Go beyond. to realities unexplored. Comprehensive workflows, integrated solutions

Transcription

1 Go beyond to realities unexplored Comprehensive workflows, integrated solutions

2 We re committed to helping researchers and scientists in academia and industry harness the power of metabolomics to gain in-depth biological insight, facilitate biomarker discovery, and advance scientific research. By collaborating with key opinion leaders and others within the scientific community, we ve developed leading solutions in the areas of separation, detection, and software. And we ll continue to pursue advancements to delve deeper into the metabolome, so you can go beyond the edge of what we ever thought possible.

3 The total package Moving metabolomics forward requires an all-in commitment. That s why we ve invested heavily in developing new metabolomics software that lives up to the high standards set by our leading instrumentation. Now you can have the total package needed to perform increasingly complex analysis, and easily translate your metabolomics data into valuable discoveries for your next step.

4 A multitude of discoveries Driving advancements across a broad range of applications With its close link to phenotype, metabolomics is an ideal approach in areas of disease research to understand mechanisms of drug metabolism, biomarker discovery, and drug safety studies. Metabolomics approaches have also found wide applicability to other research areas such as the food and beverage industry, biofuels, and biochemicals. 4

5 We collaborate with leading metabolomics researchers across these industries to deliver results for diverse applications. Human health Metabolomics expands understanding in areas such as disease research, systems biology, and metabolic profiling, leading to insights into biomarker discovery and mechanisms for treatment, and driving new research directions. Plant science Metabolomics can help researchers understand the factors contributing to plant traits that influence yield, stress tolerance, seed quality, disease resistance, and mode of action of pesticides, knowledge of which will help to optimize crop development and production. Food science Through metabolomics, food scientists can rapidly obtain valuable information about the biochemical composition of foods. This helps in the derivation of components for food flavors and shelf life, leading to better control over food quality, among other factors. Nutrition and diet Using metabolomics, the biochemical profile of food components and its interaction with the biochemical networks of living organisms can be studied to provide information regarding the potential role of nutrition in health. This can help guide diet-based disease treatments and prevention. Drug development Metabolic profiling has the potential to identify markers arising from drug-induced toxicity leading to improvements in public safety and optimizing drug development. Environmental science Environmental metabolomics is an emerging approach to characterizing the effects of disease and toxic chemicals on sentinel species to understand their possible impact on humans. It is also a useful tool for carrying out chemical risk assessment of pharmaceuticals, pesticides, and other chemicals, and maintaining healthy livestock and fish. Biofuels and bioengineering The techniques of metabolomics can help researchers identify microbial strains and engineer novel metabolic routes for biomass production and conversion. This could potentially increase production of biofuels needed to meet future energy demands. 5

6 The core of successful metabolomics powered by Orbitrap mass spectrometry The success of metabolomics research lies with proven Thermo Scientific Orbitrap technology. Workflows that include Orbitrap mass spectrometers have already been widely adopted for life science applications such as proteomics. Based on published reports, the same technology is also suited for metabolomics. The Orbitrap mass spectrometers represent the leading mass spectrometry (MS) technology for analyzing samples that are complex both in the sheer number of analytes present and in the type of sample matrix, such as blood, food, urine, etc. Additionally, with a breadth of chromatographic separations and integrated data analysis tools, the Orbitrap mass spectrometer enables researchers to successfully analyze physicochemically diverse samples in metabolites making us the leader in metabolomics, delivering solutions to keep you at the forefront of science. 6

7 Comprehensive coverage of the metabolome Harnessing the power of chromatographic separation Since endogenous metabolites are diverse in their physicochemical properties and abundance, a true comprehensive metabolomics study will require multiple separation techniques. This complexity demands sophisticated methods of separation and detection high-resolution separation that is designed for high-resolution detection by Orbitrap mass spectrometers. The integration of each of the chromatographic separation methods (liquid, gas, and ion) with the high-resolution detection system (Orbitrap mass spectrometer) on one single platform enables broader, deeper, and faster analysis of complex and diverse metabolites offering a robust system that is more effective than the sum of its parts. Seamless integration with Orbitrap mass spectrometers Thermo Scientific products deliver comprehensive breadth in separation of analytes with gas, liquid, and ion chromatography systems. When combined with the leading Orbitrap mass spectrometer, our technologies enable broader and deeper analysis into the metabolome. With high robustness and technical reproducibility, these solutions provide novel yet stringent results for high-impact discoveries. LC-MS: Thermo Scientific Vanquish Flex Binary UHPLC system with Q Exactive HF MS GC-MS: Thermo Scientific Q Exactive GC Orbitrap GC-MS/MS system with proven TRACE 13 Series GC injectors IC-MS: Thermo Scientific Dionex ICS- 5+ Capillary HPIC System with Q Exactive HF MS 7

