Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 1. Chemical structures of MK-2662 and its internal standard, [ 13 C18, 15 N2] MK-2662 (ISTD). The underlined region indicates the N-terminal tryptic peptide used as surrogate for quantification. is time-consuming; therefore, reagent availability could become a rate-limiting step. In the case of MK-2662 analysis, ELISA was abandoned because a drug-specific antibody was not available at the time of assay development. Bioassay is a cell-based or enzyme-based assay that measures the biologic activity of a specific biological process. 7 Since bioassay measures the total activity of the sample irrespective of the chemical structures, the assay specificity is usually less than that of ELISA. It could provide a measure of pharmacodynamic (PD) properties 7 but may not be appropriate for pharmacokinetic (PK) assessment. Liquid chromatography-mass spectrometry (LC-MS) has been increasingly used to quantify peptides and proteins in biological matrixes 9-11 because of its selectivity. Despite commonly used sample cleanup techniques, such as protein precipitation (PPT), 9 solid-phase extraction (SPE), 12-14 two-dimensional (2D) SPE, 15 and 2D high-performance liquid chromatography (HPLC), 16,17 the immunoaffinity purification (IAP) coupled with LC-MS/MS represents an emerging strategy for peptide and protein bioanalysis. The IAP strategies include immunoaffinity depletion that can be used to remove abundant proteins from biological matrixes 18,19 and immunoaffinity capture that utilizes a single antibody to isolate and enrich the target peptides or (9) van den Broek, I.; Sparidans, R. W.; Schellens, J. H. M.; Beijnen, J. H. J. Chromatogr., B 2008, 872, 1–22. (10) Careri, M.; Mangia, A. J. Chromatogr., A 2003, 1000, 609–635. (11) John, H.; Walden, M.; Schafer, S.; Genz, S.; Forssanmm, W. G. Anal. Bioanal.Chem. 2004, 378, 883–897. (12) van den Broek, I.; Sparidans, R. W.; Huitema, A. D. R.; Schellens, J. H. M.; Beijnen, J. H. J. Chromatogr., B 2006, 837, 49–58. (13) van den Broek, I.; Sparidans, R. W.; Huitema, A. D. R.; Schellens, J. H. M.; Beijnen, J. H. J. Chromatogr., B 2007, 854, 245–259. (14) Heudi, O.; Barteau, S.; Zimmer, D.; Schmidt, J.; Bill, K.; Lehmann, N.; Bauer, C.; Kretz, O. Anal. Chem. 2008, 80, 4200–4207. (15) Yang, Z.; Hayes, M.; Fang, X.; Daley, M. P.; Ettenberg, S.; Francis, L. S. T. Anal. Chem. 2007, 79, 9294–9301. (16) Motoyama, A.; Xu, T.; Ruse, C. I.; Wohlschlegel, J. A.; Yates, J. R. Anal. Chem. 2007, 79, 3623–3634. (17) Linke, T.; Ross, A. C.; Harrison, E. H. J. Chromatogr A. 2006, 1123, 160– 169. 6878 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 proteins from biological samples. Although many applications using online immunoaffinity columns have been reported, 20-24 offline affinity purification using different carriers, such as macroporous polymeric beads, agarose or sepharose beads, 25,26 and magnetic beads, 27-29 etc., for immobilization of a variety of enzyme and/or capture antibodies enables more flexibility in terms of selection of carriers, antibodies, assay formats, and experimental conditions as compared to the online approach. This report presents a sensitive and reproducible bioanalytical approach