Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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were 50, 6, and 30 L/min. The optimized declustering potential (DP), collision energy (CE), collision cell exit potential (CXP), and entrance potential (EP) were 140, 36, 15, and 10 V. The dwell time was 100 ms for MK-2662 and ISTD. Both Q1 and Q3 quadrupoles were set at unit resolution. The total run time for each injection was 4.5 min, while the MS data acquisition window was started at 100 s after injection and kept open for 50 s. Peak area ratios were calculated using Analyst software version 1.4. A calibration curve was obtained by weighed (1/x 2 ) least-squares linear regression of the peak area ratio of the analyte to the IS versus the nominal concentration (x) of analyte. Method Validation. The selectivity of the assay was confirmed by processing control plasma from six different lots. Intraday precision and accuracy were determined by analyzing six sets of standard curve samples, each prepared in a different lot of control plasma. Assay accuracy was calculated from a least-squares regression curve constructed using all six replicate values at each concentration, and the intraday precision (% CV) was calculated from the peak area ratio of MK-2662 versus ISTD for each concentration used to construct the standard curve. QC samples were analyzed after first freezing and thawing, and the measured concentrations were considered as the initial values. Freeze-thaw stability was evaluated using QC samples that went through three cycles of freezing and thawing, with at least one day of storage at -70 °C between each thawing. Benchtop QC stability was tested following 5hatroom temperature and comparing the measured concentrations with their initial values. The stability of processed samples in the autosampler was assessed by comparing the results of QC samples analyzed at the end of a 14 h run with those analyzed at the beginning of the run. The reinjection stability was assessed by freezing the processed sample plate at -70 °C and reinjected after 8 days of storage. In order to examine the dilution integrity, five replicates of 10-fold of upper limit of quantification (ULOQ) were diluted by 20-fold with the corresponding control matrix during sample preparation and analyzed on LC/LC-MS/ MS. RESULTS AND DISCUSSION Mass Spectrometry Detection of Surrogate Peptide of MK- 2662. Tandem mass spectrometry (MS/MS) has been increasingly used for peptide quantification in biological matrixes because of its specificity. However, in the case of MK-2662, the PEGylated peptide exhibited a complex mass spectrum obtained using turbo ion spray (TIS), due to the large molecular weight (40 kDa) and oligomeric dispersity of the conjugated PEG polymer. DePEGylation of MK-2662 could be achieved by base hydrolysis and/or enzymatic hydrolysis. For MK-2662, base hydrolysis liberated the peptide via cleavage of the linker moiety. However, the MRM sensitivity of the dePEGylated peptide was low due to weak collision-induced dissociation (CID) of highly charged precursor ions. Additionally, base hydrolysis could add a risk of chemical degradation such as deamidation or hydrolysis to the peptide. Because of these limitations of base hydrolysis, enzymatic hydrolysis was selected to remove the PEG portion from MK-2662 prior to MS/MS analysis. The N-terminus of MK-2662 is required for activity, and more importantly, it contains the non-native amino acid (Aib) substitution at the second amino acid in the peptide sequence, providing mass selectivity against possible low levels of endogenous oxyn- 6880 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 tomodulin. Digestion of MK-2662 with trypsin produces a 12 amino acid N-terminal peptide (N 1-12-mer), HAibDGTFTSDYSK, and it was selected as a surrogate for quantification of MK-2662. In comparison to the other tryptic cleavage sites in the MK- 2662 sequence, the single cleavage reaction at the 12th amino acid (to generate N1-12-mer) demonstrated more rapid and reproducible digestion. An internal standard was synthesized by preparing a stable-isotope-labeled MK-2662 (PEGylated) that contained 13 C/ 15 N-labeled phenylalanine at the sixth amino acid in the sequence. The use of a chemically matched IS resulted in improved assay precision by compensating for variation during the sample preparation and detection process. Precursor ions for the surrogate N1-12-mers of MK-2662 and its IS were monitored as the doubly charged molecular weight (MW) ions, [M + 2H] 2+ ,atm/z 672.1 (MW ) 1342) and 676.9 (MW ) 1352), respectively. After optimizing CID, CE, and DP values, the product ion spectra of the N1-12-mers were collected, showing singly charged b- and y-ions (Figure 2). The b2 ion at 223 m/z was selected based on its ion intensity and specificity, where a non-natural amino acid (Aib) in b2 could allow MS differentiation of MK-2662 from the endogenous OXM fragments. Therefore, the MRM at m/z 672 f 223 (MK-2662) and m/z 677 f 223 (ISTD) was used for quantification of MK-2662. Two-Dimensional Chromatographic Conditions. One major challenge for supporting clinical studies was the assay sensitivity requirement. Our initial effort on developing a traditional onedimensional (1D) reversed-phase HPLC-MS/MS assay allowed us to successfully achieve a lower limit of quantification (LLOQ) of 5 nM using protein precipitation followed by tryptic digestion (Figure 3A). Since