8 Comprehensive coverage of the metabolome Enabled by orthogonal separations and deep coverage with Orbitrap mass spectrometry Relative abundance 2 1 Peak #9, F6P Peak #1, G6P Retention time Separation of 11 monophosphate isomers of sugar by IC-MS. IC: Ion chromatography (IC)-MS is best suited for charged or very polar metabolites such as sugar phosphates and amino acids that are difficult to analyze by LC-MS. IC has high resolving power, and many isomers can be separated prior to mass spectrometry analysis by this technique. RT: Day Day 1 Relative abundance Day 2 Day Time (min) Quantitation of components in rat muscle homogenate was carried out by GC. Rat muscle tissues were decayed for 3 days (top to bottom). The peaks in the highlighted region indicate the change in intensity over time. GC: Samples that are volatile and amenable to chemical derivatization are well suited for gas chromatography (GC)-MS. GC offers high resolving power for separation of isomers. Plant secondary metabolites are well suited for this technique. 8

9 +ve and ve ion mode on the MS adds another dimension Fatty acid Galactose, nucleotide sugar Pentose and ascorbate Nucleotides Secondary metabolites RP-HPLC and ESI (+) HILIC and ESI (+) RP-HPLC and ESI ( ) HILIC and ESI ( ) RP- and NP-HPLC, and ESI (+/ ) Energy Amino acid GC and CI/EI IC and ESI ( ) Lipids Xenobiotics biodegradation Carbon fixation TCA Amino acid Amino acid Amino acid +ve and ve ion mode on the MS adds another dimension Ionic Ion chromatography Ionic property Liquid chromatography: HILIC Liquid chromatography: RP C18 Neutral Polar Polarity Nonpolar LC: Liquid chromatography (LC)-MS offers the broadest coverage of metabolites through the ability to change column chemistries, such as reversed-phase high-performance liquid chromatography (RP-HPLC) for nonpolar to moderately polar metabolites, and hydrophobic interaction liquid chromatography (HILIC) for ionic and polar compounds not retained by RP. Ultra high-performance LC (UHPLC) systems and columns are available for high-resolution separations. 9

10 Leading-edge metabolomics Powered by Orbitrap mass spectrometry When combined with advanced separation techniques, industry-leading Orbitrap system based mass spectrometers provide high-resolution accurate mass (HRAM) measurements and sensitivity required for metabolomics. This powerful technology enables high-throughput and quantitative capabilities, expanding the scope of our understanding of metabolites involved in cell metabolism and biological pathways, and in putative biomarker discovery. Key attributes of Orbitrap HRAM for successful metabolomics HRAM: With up to 5K resolution, Orbitrap analyzers enable more confident identification and quantitation due to high specificity allowing accurate mass assignments, especially with metabolites of similar mass, and isotope-labeling studies. The very high resolving power of Orbitrap HRAM analyzers resolves isobaric metabolites from a complex mixture in (A) plasma extract and (B) a standard mix of D-glucose and paraxanthine at concentrations of 5 mm and.1 mm, respectively. A B Relative abundance Relative abundance Relative abundance D-Glucose (m/z ) or Paraxanthine (m/z )? R = 6, R = 12, R = 24, R = 5, m/z m/z m/z m/z m/z m/z m/z m/z Peak intensity x1 6 5, 4, 3, 2, 1, A R 2 = pg 2 ng on column (1 fmol 1 pmol on column) 11 levels 5, 1, Amount (pg) 15, 2, Endogenous metabolites concentration range (mol/l) Peak intensity 1, 8, 6, 4, 2, B 3,89 pg n = 18 UM1 Citric acid 2,372 pg n = 24 UM2 7,779 pg n = 1 UM5 UM1 cells, very invasive UM2 cells, less invasive UM5 cells, highly invasive UM6 cells, less invasive 3,13 pg n = 12 UM6 Sensitivity: Metabolomics is and always has been a quantitative science. There are ordersof-magnitude differences between concentrations of different endogenous metabolites that must be detected for a comprehensive and meaningful view of their biological states. Orbitrap technology enables quantitation down to low-femtomole concentrations, with up to 5 orders of linear dynamic range with tight coefficients of variation (CV). 1 Sensitivity of HRAM analyzer: (A) Quantitation of citric acid was achieved at low-femtomole sensitivity using full scan MS. (B) shows the amount of endogenous citric acid obtained from each plate containing ~3 x 1 6 cells.

