AbsoluteIDQ Kit: Targeted Metabolomics Identification and Quantification

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AbsoluteIDQ Kit: Targeted Metabolomics Identification and Quantification Accurate, Reproducible, Metabolomics Studies The word metabolome refers to the complete set of small molecule metabolites arising from specific cellular processes that are on-going in biological cells, tissues, organs or organisms. Studying the metabolome can lead to an instantaneous snapshot into the physiology of the cell or organism. While the metabolome is dynamic and complex, there are a large number of metabolites that are already identified and understood as intermediates or end products of key biological pathways. This fact makes a targeted approach to metabolomics an excellent strategy for many areas of research. Targeted assays typically enable higher throughput, higher quality quantitation and easier down-stream data processing. Additionally, biological answers can often be obtained in a more rapid, seamless manner AB SCIEX Triple Quad and QTRAP systems are ideal instruments for targeted metabolomics workflows using Multiple Reaction Monitoring (MRM). With the unique LINAC Collision Cell, large numbers of analytes can be analyzed across a broad dynamic range with high accuracy and precision. Features of AbsoluteIDQ Kits for Targeted Metabolomics Accurately identifies and quantifies over 160 metabolites in only a few minutes per sample (Table 2) Covers four classes of metabolites (Table 1) 1.Amino acids 2.Acylcarnitines 3.Glycerophospho- and Sphingolipids 4.Hexose Requires very small sample volumes (10 µl) Includes software to control sample management, data collection, data validation, and analysis AbsoluteIDQ Kit Contents Figure 1. MetVal Module in the MetIQ Software. Evaluation of analyte concentrations in human plasma is shown. AbsoluteIDQ kit plate Silicone mat cover for kit plate Reagent buffer vials BIOCRATES solvent I 96 deep-well plate with attached filter plate Standards and quality control (QC) samples Kit user manual Analytical specifications MetIQ Software AB SCIEX MS acquisition methods p 1

1. Register the Assay in MetLIMS Import and register samples, generate 96-well plate overview and.csv file for Analyst Software 2. Assay Preparation Perform sample preparation in the laboratory 3. Process Assay in the Mass Spectrometer Analyze extracts in the MS instrument 4. Convert Mass Spectrometer Data Analyst.wiff files are converted and the concentrations are automatically calculated 5. Validate the Kit Plate Automated quality assessment of Standards, Quality Controls and Internal Standards 6. Evaluate and Export Data The results are evaluated and can be exported into other programs New module! 7. Data Analysis Box plots, Scatter plots Univariate tests (p-values) ROC curves Figure 2. AbsoluteIDQ Kit Workflow. The kit provides the whole workflow, from sample preparation, through data acquisition to data processing and reporting. The complete workflow for the AbsoluteIDQ kit is shown in Figure 2. The assay preparation is very simple. Samples are added to the upper filter spot on the specially adapted 96-well kit plate. Internal standards are incorporated into each well of this plate. Phenylisothiocyanate (PITC) is then added to each well to derivatize any amino acids. The plate is then dried and the metabolites are extracted with the organic extraction solvent. After centrifugation, the lower deep well capture plate containing the metabolites is analyzed by LC/MS/MS. Only about 60 minutes of hands-on preparation time is required for each 96- well microtitre plate. Each sample is then analyzed by a 3 minute flow injection analysis (FIA) in both positive and negative ion mode enabling a fast run time for the entire plate. Combined with the AB SCIEX Triple Quad or QTRAP system, the AbsoluteIDQ kits enables high throughput, quanitative, targeted metabolomics experiments to be performed routinely in any laboratory setting. Figure 3. Unique Design of the AbsoluteIDQ Kit Plate. These plates are designed so that all sample preparation is performed directly within the microtitre plate. This simplifies and streamlines the workflow, making the assay amenable to either manual or automated sample preparation. p 2

Table 1. Overview of Metabolite Classes. A number of major classes of metabolites are covered in the AbsoluteIDQ Kit. Details of analyzed metabolites in each class are listed in Table 2. MetIQ Software Included with each kit, BIOCRATES software converts complex metabolomics data into powerful clinical knowledge. The MetIQ Software package is an integral part of the AbsoluteIDQ solution and provides an integrated solution for the complete targeted metabolomics analytical process. Four separate modules, MetLIMS, MetConc, MetVal and MetStat handle separate steps in the process, from project setup to data conversion, to calculation of metabolite concentrations. MetLIMS laboratory management system to define projects, import sample information, administer standards and quality controls, print barcodes. MetConc automatically reads Analyst Software data files and calculates metabolite concentrations. MetVal validates datasets by analyzing internal standards, blanks, quality controls, and full plate results. MetStat finalizes results for the project by combining unknowns and validation results. Multiple data display and export options are available. Conclusions BIOCRATES and AB SCIEX have teamed up to deliver a metabolomic assay preparation kit with a simplified workflow that produces a robust assay and reliable data. The AbsoluteIDQ Kit, sold and supported exclusively by BIOCRATES Life Sciences AG, is designed for use on the AB SCIEX API 4000, 4000 QTRAP, API 5000, AB SCIEX Triple Quad 5500, and QTRAP 5500 systems. The AbsoluteIDQ Kit, combined with AB SCIEX instrumentation delivers the deepest and broadest metabolomic information available from a single targeted assay, enabling you to accelerate your research while simultaneously reducing your cost of performing targeted, quantitative metabolomics. p 3

