Based on the calibration function (established for each binding experiment), the concentrations of NO711 resulting from the corresponding binding samples were calculated using Analyst v.1.6.3 (Sciex, Darmstadt, Germany) as described previously (Zepperitz et al., 2006 (link)). Remaining bound reporter ligand was calculated as the percentage of the specifically bound reporter ligand in presence of a sublibrary compared to specifically bound reporter ligand in the absence of a sublibrary. All binding experiments were performed in triplicates and results are given as means ± SD. For hit identification, all chromatographic parameters and normalized peak areas (area sublibrary component/area internal standard) of each individual sublibrary component in the corresponding MRM chromatograms of samples representing total and non-specific binding were calculated via Analyst v.1.6.3 (Sciex, Darmstadt, Germany). Consequently, all library components showing significant specific binding as difference of the normalized areas of total and non-specific binding (one site student t-test, n = 3, CL = 97.5%) were classified as hits.
Analyst v1
Manufactured by AB Sciex
Sourced in United States, Canada, Germany
Analyst v1.6.0 software is a data acquisition and processing application for mass spectrometry instruments. It provides tools for controlling the instrument, acquiring data, and analyzing the acquired data. The software supports a range of mass spectrometry techniques and can be used with various AB Sciex instrument models.
Automatically generated - may contain errors
Lab products found in correlation
4000 qtrap system, by AB Sciex (13 mentions)
Chemoview 2, by AB Sciex (12 mentions)
Isotope labeled internal standard samples, by Cambridge Isotopes (3 mentions)
Qtrap 4500 mass spectrometer, by AB Sciex (2 mentions)
Kinetex c18 column, by Phenomenex (2 mentions)
Multiquant v2, by AB Sciex (1 mentions)
Prominence chromatographic system, by Shimadzu (1 mentions)
Triple tof 5600 system, by AB Sciex (1 mentions)
Nanospray 3 source, by AB Sciex (1 mentions)
Simca p v12, by Sartorius (1 mentions)
Standard samples of the amino acids, by Chromsystems (1 mentions)
4000 qtrap, by AB Sciex (1 mentions)
Millipore multiscreen hv 96 well plate, by Merck Group (1 mentions)
Twin tec 96, by Eppendorf (1 mentions)
Lipidview software, by AB Sciex (1 mentions)
Qtrap 5500, by AB Sciex (1 mentions)
Triversa nanomate, by Advion (1 mentions)
Peaks 7, by Bioinformatics Solutions (1 mentions)
Agilent 1200 hplc, by Agilent Technologies (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
29 protocols using analyst v1
1
Quantitative Ligand Binding Analysis
2
Lipidomic Analysis by LC/ESI-MS/MS
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Lipids were separated on a reverse-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) platform using a Prominence chromatographic system (Shimadzu Corporation, OR, USA). Instrument control and data processing were done with Analyst v1.6 and Multi-Quant v2.1 software (AB Sciex, MA, USA). The separation was achieved on a Zorbax C18, 1.8 μm, 50 × 2.1 mm column (Agilent Technologies, Mississauga, ON). The flow rate was set to 300 μL/min using a linear gradient of mobile phase A and mobile phase B. Mobile phase A and B consisted of tetrahydrofuran, methanol, and water in the ratio 20:20:60 (v/v/v) and 75:20:5 (v/v/v), respectively. Both A and B contained 10 mM ammonium formate. The elution program was as follows: start with 0% solvent B; increase to 100% B at 8.00 min; maintained at 100% B for 2.5 min; and back to 0% B over 0.5 min. The column was finally re-equilibrated to the starting conditions (0% mobile phase B) for 3 min before the next sample injection. Diacylglycerol (DG) and triacylglycerol (TG) species were separated in an isocratic fashion (100 μL/min) by employing 85% mobile phase B over 8 min. The analytical column and autosampler were maintained at 50°C and 25°C, respectively, during the analysis. The injection volume was 5 μL.
3
Analytical Method Validation Protocol
4
Quantitative Proteomics by TripleTOF MS
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The spectrometric data were acquired using a TripleTOF 5600 System (SCIEX, Framingham, MA, USA) fit to a Nano-Spray III source (SCIEX, Framingham, MA, USA) and a pulled quartz tip-type emitter (New Objectives, Woburn, MA, USA), which was controlled with the franchise software Analyst v1.6 (AB-SCIEX, Concord, ON, Canada). The MS data procurements were undertaken as per the following conditions: the ion spray voltage was set to 2300 V, the curtain gas was set to 30, the nebulizer gas was set to 15, and the interface heater temperature was 150 °C. High sensitivity mode was used for the whole data acquisition process. The MS1 accumulation time was set to 250 ms, while 350–1500 Da was the allowed mass range. At least 30 product ion scans were collected based on the MS1 survey intensity, exceeding a threshold of 120 counts/s and a 2+ to 5+ charge-state. A value of ½ peak width was set for the dynamic exclusion. The collision energy was adjusted to all precursor ions for the collision-induced dissociation for the iTRAQ data acquisition, and the Q2 transmission window for 100 Da was at 100%. Three independent biological replicates were included for each sample in the experiment.
