Agilent Metabolomics Laboratory: The breadth of tools you need for successful metabolomics research

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1 AT A GLANCE Metabolomics is a logical complement to large-scale expression profiling and proteomics studies, offering valuable insight into the biochemistry of organisms. But metabolomics also presents significant analytical challenges. Agilent Technologies can help you meet those challenges with the knowledge, instruments, and data analysis tools you need for success in metabolomics. Agilent Metabolomics Laboratory: The breadth of tools you need for successful metabolomics research

2 A powerful approach to understanding biological systems Metabolites play vital roles in biological systems. Metabolomics, the comparative analysis of metabolites found in sets of similar biological samples, can identify potential biomarkers, identify effects of drugs or diseases on known biological pathways, or detect effects of drugs or diseases in unexpected biological pathways. Metabolites as vital components in biological systems Metabolites play vital roles in biological systems. They transport energy, facilitate cellular communication and control, and function as building blocks for other processes. Changes in metabolites provide valuable insight into underlying biochemistry. They can serve as markers for disease, and point to correlations between genes and function. Metabolomics provides a useful avenue to the understanding of biological systems. It can be a powerful complement to genomic and proteomic analyses. Metabolomics an analytical challenge Metabolites exhibit vast chemical diversity, with tremendous variation in structures, functional groups, and physicochemical properties. This, and their very wide abundance range, makes metabolomic analyses very challenging. To further complicate the task, analytical requirements vary, depending on whether you are finding compounds, identifying them, quantifying them or all of the above. No single instrument or technology is suitable for all metabolomic analyses. Mass spectrometry, coupled to either gas or liquid chromatography (GC/MS and LC/MS, respectively), is the most widely used analytical technique. Capillary electrophoresis combined with mass spectrometry (CE/MS) offers an alternate solution for hydrophilic compounds. Metabolomic studies generally involve large numbers of samples and complex data processing. Therefore, powerful data analysis capabilities are also essential. Superior tools for metabolomic investigation Agilent Technologies offers the industry s most complete selection of tools for metabolomic investigation including GC, LC, CE, GC/MS, LC/MS, and CE/MS as well as powerful software tools for metabolite identification, quantitation, and statistical analysis. With Agilent, you get the confidence of having a single, reliable source for hardware, software, consumables, service, and support. You also get a knowledgeable team of application scientists who understand your work, and can help you derive more biologically relevant information from every experiment. Agilent offers the widest range of instruments and software for metabolomics, including complete GC/MS and LC/MS systems. 2

3 Agilent Metabolomics Laboratory Metabolomics workflow: from sample to data to biological meaning The workflow for metabolomics analysis is a multistep process with different analytical challenges and multiple analytical choices at each step. Comprehensive metabolic analysis is broken into multiple steps. Details will vary depending on the samples and metabolites involved, but the general process is: 1. Profiling finding metabolites with statistically significant variations in abundance within a small set of experimental and control samples. 2. Identification identifying the metabolites flagged during profiling. 3. Validation validating the statistical significance of the identified metabolites in the original samples and then validating again using much larger sets of samples to eliminate the effect of natural variations. 4. Interpretation evaluating the discovered metabolomic markers in the context of the relevant biological systems. 3

4 Profiling finds statistically significant metabolites Profiling involves finding metabolites with statistically significant variations in abundance between the experiment and control groups. Profiling can be comprehensive, if you have clear goals but limited understanding of the system involved, or targeted, if you are working with a well-understood system. Often, the best approach includes both, with targeted profiling of known metabolites but comprehensive feature extraction to find unexpected metabolites. A common workflow for profiling is: Select approach evaluate your system knowledge and goals and select the best approach to profiling Sample preparation sampledependent extraction, protein precipitation, and prefractionation Analysis separation and detection of metabolites, usually by GC/MS or LC/MS Feature extraction find and quantify all the metabolites in the sample Data normalization correct for any retention time or response drift Statistical analysis discover statistically significant differences between sample sets Analytical reproducibility is of great importance for expression profiling work. The combination of normal sample variance and analytical variance determines the number of sample replicates required to conclude that differences between samples or sample sets are statistically significant. The smaller the analytical variance, the fewer replicates are required. Hierarchical clustering groups samples based on the similarity of their mass abundance profiles. It is a good way to assess data quality. Replicate samples from the same experimental group should cluster more closely to each other than to samples from a different group. In this example, hierarchical clustering of rice data allows us to immediately distinguish between the wild rice line (TP309) and a transgenic line (TP309 Xa21 + ). 4

