ichemistry SOLUTIONS Integrated chemistries to boost mass spectrometry workflows

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1 ichemistry SOLUTIONS Integrated chemistries to boost mass spectrometry workflows ichemistry SOLUTIONS

2 ichemistry solutions ichemistry solutions are the world s only reagents and consumables that are customdesigned to boost the performance of your Extend and improve mass spectrometry quantitation All analytical workflows have their limits. ichemistry solutions are designed to boost the performance of workflows beyond conventional limits and enable new applications through advanced chemistries. mass spectrometer and improve sensitivity, productivity, and data precision. But success needs more than just a reagent. Combined with the AB SCIEX mass spectrometry systems and the resources of the ichemistry solutions designed to improve specific applications: Biomarkers and Omics global AB SCIEX service and support network, ichemistry solutions enable you to push the Clinical and Forensic limits of mass spectrometry by improving the quality and performance of your workflows. MS Qualification Food and Environmental

3 ichemistry for MS qualification solutions ichemistry solutions for biomarker and omics applications Optimize before you analyze Save time, save money, stay compliant and be confident that your mass spectrometer is performing at its best. ichemistry MS Performance Calibrants and Standards ensure your AB SCIEX mass spectrometer is tuned for optimal performance and delivers reproducible results. Avoid risking the integrity of your workflow Save time and resources Troubleshoot your mass spec in minutes Make sure you have the standards you need to qualify your instrument for IQ/OQ/PQ any day, any time ichemistry solutions for Biomarkers and Omics applications enable you to: Reduce sample complexity Analyze multiple samples simultaneously Provide a global internal standard for peptide quantitation Directly detect and quantify proteins and their isoforms Reduce sample complexity Cleavable ICAT reagents: selective fractionation of cysteinecontaining proteins reduces sample complexity Cleavable ICAT reagents are cysteine-specific, affinity-enabled mass tags that provide a powerful mass spectrometry-based method for separating and quantitatively analyzing protein expression differences from complex samples. With cleavable ICAT reagents, you can readily identify and quantify important classes of proteins such as membrane and low-abundance proteins that are often difficult to analyze by traditional methods. Cleavable ICAT reagents and workflows are easily integrated with AB SCIEX instrumentation and application-specific software for rapid, unattended quantitation and identification of biologically significant proteins. Achieve cysteine-specific protein labeling with precise MS quantitation Identify membrane and low-abundance proteins Integrate workflows into analytical platforms and software 1 Sample 1 (light) combine and proteolyze Quantify (MS) light heavy labeled cysteines multi-dimensional separation mass analysis m/z NH 2 -EACDPLR-COOH Sample 2 (heavy) Identify (MS/MS) m/z Relative quantitation workflow: Two samples are reduced and labeled in parallel, each with two chemically identical reagents, but differentiated in mass by 9 Da, then combined and digested. The labeled cysteine peptides are isolated via biotin-avidin interaction. The cleaved peptides are analyzed in MS mode to obtain quantitative information and then subjected to MS/MS for identification.

4 Analyze multiple samples simultaneously A global internal standard for peptide quantitation itraq reagents: the industry standard cited in over 84 peer-reviewed papers The ability to simultaneously analyze multiple samples readily enables important research into dosing, temporal investigations, biomarker elucidation, and absolute quantitation of target proteins. itraq reagents are a multiplexed set of four or eight isobaric By combining itraq reagents with AB SCIEX instrumentation and application-specific software such as ProteinPilot software you ll achieve the meaningful, highly accurate results for confident quantitative protein expression analysis. Peptide quantitation mtraq reagents To get quantitation results you can trust it is always better to use an internal standard. But synthesizing internal standards can take up to 6 weeks, and not all of them will be appropriate for MRM analysis. The mass differential mtraq Reagents are specifically designed for peptide quantitation using MRM and are significantly more economical when larger numbers of peptides need to be monitored. (same mass), amine-specific tags that yield labeled peptides that are easily quantitated using mass spectrometry. Achieve identification and quantitation of multiple samples simultaneously Available as duplex and triplex to radically increase sample throughput The simple peptide-based itraq workflow eliminates many of the restrictions and difficulties presented by other strategies, allowing protein coverage that includes post-translational modifications, affinity pull-downs, and membrane proteins. Maximize protein/proteome coverage Increase statistical confidence and relevance quantitate up to eight samples at a time High selectivity, MRM-based relative quantitation of every protein, peptide, and PTM with a single workflow Up to 1x more cost effective than synthetic internal standards S1 S5 S2 S6 S3 S7 S4 S8 Parallel Denature & Digest Mix MS MS/MS 8 samples identical m/z Global Internal Standard Parallel Denature & Digest itraq Reagents Quantitation Identification Mix 1:1: % Intensity Pool aliquot for Global Internal Standard (GIS) 8 MRM mtraq reagent 8 mtraq reagent mtraq reagent 8 mtraq reagent Mass (m/z) Addresses mass space limitation allows for multiplexing Multiplexed workflow: Up to 8 samples are reduced and alkylated, digested, and labeled in parallel, each with reagents that are chemically identical and isobaric. The peptide pool from all the samples is combined and subjected to LC/MS/MS analysis. Each precursor peak in MS mode represents up to 8 samples. Both quantitation and identification is obtained in MS/MS mode. = protein reactive group. All biological samples are prepared in parallel and digested. An aliquot of each sample is collected and reacted with mtraq reagent 8 to form the Global Internal Standard (GIS). This 8 GIS solution is then mixed with the and 4 labelled samples at a 1:1:1 ratio, and the MRM transitions are monitored for all 3 versions of the peptides.