5
Comprehensive Amino Acid Profiling by LC-MS
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Details of the metabolomics assessment method were published previously (28 (link)). Briefly, 8 h of fasting blood sample was collected at admission. A total of 23 amino acids, i.e., alanine (Ala), asparagine (Asn), leucine (Leu), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), valine (Val), arginine (Arg), glycine (Gly), proline (Pro), threonine (Thr), citrulline (Cit), glutamine (Gln), histidine (His), lysine (Lys), methionine (Met), serine (Ser), ornithine (Orn), glutamate (Glu), aspartate (Asp), piperamide (Pip), cysteine (Cys), and homocysteine (Hcy), were detected via LC-MS. AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA) was used to conduct direct injection MS metabolomic analysis. Analyst v1.6.0 software (AB Sciex) was used for data collection. ChemoView 2.0.2 (AB Sciex) was used for data preprocessing. Isotope-labeled internal standard samples were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Standard samples of the amino acids were purchased from Chrom Systems (Grafelfing, Germany).
6
Direct Injection MS Metabolomic Analysis
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The direct injection MS metabolomic analysis was conducted by using an AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA). The equipped ion source was an electrospray ionization source. A 20 μl sample was injected for each run. The mobile phase was 80% acetonitrile aqueous solution. The initial flow rate was 0.2 ml/min. Afterward, the flow rate was set to 0.01 ml/min within 0.08 min, kept constant until 90 s, returned to 0.2 ml/min within 0.01 min, and held constant for another 30 s. The ion spray voltage was 4.5 kV. The curtain gas pressure was set at 20 psi. A 35 psi pressure was applied to the ion source gas 1 and gas 2. The auxiliary gas temperature was maintained at 350 °C. Analyst v1.6.0 software (AB Sciex) was used for system control and data collection. ChemoView 2.0.2 (AB Sciex) was used for data preprocessing. Partial least squares discriminant analysis (PLS-DA) was performed by using SIMCA-P v12.0 (Umetrics, Umeå, Sweden).
7
Amino acid profiling by LC-MS
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Details of the amino acids measured were published previously.19 (link) Briefly, liquid chromatography-mass spectrometry metabolomic analysis was conducted by using an AB Sciex 4000 QTrap system (AB Sciex, Framingham, Massachusetts, USA). Eight-hour fasting blood was taken and stored as dried blood spot. Each dried sample was dissolved in 100 µL fresh mobile phase solution. The mobile phase was 80% acetonitrile aqueous solution. Amino acid quality control standards were provided by Chromsystems (Grafelfing, Germany). The standards were mixed and dissolved in 2 mL pure methanol and stored at 4°C. Working solution was prepared through 100-fold dilution for metabolite extraction. Analyst V.1.6.0 software (AB Sciex) was used for system control and data collection. ChemoView V.2.0.2 (AB Sciex) was used for data preprocessing.
8
Metabolomics Assessment of Amino Acids
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Details of the metabolomics assessment method were published previously (22 (link)). Briefly, 8 h of fasting blood sample was collected at admission. A total of 23 amino acids, i.e., alanine (Ala), asparagine (Asn), leucine (Leu), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), valine (Val), arginine (Arg), glycine (Gly), proline (Pro), threonine (Thr), citrulline (Cit), glutamine (Gln), histidine (His), lysine (Lys), methionine (Met), serine (Ser), ornithine (Orn), glutamate (Glu), aspartate (Asp), piperamide (Pip), cysteine (Cys), Homocysteine (Hcy) were detected via LC-MS. AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA) was used to conduct direct injection MS metabolomic analysis. Analyst v1.6.0 software (AB Sciex) was used for data collection. ChemoView 2.0.2 (AB Sciex) was used for data preprocessing. Isotope-labeled internal standard samples were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Standard samples of the amino acids were purchased from Chrom Systems (Grafelfing, Germany).
9
Comprehensive Metabolomic Profiling of Amino Acids
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Details of the metabolomics assessment method have already been published [19 (link)]. Briefly, 8 h of fasting blood sample was collected at admission. A total of 22 amino acids were detected via LC-MS, i.e., asparagine (Asn), alanine (Ala), arginine (Arg), citrulline (Cit), Leu, lysine (Lys), Trp, Tyr, Thr, Val, glycine (Gly), proline (Pro), Phe, glutamine (Gln), His, methionine (Met), serine (Ser), ornithine (Orn), glutamate (Glu), aspartate (Asp), piperamide (Pip), and cysteine (Cys). AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA) was used to conduct direct injection MS metabolomic analysis. Analyst v1.6.0 software (AB Sciex) was used for data collection. ChemoView 2.0.2 (AB Sciex) was used for data preprocessing. Isotope-labeled internal standard samples were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Standard samples of the amino acids were purchased from Chrom Systems (Grafelfing, Germany).
10
Metabolomic Profiling of Amino Acids and Acylcarnitines
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Details of metabolomic approach for AA and AcylCN were published previously [18 (link)]. Briefly, dry blood spot (DBS) samples of patients were collected after 8 hours of fasting. The sample preparation process involved punching wells from DBS paper, adding working and quality control (QC) solutions, derivatizing and drying, and finally, dissolving the dried sample in fresh mobile phase solution. An AB Sciex 4000 QTrap system (AB Sciex, Framingham, MA, USA) was used to carry out the mass spectrometry metabolomic analysis. The ion source was electrospray ionization source. Analyst v1.6.0 software (AB Sciex) was used for system control and data collection. Isotope-labeled internal standards of AA (NSK-A) and AcylCN (NSK-B) from Cambridge Isotope Laboratories (Tewksbury, MA, USA) were used for preparing working solutions. AAs and carnitine QC standards were provided by Chromsystems (Grafelfing, Germany). Acetonitrile (high-performance liquid chromatography grade) was obtained from Thermo Fisher (Waltham, MA, USA).
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