264:
CONCLUSIONS In this paper we have i
Page 265 and 266:
electroactive-species loaded liposo
Page 267 and 268:
Figure 2. Qdot-based FLFTS response
Page 269 and 270:
Figure 6. Fluorescence imaging of Q
Page 271 and 272:
Anal. Chem. 2010, 82, 7015-7020 Sel
Page 273 and 274:
Table 1. Effect of 1 D-LC Condition
Page 275 and 276:
Figure 3. Peak-production rate vers
Page 277 and 278:
Anal. Chem. 2010, 82, 7021-7026 Cel
Page 279 and 280:
luciferase (T7 control vector) was
Page 281 and 282:
Figure 3. Inhibitory effects of luc
Page 283 and 284:
Anal. Chem. 2010, 82, 7027-7034 Pat
Page 285 and 286:
Electrochemistry. Electrochemical m
Page 287 and 288:
Figure 2. SEM images of Au substrat
Page 289 and 290:
Table 3. Film Thickness Measurement
Page 291 and 292:
Anal. Chem. 2010, 82, 7035-7043 Qua
Page 293 and 294:
Figure 1. (A) FL spectra of BSPOTPE
Page 295 and 296:
Figure 4. (A) Variation in the FL i
Page 297 and 298:
Figure 6. (A) FL spectra of BSPOTPE
Page 299 and 300:
Scheme 1. Proposed Mechanism for Fl
Page 301 and 302:
one or more drawbacks including poo
Page 303 and 304:
Figure 2. Free fluorophores do not
Page 305 and 306:
Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308:
Figure 2. Comparative analysis of d
show all

Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Create successful ePaper yourself

Delete template?

Save as template?