the clinical assay required a LLOQ of 1 nM, addition assay development efforts were focused on reducing background and increasing signal-to-noise ratio. An attempt to use SPE to extract full length of MK-2662 from plasma was not successful (very low recovery), mostly likely due to the fact that the large PEG portion blocked or interfered with the interaction of the peptide with the SPE sorbent. Other approaches, such as optimizing the sample recovery and digestions conditions or evaluating different CID transitions, were unsuccessful toward obtaining an LLOQ below 5 nM, due to high background signal in the LC-MS/MS chromatogram. Multidimensional protein identification technology (MudPIT) 30-33 has been widely used in the proteomics area. Applying the same concept, a 2D LC-MS/MS was developed to separate a surrogate peptide of MK-2662 (after trypsin digestion) from the background using two different separating chromatographic mechanisms. The 2D HPLC was performed on a Cohesive Flux 2300 under quick elution mode with built-in switching valves. A strong cation-exchange column, BioBasic SCX (50 mm × 2.1 mm, 5 µm), was used as the first dimension. An ∼60 s salt gradient from 50 to 200 mM ammonium formate was used at a flow rate of 0.5 mL/mL to maintain a net positive charge on the N-terminal tryptic peptide and separate N1-12-mer from other charged components in the digest mix. A small percentage of organic (30) Lohrig, K.; Wolters, D. Methods Mol. Biol. 2009, 564, 143–153. (31) Florens, L.; Washburn, M. P. Methods Mol. Biol. 2006, 328, 159–175. (32) Cagney, G.; Park, S.; Chung, C.; Tong, B.; O’Dushlaine, C.; Shields, D. S.; Emili, A. J. Proteome Res. 2005, 4, 1757–1767. (33) Krapfenbauer, K.; Fountoulakis, M. Methods Mol. Biol. 2009, 566, 165– 180.
Figure 2. Product ion mass spectra of tryptic peptides (A) HAibDGTFTSDYSK from MK-2662, [M + 2H] 2+ , and (B) HAibDGTF[ 13 C9, 15 N]TSDYSK from [ 13 C18, 15 N2] MK-2662 (ISTD), [M + 2H] 2+ . solvent (10% acetonitrile, v/v) was necessary to maintain good chromatographic peak shape and high recovery from the SCX column. Around the retention time of the N1-12-mer on the SCX column, a narrow peak window (about 0.3-0.4 min) was transferred from the SCX column to a reversed-phase (RP) columnsthe second dimensionsfollowed by continued organic gradient mobile phase to further separate the surrogate peptide from background noise, and therefore, to provide a clean chromatogram (Figure 3B). During method development, several reversed-phase columns were tested for the second dimension, and a Thermo Scientific Hyperil Gold, PFP column (50 mm × 2.1 mm, 5 µm) was found to provide the best retention. The analyte focusing was achieved by introducing 10 mM ammonium formate (pH 3) into 20% acetonitrile as a mobile phase. The chromatographic conditions, including mobile phase selection, gradient, and the timing for column switching (SCX to RP and switching back), were optimized so as to provide a good separation within a reasonable run time. Figure S1 (see the Supporting Information) is a schematic of the column and switching valve arrangement for online 2D LC, and Table S1 (see the Supporting Information) details the chromatographic conditions including the gradients, flow rates, and event timing. A RP column heater was set at 40 °C to produce a symmetric and well-resolved chromatographic peak. An SCX guard column was used to protect the SCX column which resulted in over 2000 injections per set of SCX/RP columns. The acquisition window on the MS was set for 50 s so that the MS ion source was kept clean and well-maintained. The overall run time was 4.5 min per sample. Pretreatment of Clinical Plasma Samples Using DPP-IV Inhibitors as Stabilizer. Clinical samples were collected in blood collection tubes (BD-P700) containing proprietary DPP-IV inhibitors, however the composition and quantity of the enzyme inhibitor was unknown. Since BD-supplied inhibitor can only be obtained by purchasing tubes from the vendor, it presented a practicality Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6881
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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
Page 85 and 86: Figure 6. Characterization of PSMs
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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
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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
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Page 133 and 134: Anal. Chem. 2010, 82, 6877-6886 Imm
Page 135: NaCl, phosphate buffer saline (PBS)
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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
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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
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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
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Page 177 and 178: the ×10 objective, to have a large
Page 179 and 180: Figure 2. With a suitable removal o
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Page 183 and 184: Scheme 1. Reactions of Selenium Rea
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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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Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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