5 Agilent Metabolomics Laboratory Identification of metabolites of interest After profiling, statistically interesting metabolites must be identified. There are four common approaches: Spectral library searching can eliminate the need for reanalysis and provide positive identification. This approach is most often used with reproducible EI spectra from GC/MS. Database searching is frequently used with LC/MS data to find likely identities. A good database makes it relatively easy, but for positive identification, standards must be acquired and analyzed. De novo spectral interpretation is possible especially when investigating well-understood systems but it requires great skill. Standards must still be acquired and analyzed to achieve positive identification. Purification and analysis by NMR offers a high degree of confidence, but can be difficult, time-consuming, and expensive. x Abundance CH 2 H N CO MS/MS spectrum of precursor ion m/z H H O + N + NH O -CH 2 O 2 O C OH (measured) Mass-to-charge ratio (m/z) A METLIN database search using accurate-mass TOF data yielded six possible identities for a metabolite with a mass of (molecular ion). The MS/MS spectrum generated by subsequent Q-TOF analysis narrowed the identity to one of two enantiomers: pyroglutamic acid or pyrrolidonecarboxylic acid. A library search of GC/MS spectra can often produce fast, positive identification. In this example, the compound was positively identified as a particular propanoic acid. 5

6 Validation confirms significance To account for natural variations between individual organisms, and for exogenous chemical noise, tentative conclusions from previous steps must be validated. In general, this is a twostep process: Targeted quantitative reanalysis of the original samples to verify the statistical significance of the metabolites tagged in the profiling step Quantitative analysis of much larger sample sets to validate the original profiling and eliminate natural variations While validation analyses are more routine than profiling, hundreds or thousands of samples must be processed. Protocols must be optimized to reduce the cost per analysis. Generally, these quantitative analyses are performed by selected ion monitoring (SIM) with a GC/MS system or by targeted MS/MS (MRM) with a triple quadrupole LC/MS system. Internal standards are usually used to ensure accurate quantitation. Interpretation within the biological context After finding, identifying, and verifying significant metabolites, you must still evaluate them within the relevant biological context. This may involve pathway analysis, evaluation of turnover, or correlation between genes and the functional phenotype of the organism. It may require integration of metabolomic data with genomic and proteomic data. Quantitative validation can involve concentration comparisons (calibration curves) when target compound standards are available, or area comparisons when sample standards are unavailable. 6

7 Agilent Metabolomics Laboratory The tools you need to get the data you need Metabolomes exhibit significant natural variation even in normal organisms. Real differences can only be seen by analyzing large numbers of samples. This puts a premium on analytical instruments that have low error rates and facilitate high throughput. Agilent GC/MS and LC/MS systems deliver outstanding analytical performance with the reliability and ease of use required for high-throughput metabolomic analyses. GC/MS offers sensitive, economical profiling and identification GC/MS offers you high separation power and high sensitivity for volatile compounds and compounds that can be converted to volatile derivatives. It has the potential for sample identification through searching of EI spectral libraries. It is also by far the most economical approach to metabolomic analyses. Agilent is the world s leading supplier of GC/MS systems and knowledge. The best-selling Agilent 6890N gas chromatograph and innovative Agilent 5975B Series MSD quadrupole mass spectrometer deliver an unbeatable combination of performance and reliability: Best-in-class sensitivity Repeatable and library-searchable EI spectra Automation that makes the optional chemical ionization as easy as electron ionization Up to 6 orders of dynamic range to analyze metabolites with widely varying abundances Fast (10,000 u/s) scanning can keep up with fast chromatography for higher throughput Superlative mass axis stability reduces instrument error Chemically inert ion source eliminates degradation of metabolites Agilent s J&W Scientific brand of technically advanced GC columns are designed and manufactured to offer excellent and reproducible performance for even the most difficult types of samples. These columns feature the lowest bleed levels, the best inertness for acids/bases/mixed functional compounds, and the tightest columnto-column reproducibility available. Agilent GC/MS systems provide high separation power and high sensitivity for metabolomic analyses. Librarysearchable EI spectra greatly simplify identification. 7