11 Resolution Accurate mass Technical reproducibility: Due to its sensitivity and selectivity, Orbitrap mass spectrometry techniques have become the method of choice for metabolomics research. To find meaningful answers with a large number of datasets, typically found with metabolomics, the mass spectrometer must provide accurate and reproducible data from run to run, without the need for internal calibration. 25 hr post-external calibration Relative abundance Mean Area 96,686, % CV 95,693,541 1.% 96,475,711.2% 95,147, % 98,394, % x Mean ppm ppm ppm ppm ppm Mean ppm ppm ppm ppm ppm 25 hr post-external calibration ,722, %.43 ppm ppm Time (min) m/z Relative Abundance scan points across a peak Full scan MS R = 7, Peak width = 3.6 s, mean 14.5 scans D3-hippuronic acid, theoretical m/z = Positive Relative abundance Time (min) Negative Fast polarity switching: A combination of separation techniques (GC, IC, and LC (RP or HILIC)) can help achieve a more comprehensive view of the metabolome. Additionally, more coverage can be obtained using both positive and negative ionization modes on the mass spectrometer. Polarity switching on an Orbitrap mass spectrometer can be achieved in a single run, enabling a more productive analysis. Base peak chromatograms of positive and negative ion modes of bovine heart lipid extract (1 µg on column) in a single run. Time (min) MS n capabilities for the structural elucidation of metabolites: Multiple-stage mass spectrometry (MS n ) offers a solution for de novo identification and structural elucidation. MS n is usually achieved using ion traps and Tribrid mass spectrometers such as the Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. This is an advanced technique requiring high resolution, accurate mass (HRAM), and speed on an HPLC time scale, and a variety of dissociation modes such as high-energy collisional dissociation (HCD) and collision-induced dissociation (CID). Relative abundance Isoleucine Leucine sec HCD CID CID MS HCD 1 MS Orbitrap scan R = 12, CID CID 2 MS/MS HCD-ion trap scans 4 MS/MS Orbitrap scan R = 3, MS n improves the ability to confidently identify isomeric metabolites and elucidate structures of unknowns. 11

12 Numerous targets, unlimited possibilities Solutions designed to meet your metabolomics goals Several approaches to metabolomics research are available depending on the goals. The untargeted discovery approach can be used to identify putative biomarkers, understand metabolism on a system-wide scale, and detect unknown metabolites in screening applications. Targeted approaches are best suited when there is a focused study on a subset of known compounds, to verify and/or validate results from the discovery or hypothesis-driven phase, or for the routine detection and quantitation of compounds. We provide a range of solutions and workflows that are specifically designed to meet your needs Chromatographic separations Mass spectrometry Data analysis GC Untargeted metabolomics What is the global metabolite profile of a sample? HRAM detection by Orbitrap mass spectrometry Profiling Statistical analysis Identification Pathway analysis LC: HILIC Discovery phase or hypothesis driven LC: RP-HPLC (C-18) HRAM detection by Orbitrap mass spectrometry Profiling Quantitation Statistical analysis IC Targeted metabolomics What are the levels of specific metabolites in a sample? Selected reaction monitoring (SRM) by triple quadrupole mass spectrometry Quantitation Statistical analysis Reporting 12