Table 2. List of Analyzed Metabolites. Aminoacids Glycerophospholipids 42 Arg-PTC Arginine 57 lysopc a C6:0 103 PC aa C40:6 43 Gln-PTC Glutamine 58 lysopc a C14:0 104 PC aa C42:0 44 Gly-PTC Glycine 59 lysopc a C16:0 105 PC aa C42:1 45 His-PTC Histidine 60 lysopc a C16:1 106 PC aa C42:2 46 Met-PTC Methionine 61 lysopc a C17:0 107 PC aa C42:4 47 Orn-PTC Ornithine 62 lysopc a C18:0 108 PC aa C42:5 48 Phe-PTC Phenylalanine 63 lysopc a C18:1 109 PC aa C42:6 49 Pro-PTC Proline 64 lysopc a C18:2 110 PC ae C30:0 50 Ser-PTC Serine 65 lysopc a C20:3 111 PC ae C30:1 51 Thr-PTC Threonine 66 lysopc a C20:4 112 PC ae C30:2 52 Trp-PTC Tryptophan 67 lysopc a C24:0 113 PC ae C32:1 53 Tyr-PTC Tyrosine 68 lysopc a C26:0 114 PC ae C32:2 54 Val-PTC Valine 69 lysopc a C26:1 115 PC ae C34:0 55 xleu-ptc xleucine 70 lysopc a C28:0 116 PC ae C34:1 Hexoses 71 lysopc a C28:1 117 PC ae C34:2 56 H1 Hexose 72 PC aa C24:0 118 PC ae C34:3 73 PC aa C26:0 119 PC ae C36:0 Sphingolipids 74 PC aa C28:1 120 PC ae C36:1 149 SM (OH) C14:1 75 PC aa C30:0 121 PC ae C36:2 150 SM C16:0 76 PC aa C30:2 122 PC ae C36:3 151 SM C16:1 77 PC aa C32:0 123 PC ae C36:4 152 SM (OH) C16:1 78 PC aa C32:1 124 PC ae C36:5 153 SM C18:0 79 PC aa C32:2 125 PC ae C38:0 154 SM C18:1 80 PC aa C32:3 126 PC ae C38:1 155 SM C20:2 81 PC aa C34:1 127 PC ae C38:2 156 SM C22:3 82 PC aa C34:2 128 PC ae C38:3 157 SM (OH) C22:1 83 PC aa C34:3 129 PC ae C38:4 158 SM (OH) C22:2 84 PC aa C34:4 130 PC ae C38:5 159 SM C24:0 85 PC aa C36:0 131 PC ae C38:6 160 SM C24:1 86 PC aa C36:1 132 PC ae C40:0 161 SM (OH) C24:1 87 PC aa C36:2 133 PC ae C40:1 162 SM C26:0 88 PC aa C36:3 134 PC ae C40:2 163 SM C26:1 89 PC aa C36:4 135 PC ae C40:3 90 PC aa C36:5 136 PC ae C40:4 91 PC aa C36:6 137 PC ae C40:5 92 PC aa C38:0 138 PC ae C40:6 93 PC aa C38:1 139 PC ae C42:0 94 PC aa C38:3 140 PC ae C42:1 95 PC aa C38:4 141 PC ae C42:2 96 PC aa C38:5 142 PC ae C42:3 97 PC aa C38:6 143 PC ae C42:4 98 PC aa C40:1 144 PC ae C42:5 99 PC aa C40:2 145 PC ae C44:3 100 PC aa C40:3 146 PC ae C44:4 101 PC aa C40:4 147 PC ae C44:5 102 PC aa C40:5 148 PC ae C44:6 p 4

Table 2. List of Analyzed Metabolites, continued. Availability The AbsoluteIDQ Metabolomics kits are sold and supported by BIOCRATES Life Sciences AG. To purchase kits or for more details about the kit workflow, contact BIOCRATES at sales@biocrates.com. The AB SCIEX mass spectrometers are sold and supported by AB SCIEX. Please contact your local AB SCIEX sales representative or visit www.absciex.com for more information. For Research Use Only. Not for use in diagnostic procedures. 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. Publication number: 0430310-01 p 5

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