8 LC/MS systems address many metabolomic challenges Liquid chromatography/mass spectrometry (LC/MS) can be used to analyze a wide range of metabolites. It is especially useful for classes of metabolites that are not volatile and cannot be derivatized. LC/MS offers great selectivity and sensitivity, and generates both molecular ion and structural information. Agilent offers a wide range of reliable, high-performance LC/MS systems. Each is targeted at a different phase of the metabolomics workflow, and each combines exceptional performance with Agilent s legendary reliability and ease of use. In a multi-instrument metabolomics laboratory, consistent cross-platform performance is needed to maximize throughput and reduce analytical variability. Shared technology such as the collision cell in the Q-TOF and triple quadrupole instruments and the TOF components in the TOF and Q-TOF instruments makes it easier to move experiments from one instrument to another and ensures more consistent results. Interchangeable LC/MS ion sources ensure consistent ionization at all steps in the workflow; removing a significant source of analytical variability. Fast, repeatable separations with the 1200 Series Rapid Resolution LC Good sample separation is essential to metabolomic analyses. The Agilent 1200 Series Rapid Resolution LC is particularly well suited to metabolomic analyses. Its higher maximum operating pressure allows the use of longer columns with smaller particles for enhanced chromatographic separation. Higher pressure also facilitates the use of methanol instead of acetonitrile in the mobile phase. Acetonitrile interferes with APCI, while methanol facilitates it. APCI, either with a single-mode source or with the multimode source, can be essential to achieving greater metabolite coverage. The 1200 Series offers: Complete systems from analytical to preparative scale Vial and well-plate autosamplers Temperature-controlled column compartments and autosamplers Fraction collectors ZORBAX RRHT columns maximize compound separation Agilent s ZORBAX Rapid Resolution High Throughput (RRHT) columns are specifically designed to work with the 1200 Series Rapid Resolution LC to achieve ultra-fast HPLC separations with more resolving power. ZORBAX RRHT columns use 1.8 µm particles to maximize resolving power. Short column lengths (15 50 mm) enable high-speed analyses while longer lengths (100 or 150 mm) maximize resolution. Fast, high-resolution separations by the 1200 Series Rapid Resolution LC increase both throughput and the number of metabolites found. 8

9 Agilent Metabolomics Laboratory 6210 Time-of-Flight LC/MS provides fast, accurate profiling The Agilent 6210 TOF LC/MS is an easyto-use and very stable system ideal for metabolomic profiling. Continuous introduction of a reference mass maximizes mass accuracy and ensures repeatability, reducing the number of samples required for statistical reliability. The 6210 features: 2-ppm mass accuracy for more accurate comparison of metabolites found in different samples Unmatched sensitivity in a bench-top TOF instrument Greater than 13,000 resolving power, to distinguish metabolites with similar masses Fast spectral acquisition for more accurate peak area measurements and more accurate quantitation Superb stability and repeatability You can equip the 6210 TOF with a wide range of ionization sources, including the highly versatile multimode source Q-TOF LC/MS offers more confident metabolite identification The Agilent 6510 Quadrupole Time-of- Flight LC/MS is a highly versatile instrument, perfect for metabolite profiling and identification. It combines the rich structural information of MS/MS with very accurate mass measurements for more confident metabolite identification. The 6510 features: Fast spectral acquisition 1 MS and 5 MS/MS spectra per second provides more data for identification Advanced collision-cell technology eliminates chemical crosstalk Routine 2-ppm MS and 5-ppm MS/MS mass accuracy for more confident metabolite identification Greater than 13,000 resolving power, to distinguish metabolites with similar masses Automated tuning and mass calibration for maximum performance with minimum effort Repeatability and mass accuracy of the 6210 TOF make it an outstanding tool for profiling Q-TOF s combination of accurate-mass measurements and MS/MS spectra yield more confident metabolite identification. 9

10 6410 Triple Quadrupole LC/MS for high-throughput validation Validation studies involving hundreds or thousands of samples require accurate, high-throughput quantitation. Triple quadrupole MRM is the standard for targeted quantitation. The Agilent 6410 Triple Quadrupole LC/MS gives you femtogram detection levels, as well as 24/7, day-after-day reliability. The quantitation software is extremely easy to use thanks to features such as compound- or sample-centric navigation and parameterless integration. Femtogram-level sensitivity Fast MRM transitions increase the number of analytes that can be analyzed in a single run Advanced collision-cell technology eliminates chemical crosstalk for better sensitivity and linearity Simple method setup and parameterless integration save time Automated tuning ensures maximum performance with minimum effort Interchangeable LC/MS ion sources maximize flexibility and ensure consistency The vast chemical diversity of metabolites means no single ionization mode is sufficient for all analyses. Agilent offers the industry s widest selection of interchangeable ion sources. You can match ionization mode with instrument as necessary. Ionization choices include: Electrospray (ESI) at standard, microliter, and nanoliter flow rates Atmospheric pressure chemical ionization (APCI) Multimode ESI/APCI Atmospheric pressure photoionization (APPI) Agilent LC/MS ion sources use orthogonal nebulization and high-temperature, counter-flow drying gas to maximize performance and repeatability, and minimize chemical noise. Of particular note is the multimode source, which can simultaneously generate ions by electrospray and APCI, with no significant loss of sensitivity compared to single-mode sources. This can double sample throughput for profiling and validation studies. Interchangeable LC/MS ion sources allow you to match your ionization mode to your expected metabolites and ensure consistent ionization throughout the metabolomics workflow Triple Quad delivers the reliable MRM quantitation required for highsample-volume validation. 10