for quantification of a PEGylated peptide in plasma. To support human clinical studies for MK-2662, a 2D HPLC-MS/ MS method using a turbo ion spray (TIS) interface, monitoring the surrogate N-terminal peptide (HAibDGTFTSDYSK) of MK- 2662 after tryptic digestion, has been developed and validated to quantify MK-2662 in human plasma. A stable-isotope-labeled internal standard, [ 13 C18, 15 N2] MK-2662, was employed. Two sample preparation methods, PPT versus IAP using an anti- PEG antibody, followed by trypsin digestion in a 96-well format using 0.2 mL plasma sample, have been evaluated and applied to clinical sample analysis. The potential caveats of the protein precipitation approach and the benefit of immunoaffinity purification in regard to assay specificity are discussed. To our best knowledge, this is the first report describing use of anti- PEG antibody to capture a PEGylated peptide in combination with LC-MS/MS analysis. This approach is potentially applicable to analysis of other PEGylated peptides or proteins. EXPERIMENTAL SECTION Chemicals and Reagents. MK-2662 and stable-isotope-labeled internal standard (ISTD, Figure 1) were synthesized at the Merck Research Laboratories, Merck & Co. (Rahway, NJ). HPLC grade acetonitrile, HPLC grade methanol, laboratory grade formic acid (88%), certified ammonium formate, and certified ammonium bicarbonate were obtained from Fisher Scientific (Pittsburgh, PA). Human control plasma (K2EDTA as anticoagulant) was purchased from Biological Specialty Co. (Colmar, PA). Water was purified by a Milli-Q ultrapure water system from Millipore (Bedford, MA). Bovine serum albumin (BSA) at 22% in 0.85% (18) Hagman, C.; Ricke, D.; Ewert, S.; Bek, S.; Falchetto, R.; Bitsch, F. Anal. Chem. 2008, 80, 1290–1296. (19) Dekker, L. J.; Bosman, J.; Burgers, P. C.; van Rijswijk, A.; Freije., R.; Luider, T.; Bischoff, R. J. Chromatogr., B 2007, 847, 65–69. (20) Radabaugh, M. R.; Nemirovskiy, O. V.; Misko, T. P.; Aggarwal, P.; Rodney Mathews, W. Anal. Biochem. 2008, 380, 68–76. (21) Li, W. W.; Nemirovskiy, O.; Fountain, S.; Rodney Mathews, W.; Szekely- Klepser, G. Anal. Biochem. 2007, 369, 41–53. (22) Berna, M.; Schmalz, C.; Duffin, K.; Mitchell, P.; Chambers, M.; Ackermann, B. Anal. Biochem. 2006, 356, 235–243. (23) Zhang, X.; Martens, D.; Kramer, P. M.; Kettrup, A. A.; Liang, X. J. Chromatogr., A 2006, 1133, 112–118. (24) Edinboro, L. E.; Karnes, H. T. J. Chromatogr., A 2005, 1083, 127–132. (25) Huang, L.; Harvie, G.; Feitelson, J. S.; Gramatikoff, K.; Herold, D. A.; Allen, D. L.; Amunngama, R.; Hagler, R. A.; Pisano, M. R.; Zhang, W. W.; Fang, X. Proteomics 2005, 5, 3314–3328. (26) Hoofnagle., A. N.; Becker, J. O.; Wener, M. H.; Heinecke, J. W. Clin. Chem. 2008, 54, 1796–1804. (27) Lee, Y. C.; Block, G.; Chen, H.; Folch-Puy, E.; Foronjy, R.; Jalili, R.; Jendresen, C. B.; Kimura, M.; Kraft, E.; Lindemose, S.; Lu, J.; McLain, T.; Nutt, L.; Ramon-Garcia, S.; Smith, J.; Spivak, A.; Wang, M. L.; Zanic, M.; Lin, S. H. Protein Expression Purif. 2008, 62, 223–229. (28) Dubois, M.; Becher, F.; Herbet, A.; Ezan, E. Rapid Commun. Mass Spectrom. 2007, 21, 352–358. (29) Berna, M. J.; Zhen, Y.; Watson, D. E.; Hale, J. E.; Ackermann, B. L. Anal. Chem. 2007, 79, 4199–4205.