5 Direct detection of proteins and their isoforms ichemistry Solutions for food and environmental applications Cytochrome P45 protein assay Immunoassays can be expensive and often lack specificity for proper protein and protein isoform detection. LC/MS/MS is an ideal alternative, allowing direct detection of CYP45 protein The ichemistry CYP45 Protein Assay enables direct measurement of protein expression changes of 4 individual CYP45 isoforms (1A2, 2B6, 3A4, and 3A5) or 7 individual isoforms (1A2, 2B6, Accelerate contaminant analysis Simplified preparation of contaminant standards with idquant standards kits So you can verify and validate the performance of your method faster, improve efficiency, and speed up sample turnaround time. isoforms with high specificity and sensitivity. 2C9, 2C19, 2E1, 3A4, and 3A5) with high specificity, sensitivity, and accuracy for induction studies: New idquant standards kits for contaminant analysis streamline the preparation of contaminant standard solutions, providing Kits contain up to 2 chemical contaminant residues that are commonly found or are of high concern to regulatory agencies Directly measure CYP45 induction at the protein level Eliminate the need for expensive antibodies Get results that are equivalent to traditional mrna or activity assays the ultimate solutions for residue analysis and delivering faster results. The idquant standards kits contain certified reference materials (ISO Guide 34, ISO/IEC 1725 and ISO 91:28) and eliminate the need to source individual chemical contaminants or reagents and to measure each one manually. Solutions are provided as multiple mixes of 1-2 compounds per mix Concentrations are certified for internal calibration or as an external calibration reference material Stability conditions are provided to maintain confidence in reagent integrity Relative Fold CHange Protein Expression A2 2B6 3A4 3A Short and Simple Workflow Amenable to 1s of Samples per Day Starting sample of microsomes or S9 cells Reduce and alkylate sample for 1 hour at 6ºC Digest sample with Trypsin for 4 hours at 37ºC Add isotope-labeled peptides to the digested solution Perform LC-MS/MS analysis Routine workflow WITHOUT idquant Standards Kit 9 steps to analysis 1. Source neat materials of individual compounds 2. Order neat materials of individual compounds 3. File and document Certificates of Analysis 4. Prepare stock solutions of each compound Routine workflow WITH idquant Standards Kit 4 steps to analysis 1. Order IDQuant Standards Kit 2. File and document Certificate of Analysis 3. Prepare working mixes and calibration standards 4. File and document calibration standard preparation records (-) Control 3-MC Treatment PB RIF Protein-level measurement Specificity Sensitivity Quantitation of many isoforms 5. File and document stock solution preparation records 6. Prepare intermediate mixes of compounds Total time to analysis: < 2 Days Control (.1%DMSO) Rif 1 µm PB 1 µm 3MC 2 µm Control (.1%DMSO) Rif 1 µm PB 1 µm 3MC 2µM Control (.1%DMSO) 3MC 2µM PB 1 µm Rif 1 µm mrna techniques Activity studies Western blots 7. File and document intermediate mix solution preparation records 8. Prepare working mixes and calibration standards 7x time savings! CYP 2B6 CYP 3A4 CYP 1A2 CYP45 MRM assays 9. File and document calibration standard preparation records Enzyme activity and expression: A strong correlation between enzyme activity, mrna expression, and protein expression measured by LC-MRM were observed following treatment of human hepatocytes with prototypical CYP inducers. Both the baseline and inducible levels of the CYP isoforms were easily detected by the LC-MRM protein quantitation method. The method requires no antibodies and avoids the drawbacks of Western Blotting, which is semi-quantitative at best. Total time to analysis: > 2 weeks * Total time to analysis includes estimate of time required for delivery of individual materials from suppliers.