13 Untargeted metabolomics Explore all metabolites and compounds present in many complex samples. Identify putative biomarkers Understand cellular metabolism Identify trends between samples and populations Study the most challenging samples and overcome the technical limitations associated with them Both the separation techniques and HRAM Orbitrap MS are proven to be highly reproducible, have high sensitivity, and are capable of measuring a wide range of metabolites, ranging from very hydrophilic to hydrophobic, for comprehensive coverage. Our metabolomics software is specially designed to examine the rich HRAM Orbitrap data to convert large datasets into meaningful insights. Targeted metabolomics Validate previously identified metabolites using a quantitative approach. Large-scale targeted profiling Target selected metabolites and compounds from either untargeted discovery or hypothesis-driven data Apply high-throughput and sensitive techniques Target and verify putative biomarkers or compounds indicative of quality, origin, and toxicity before developing routine assays Large-scale metabolomics quantitation using Orbitrap HRAM MS provides a workflow to quickly evaluate and screen known metabolites. As untargeted analyses are performed using HRAM Orbitrap systems, the same system can be used for targeted profiling with the same high selectivity, post-acquisition flexibility, and retrospective data analysis of compounds by MS/MS. Routine quantitation Target known, well-characterized metabolites and compounds Apply high-throughput and sensitive techniques Customize reports For targeted lists of known, well-characterized metabolites and compounds, the Thermo Scientific TSQ quadrupole mass spectrometers offer sensitive, fast, and robust quantitation of metabolites. 13

14 Tools for translational research Large-scale metabolic phenotyping As metabolism is closely associated with the dynamics of the human phenotype, metabolic phenotyping (phenome studies) can be applied to large-scale analysis of thousands of samples per study to identify a single metabolite or metabolite signatures that relate to disease onset and progression, or response to treatment. Several research initiatives are underway worldwide using this approach. SNP CNV LOH DNA methylation Histone modification TF binding Gene expression Alternative splicing Small RNA Protein expression PTM Cytokine array Metabolite profiling Metabolite flux Genome Epigenome Transcriptome Proteome Metabolome Phenome Transcription Expression Translation Function Aging Metabolicsyndrome Cardiovascular disease Cancer Due to its sensitivity, dynamic range, broad coverage, and high throughput, Orbitrap mass spectrometers are ideally suited to both small-scale metabolomics and large-scale metabolic phenotyping. The Phenome Centre Birmingham, UK, employs multiple Orbitrap LC-MS systems with the aim to complete more than 15, assays per year to study human metabolism and its role in health and disease. One such study applied a nontargeted discovery based metabolomics approach to investigate metabolic changes that relate to phenotypic changes, following burn trauma in an adult population. The study showed expected (e.g., fatty acid and carbohydrate metabolism) and unexpected (e.g., tryptophan metabolism) metabolic changes longitudinally, which may allow the development of improved nutritional support following trauma (A in figure). The study also identified putative prognostic markers of outcome, such as M3328 (e.g., later sepsis onset), which, following validation and replication, may be used to provide stratification and personalized treatment paths (B in figure). A A:D1-2 B 1, B:D3-4 C:D5-9 N6236 D12-15 D19-24 D26-29 G:M2 H:M2 I:M6 Triacylglycerides (TAG) Hypermetabolism is observed across many different metabolic classes in an adult sample population following burn trauma. Later onset of sepsis p =.72, FC = 5.2 M A:D B:D3-4 C:D5-9 N5131 D12-15 D19-24 D26-29 G:M2 H:M2 I:M6 Diacylglycerides (DAG) Time of sepsis p =.3, FC = 13.6 M A:D1-2 1, B:D3-4 C:D5-9 N274 D12-15 D19-24 D26-29 G:M2 H:M2 I:M6 Fatty acids Multiple organ failure p =.47, FC = 2.9 M3328 Data courtesy of the Phenome Centre Birmingham, UK. 14 Case Control Case A single metabolite shows promise as a biomarker for stratification, as it shows statistically significant changes before sepsis and during sepsis, and is also predictive of multiple organ failure. Control Case Control