NaCl, phosphate buffer saline (PBS), phosphate buffer saline with 0.05% Tween-20 pH 7.4 (PBST), and Triton-X 100 were purchased from Sigma-Aldrich (Milwaukee, WI). Linco DPP- IV inhibitor was purchased from Millipore Corporation (St. Charles, MO). BD P700 blood collection tubes that contain proprietary DPP-IV inhibitors were purchased from BD Bioscience (Franklin Lakes, NJ). Lyophilized sequence grade modified trypsin (TRSEQZ at 1 mg/vial) was purchased from Worthington Biochemical Corporation (Lakewood, NJ). Dynabeads M-280-streptavidin was purchased from Invitrogen Corporation (Carlsbad, CA). Biotinylated anti-PEG rabbit monoclonal antibody, anti-PEG-biotin (or PEG-B-47-bio), was purchased from Epitomics, Inc. (Burlingame, CA). Instrumentation. Dynal MPC-L and MPC-96 (Invitrogen Corporation, Carlsbad, CA) were used to capture magnetic beads in the vial and 96-well plate, respectively. A TomTec Quadra 96 workstation, model 320 (TomTec Corporation, Hamden, CT) was used to perform automated liquid transfer for protein precipitation. A MaxQ mini 4450 benchtop incubating orbital shaker (Thermo Scientific, Waltham, MA) and an SPE-Dry 96 micropate sample concentrator (Jones Chromatography, Lakewood, CO) were used for sample preparation. A Cohesive Aria 2300 system (Cohesive Technologies Inc., Franklin, MA), which included two quaternary Flux pumps, a valve module, and a CTC HTS autosampler, was used for 2D HPLC. A Sciex API 5000 triple-quadrupole mass spectrometer with a Sciex turbo ion spray interface (Sciex, Toronto, Canada) was used as a detector. The data were collected and processed through Analyst 1.4 software. Preparation of Standards and Quality Control (QC) Samples. MK-2662 stock solutions at about 40 µM were prepared from two separate weighings and dissolved in acetonitrile/water (30/70, v/v). One set of analyte stock solutions was used to prepare calibration standards, and the other set was used to make QC samples. Working standards, containing MK-2662 at the different concentration levels, were prepared by serial dilutions of analyte stock solution with 1% BSA, aliquoted to 1.5 mL polypropylene microcentrifuge tubes and stored at -70 °C. A 500 nM ISTD working solution was obtained by dilution of an ∼40 µM ISTD stock solution in 1% BSA. Plasma calibration standards were prepared daily by adding 40 µL of working standard and 40 µL of 500 nM IS into 200 µL of control plasma to provide final concentrations of MK-2662 ranging from 1.0 to 1000 nM for the PPT process and 2-200 nM for the IAP process. The plasma QC samples were prepared in human control plasma that contained Linco DPP-IV inhibitor (20 µL inhibitor solution per milliliter of plasma) at 3, 50, 800, and 10 000 nM (for testing dilution integrity) MK-2662 for the PPT assay and 3, 6, 50, and 150 nM for IAP. All QC aliquots were stored in a -70 °C freezer. Sample Preparation: Protein Precipitation Followed by Trypsin Digestion. Clinical samples and QCs were thawed at room temperature, mixed, and centrifuged at 4000 rpm (∼1300 g RCF), at 10 °C for 10 min. A 200 µL aliquot of sample was then mixed well with 40 µL of 1% BSA (to match the volume of standards), 40 µL of 500 nM ISTD, and 500 µL of 0.1% formic acid in acetonitrile/methanol (90/10, v/v) ina2mL96-well deepwell plate. After centrifuging at 2000 RCF for 5 min, 600 µL of supernatant was transferred into a clean 1.2 mL 96-well plate using a TomTec automated liquid handling system and dried on an SPE Dry-96 under a stream of nitrogen at 40 °C. After resuspending the residue in with 140 µL of 50 mM ammonium bicarbonate (pH 8.0) containing 67 µg/mL trypsin, the samples were incubated at 37 °C for3hinaMaxQmini shaker to allow trypsin digestion, and then the reaction was quenched by adding 10 µL of 44% formic acid to each well at the end of incubation. After mixing and centrifugation, 20 µL of sample was injected into the LC/LC-MS/ MS for analysis. Sample Preparation: Immunoaffinity Capture Using Anti- PEG Antibody Followed by On-Bead Trypsin Digestion. DynaBeads M280-steptavidin (magnetic beads) were