6 ichemistry solutions for clinical research applications Easy compound derivatization for up to 1x boost in intensity ichemistry solutions for amino acid analysis Faster performance. Better accuracy. Pushing the limits in sensitivity Mass spec is a great tool for compound detection and quantitation, but sensitivity can sometimes be negatively impacted by matrix suppression, high background noise levels, poor ionization efficiency, or poor compound fragmentation. Amplifex Reagents are the only derivatizing agents intelligently designed, synthesized, and tested to boost ionization efficiency and improve fragmentation and chromatographic properties. Amplifex Reagents take the complexity out of compound derivatization with a simple (one-step), fast (3 minute, reproducible, and robust) derivatization procedure. Just mix the sample, incubate, and you re ready for analysis. Amplifex reagents are mass tags designed to react universally with a specific functional group. For example: Amplifex -Diene Reagent can be used to derivatize any molecule with a cis-diene system, including compounds such as Vitamin D3 and Vitamin D2 analogs, among others. The result is a signal enhancement of up to 1-fold. Amplifex -Keto Reagent is universal and reacts with keto or aldehyde compounds, including ketosteroids such as Testosterone, among others. Signal enhancements improve mass limits of detection by up to 5-fold. Derivatization is no longer a painful day-long process. The most diverse of the ichemistry solutions, atraq reagent is the ideal solution to quantitate amino acids in samples ranging from protein hydrolysates for omics applications, food and consumer products, and physiological fluids for clinical research and forensic toxicology samples. atraq reagents save time and money with faster, more complete analysis. With atraq, you can quantitate up to 45 amino acids and amines from a wide variety of samples, including physiological fluids, protein hydrolysates, food and beverages, and culture media more than 3 times faster than conventional amino acid analyzers. Get confident identification, superior accuracy, and precision with internal standards for every amino acid Achieve higher throughput with an analysis time of 18 minutes per sample Add custom amines, amino acids and small peptides, and create corresponding internal standards Amplifex Diene Signal Enhancement Amplifex Keto Signal Enhancement 1,25-Dihydroxyvitamin D3 2.3e5 2.2e5 2.e5 1.8e5 1.6e5 1.4e5 1.2e5 1.e5 8.e4 x165 4 pg DHVD3 1.6e4 1.e Cholesten-3-one 1 pg 4-Cholesten-3-one x58 Amino acid sample atraq NHS NH PSer NH ASn MRM AA MW 113 AA MW 121 atraq - sample atraq - standard 6.e4 4.e4 2.e4 4 pg DHVD pg 4-Cholesten-3-one Internal standards NH Ala NH Pro NH Leu PSer ASn Pro Ala... Leu 3.2e6 3.e6 2.5e6 2.e6 1.5e6 1.e6 5.e5 Ergosterol x ng Ergosterol Cannot see compound without Amplifex 2.6 ng Ergosteral Testosterone 1 pg Testosterone x45 1 pg Testosterone min Amino acid analysis using atraq reagents and an LC/MS/MS workflow: Samples for amino acid analysis are combined with atraq reagent, an internal standard consisting of a panel of isotopically-labelled amino acids of known concentration. The sample mixture is analyzed by LC/MS/MS in MRM mode, and the concentration of amino acids in the unknown sample can be directly determined by the peak ratio of the unknown sample relative to the internal standard (with correction for the amino acid concentration in the internal standard). 18 min

7 You invest in our technology. We invest in your success. As the world leader in mass spectrometry, AB SCIEX creates solutions that are backed by the industry s most extensive service and support organization. As part of AB SCIEX, SCIEX ichemistry solutions receive that same level of support. With a network of service professionals, experienced compliance specialists, and over 15 PhD application scientists worldwide, we are dedicated to supporting your technical needs and helping you get the most out of your mass spectrometry systems. Our customer support network is available to provide expert assistance in the use and application of SCIEX ichemistry solutions through a comprehensive range of services, including application support, technical service, and training. Whether you access our service and support team by phone, or on-site visits, you can be confident that our organization will be there for you. For more information, visit For Research Use Only. Not for use in diagnostic procedures. 212 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. ICAT is a registered trademark of the University of Washington and is exclusively licensed to AB Sciex Pte. Ltd /212 Headquarters 5 Old Connecticut Path Framingham, MA 171 USA Phone International Sales For our office locations please call the division headquarters or refer to our website at