11 Agilent Metabolomics Laboratory CE/MS combines outstanding separations and fast analyses Capillary electrophoresis (CE) is a liquid-phase alternative to liquid chromatography. It offers alternate selectivity and outstanding separation efficiency. The combination of capillary electrophoresis and mass spectrometry (CE/MS) is less common than GC/MS and LC/MS for metabolomic analyses, but can be valuable, especially for analysis of hydrophilic metabolites. CE/MS combines the short analysis time and excellent separation efficiency of capillary electrophoresis with the molecular weight and structural information from mass spectrometry. The Agilent CE system provides excellent separation efficiency and easy connection to a mass spectrometer. CE/MS can be a valuable alternative to GC/MS and LC/MS, especially for analysis of hydrophilic metabolites. Agilent s industry-leading CE system can be combined with several mass spectrometers. For example, the combination of CE and the Agilent 6210 Time-of-Flight LC/MS blends outstanding separation efficiency with superior mass accuracy and mass resolution to create an excellent metabolite profiling solution. The grounded nebulizers used in Agilent ion sources make CE electrical settings independent from MS ion source settings, eliminating a major drawback found in many other CE/MS systems. 11

12 Software that turns data into information The goal of metabolomics investigation is a better understanding of how complex biological systems work. It takes the right software tools to turn even the highest quality data into useful, biologically relevant information. Agilent s software for metabolomics applications does exactly that. Method-specific deconvolution to find all your metabolites An undetected metabolite is a lost opportunity; it is essential to find as many of the metabolites in your samples as possible. This goes beyond simple chromatographic peak finding, because even with the best separation a peak can contain multiple components. The solution to finding coeluting metabolites is deconvolution. Deconvolution finds ions whose individual abundances rise and fall together over time, indicating that they are from the same compound. Deconvolution groups these ions to produce a reconstructed, single-component spectrum for each metabolite. GC/MS and LC/MS spectra have unique characteristics related to the chemistry and physics of their ionization processes and mass analyzers. For this reason, a single deconvolution program is not optimum for all applications. Agilent offers separate deconvolution programs designed specifically to account for the unique characteristics of GC/MS and LC/MS data. AMDIS for useful GC/MS deconvolution For deconvolution of GC/MS spectra, Agilent offers Automated Mass Spectral Deconvolution and Identification Software (AMDIS) as part of the NIST library search package. AMDIS extracts pure component spectra from complex GC/MS data, and helps you determine ion/peak associations. AMDIS produces library-searchable spectra and automatically exports a feature list. MassHunter Workstation for better LC/MS deconvolution Agilent s MassHunter Workstation software for LC/MS includes a proprietary feature extraction and correlation algorithm. Optimized for LC/MS time-offlight data, it identifies covariant ions associated with a single compound. Identifying and grouping such ions improves the accuracy of subsequent empirical formula determination or metabolite database searching. TIC peak Metabolite 1 Metabolite 2 Metabolite 3 Metabolite 1 Metabolite 2 Deconvolution Mass spectrum Metabolite 3 Deconvolution finds metabolites that are chromatographically unresolved or poorly resolved. It generates an extracted ion chromatogram and a reconstructed, singlecomponent spectrum for each metabolite. 12

13 Agilent Metabolomics Laboratory The molecular feature extraction and correlation algorithm in the MassHunter software locates all of the components in a chromatogram, not just all of the chromatographic peaks. In this example, it finds a real component in a segment of the total ion chromatogram (top) that appears to be mostly noise and baseline rise. Evaluating a single peak from a total ion chromatogram (top), the AMDIS deconvolution software finds covariant ions (center) indicative of a single component. The bottom panel shows the reconstructed mass spectrum for this peak. 13