washed with PBST (pH 7.4) three times and resuspended into the same volume of PBST. Biotinylated anti-PEG rabbit monoclonal antibody, anti- PEG-biotin (or PEG-B-47-bio), was added to the bead suspension to reach the concentration of 20 µg/mL and then gently shaken at room temperature for 1htoallow binding of biotinylated antibody to the steptavidin-coated bead. The beads with antibody were washed and resuspended with PBST three times. A 50 µL final suspension was added into a sample mix that contained 200 µL of plasma sample, 40 µL of 1% BSA (to match the volume for calibration standards), and 40 µL of 500 nM ISTD in a 96-well plate. The sample mix was shaken at 700 rpm at 30 °C for2hto allow antibody capture of MK-2662 from the plasma sample. After washing the beads with PBS (pH 7.4) that contained 0.1% Triton-X 100 and then with ammonium bicarbonate (pH 8), an aliquot of 140 µL of67µg/mL trypsin in 50 mM ammonium bicarbonate (pH 8) was added into each well, incubated at 37 °C with shaking for about 3 h. The supernatant was transferred into a 96-well plate with a 600 µL insert in each well, where 10 µL of 44% formic acid was added to quench the reaction. After mixing and centrifugation, 20 µL of sample was injected on LC/LC-MS/MS for analysis. Two-Dimensional HPLC Conditions. The 2D HPLC was conducted with three basic steps: loading, eluting, and separating, using a dual-column quick-elution mode on a Cohesive Aria system. A cation-exchange column, Thermo BioBasic SCX (50 mm × 2.1 mm, 5 µm), was used as the first dimension with a salt gradient of mobile phases A and B [A, 50 mM ammonium formate (pH 3) in 10% acetonitrile; B, 200 mM ammonium formate (pH 3) in 10% acetonitrile]. A reversed-phase column, Thermo-Hypersil Gold PFP (50 mm × 2.1 mm, 5 µm), was used as the second dimension with an organic solvent gradient of mobile phases C and D [C, 10 mM ammonium formate in 0.1% formic acid; D, 10 mM ammonium formate in 90% acetonitrile with 0.1% formic acid]. The LC method on the Cohesive system is listed in the Supporting Information Table S1. The compartment of the autosampler was set at 5 °C, and the reversed-phase column was controlled at 40 °C. Mass Spectrometry Detection and Calculation. A PE Sciex API 5000 triple-quadrupole mass spectrometer (MS/MS) with a turbo ion spray interface ionization source operated in a positive ion mode was used to quantitate MK-2662. The ion pairs (precursor ion f product ion) m/z 672.1 f 223.1 for MK-2662 and m/z 676.9 f 223.1 for ISTD were selected for multiple reaction monitoring (MRM). The instrument setting was adjusted to maximize the response for the analyte and IS, respectively. The turbo gas temperature was 650 °C. The flow settings of nebulizing gas (nitrogen), collision gas (nitrogen), and curtain gas (nitrogen) Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6879
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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
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
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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: Anal. Chem. 2010, 82, 6877-6886 Imm
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
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
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trode 28 by a finite element using
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Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
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Figure 2. Normalized product ion ab
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Figure 4. EID (a) and IRMPD (b) of
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Anal. Chem. 2010, 82, 6947-6957 Ide
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Figure 1. Schematic flowchart showi
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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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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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