15 Streamlined data analysis solutions Delivering the total package We have invested heavily in developing new metabolomics software that lives up to the high standards set by our leading instrumentation promising the same standards of quality, usability, and performance. Our suite of integrated applications is built to take you quickly from data acquisition to interactive analysis and interpretation of results. Customize your workflow with flexible architecture. Get simplified analysis of large-scale data. Now you can have the total package needed to perform increasingly complex analysis, and lead the way to high-impact discoveries. STATISTICAL ANALYSIS IDENTIFICATION Databases and libraries Untargeted metabolomics GC-MS Targeted quantitation with confirmation Targeted routine quantitation Raw data processing RELATIVE QUANTITATION ROUTINE QUANTITATION Compound Discoverer Software TARGETED PROFILING IDENTIFICATION TraceFinder Software PATHWAY ANALYSIS REPORT GENERATION Databases and libraries Targeted routine quantitation Results interpretation Biomarker discovery Dynamics of metabolism Metabolic alterations Biomarker verification and validation Verification of specific compounds and/or pathways of metabolism Flavor development and/or screening 15

16 Stringent and robust data analysis Compound Discoverer 2. Software From large datasets to pathway mapping Thermo Scientific Compound Discoverer Software is a flexible solution that lives up to the high standards of the Orbitrap mass spectrometer systems. Compound Discoverer Software offers a full suite of powerful tools for metabolomics analysis such as differential analysis, identification, and pathway mapping. It is the only analysis software that is designed to exploit the rich information of Orbitrap HRAM data. Compound Discoverer Software handles HRAM data from hundreds of samples, moving from data to biological interpretation. Powerful visualization tools and flexible workflows (standard and customizable) enable complete control of data processing. From the results obtained, customized reports are generated. STATISTICAL ANALYSIS IDENTIFICATION RELATIVE QUANTITATION Compound Discoverer Software PATHWAY ANALYSIS Detecting the components of interest in a sample is only the beginning. Compound Discoverer Software simplifies the next step determination of metabolite or compound structure by automatically annotating relevant components. This includes not only MS/MS analysis for the most stringent results, but also MS n analysis for structural elucidation of those compounds that are not confidently identified. MS n tree of prunin containing CID (white) and HCD spectra (green) with multiple collision energies from MS¹ down to the MS 5 level. Structure of prunin. 16

17 mzcloud online fragmentation library Powerful and rigorous compound identification Determining the components of interest in a complex analysis is only one piece of the metabolomics workflow. Compound identification remains one of the toughest challenges in many research applications. Compound Discoverer Software provides many tools to aid in compound identification, such as the unique, high-quality mzcloud online fragmentation library (mzcloud.org). Automatic identification of compounds using the mzcloud library without leaving Compound Discoverer Software. Map and Display features detect the potential metabolites of the biological pathways. mzcloud is a highly curated library, currently with >6, compounds with MS/MS fragmentation spectra. With major contributions from us and the metabolomics community, this number is increasing every day. Coupled with molecular weight or formula-based searches in the ChemSpider database and other user-created databases, Compound Discoverer Software gives you the power to turn unknowns into knowledge. MS 2 spectrum of prunin (HCD, 5 NCE) with annotated fragment structures. 17

18 Comprehensive portfolio Our complete Thermo Scientific instrument lineup One resource Thermo Fisher Scientific is a partner in the life sciences, providing innovative metabolomics solutions to address the challenges of biological research. We enable end-to-end workflows with column chemistries, comprehensive separation systems, mass spectrometry, and integrated software. For targeted routine analyses, the Thermo Scientific TSQ triple quadrupole mass spectrometer completes the lineup with leading HRAM Orbitrap MS systems. Dionex ICS-5+ Capillary HPIC System Vanquish Flex Binary UHPLC System Compound Discoverer Software Q Exactive HF Mass Spectrometer mzcloud online fragmentation library Orbitrap Fusion Lumos Mass Spectrometer der TraceFinder Software TSQ Quantiva Triple Quadrupole Mass Spectrometer Q Exactive GC Orbitrap Mass Spectrometer 18 TSQ 8 Evo Triple Quadrupole Mass Spectrometer

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