14 Simplified identification with the METLIN metabolite database There are several approaches to identifying metabolites. EI spectra from GC/MS are well suited to spectral library searching. For LC/MS, searching a database of metabolite information can help you narrow the list of possible candidates. Accurate mass data makes this database searching more effective by narrowing the mass window that needs to be searched, and thus reducing the number of possible identities. The METLIN metabolite database an effort by the Center for Mass Spectrometry at The Scripps Research Institute is probably the best-known and most-comprehensive metabolite database in the world today. It includes annotated lists of over 15,000 endogenous and exogenous metabolites, as well as di- and tri-peptides, with: Masses Chemical formulas Structures The METLIN database is open to the public. Searches can be performed one compound at a time over the Internet. However, it is also now possible to obtain a local, personal copy of the METLIN database. Advantages of a local copy include: Private searching Ability to add proprietary compounds to the database Automated searches from a mass list Agilent Technologies is the sole supplier of the METLIN Personal metabolite database. Agilent MassHunter Workstation software and GeneSpring MS software will include links to the METLIN Personal database, to increase throughput and ease of use. With masses, chemical formulas, and structural information for over 15,000 endogenous and exogenous metabolites, as well as di- and tri-peptides, the METLIN database is a powerful aid to metabolite identification. 14

15 Agilent Metabolomics Laboratory GeneSpring MS a unified platform for mass spectral data normalization and comparison Agilent s GeneSpring MS informatics software is a powerful solution for the discovery of metabolite biomarkers through the analysis of mass spectrometric data. You can easily import, normalize, compare, and display mass spectral data from large sample sets and complex experimental designs. This includes GC/MS data, and LC/MS or CE/MS data from Agilent TOF, Q-TOF, and triple quadrupole instruments. GeneSpring MS software includes comprehensive tools for statistical analysis, data mining, and visualization, including: Analysis of variance (ANOVA) Principal component analysis (PCA) t-tests Volcano plots Hierarchical trees (dendrograms) Self-organizing maps (SOMs) Quality threshold (QT) clustering Support vector machines (SVMs) GeneSpring MS informatics software is designed specifically to integrate well with other statistical software packages. If you have a favorite, specialized analysis package, the chances are good that GeneSpring MS software can work with it. PCA only Y 1 1-way ANOVA and PCA Y 1 TP309 line (wild) 0 TP309-Xa21 line (transgenic) 0 Infected wild and transgenic 1 Z X 1 Z 0 Uninfected wild 0 Uninfected transgenic 1 X TP309 wild rice - no treatment TP309 wild rice - mock challenge TP309 wild rice - bacteria challenge TP309-Xa21 transgenic rice - no treatment TP309-Xa21 transgenic rice - mock challenge TP309-Xa21 transgenic rice - bacteria challenge TP309-Xa21 transgenic rice - knockout bacteria challenge The complexity and variety of metabolomic analyses make it essential to have many options for statistical analysis and visualization. In this complex experiment with multiple conditions, PCA alone shows only genotype differences. Application of PCA after ANOVA, on the other hand, clearly differentiates infection status as well as genotypes of uninfected samples. 15

16 Agilent Metabolomics Laboratory Agilent services let you focus on what you do best Agilent s service organization is the most respected in the industry. We can provide essential services to keep your metabolomics instruments in top condition for dependable operation and maximum throughput. We offer the broadest range of services, including: Software updates at a fixed annual cost On-site preventative maintenance (PM) to ensure dependable operation and minimize unplanned downtime Phone support to isolate and resolve software and hardware problems Remote updates, diagnostics, and repair over secure Internet connections On-site instrument repair Agilent services can be acquired on an as-needed basis, or bundled into economical service contracts with a fixed price for protection against unexpected repair costs. The Agilent Service Guarantee We are so confident in our service quality and the reliability of our instruments that we offer the Agilent Service Guarantee: Should your Agilent instrument require service while covered by an Agilent service agreement, we guarantee repair or we will replace your instrument for free. No other manufacturer or service provider offers this level of commitment to keeping your laboratory running at maximum productivity. About Agilent Technologies Agilent Technologies is a leading supplier of life science research systems that enable scientists to understand complex biological processes, determine disease mechanisms, and speed drug discovery. Engineered for sensitivity, reproducibility, and workflow productivity, Agilent's life science solutions include instrumentation, microfluidics, software, microarrays, consumables, and services for genomics, proteomics, and metabolomics applications. Buy online: Find an Agilent customer center in your country: u.s. and canada agilent_inquiries@agilent.com europe info_agilent@agilent.com asia pacific adinquiry_aplsca@agilent.com This item is intended for Research Use Only. Not for use in diagnostic procedures. Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance or use of this material. Agilent Technologies, Inc Printed in USA January 31, EN