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High Resolution GC-MS Application: Metabolomics
Vladimir Tolstikov, PhD
Eli Lilly and Company
Sample Harvest and Storage
Biological Metadata
Sample Extraction Extraction Metadata
Sample Preparation RI internal standards, Derivatization
Sample Analysis QC, randomization
Standard Operational Procedure
Raw Data
Chromatography Metadata Mass Spectrometry Metadata
Metabolite Peak Annotation
Data normalization, background subtraction, detection limit
Analytical Protocols
Processed Data Collection and Organization Statistical Analysis Pathway Analysis
Experiment Submission
A
Volatiles Alchohols Organic acids Essential oils Amino acids Organic amines Esters Catecholamines Nucleosides Perfumes Fatty acids Nucleotides Terpenes Phenolics Oligosaccharides Carotenoids Prostanglandins Peptides Flavanoids Steroids Co-factors Perfumes Sugar phosphates Polar Lipids
LC/MS GC/MS
PEGASUS GC-HRT accurate mass TOF Gerstel ALEX/CIS MultiPurpose Autosampler Triple TOF 5600 accurate mass Triple quad 5500
Lilly Metabolomics Platform
Lilly Metabolomics Platform Data Analysis and Visualization
• Statistical analysis: An array of commonly used statistical and machine learning methods :
• univariate -fold change analysis, t-tests, volcano plot, and one-way ANOVA, correlation analysis;
• multivariate - principal component analysis (PCA), partial least squares - discriminant analysis (PLS-DA) and PCA-DA;
• clustering - dendrogram, heatmap, K-means, and self organizing map (SOM)); • supervised classification - random forests and support vector machine (SVM). • Functional enrichment analysis: The analysis is based on several libraries
containing ~6300 groups of biologically meaningful metabolite sets collected primarily from human studies;
• Metabolic pathway analysis: Pathway analysis (including pathway enrichment analysis and pathway topology analysis) and visualization for Human metabolic pathways with a total collection of 1173 pathways;
• Pathway analysis : MetPA, Ingenuity, GeneGo
Human urine GC/MS profiling
Throughput Quality
12:30.00 16:40.00 20:50.00 25:00.00 29:10.00 33:20.00 37:30.00 41:40.00 45:50.000.0e0
2.0e7
4.0e7
6.0e7
8.0e7
Time (min:sec)AIC
12:30.00 16:40.00 20:50.00 25:00.00 29:10.00 33:20.00 37:30.00 41:40.00 45:50.000.0e0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
Time (min:sec)AIC
Mouse CSF Sample volume - 2uL Methoxyamine, MSTFA 2% TMSCI 1 uL splitless, CIS C4 injector Detector EI 70ev
>60% probability score >3000 peaks deconvoluted >1200 names assigned ~ 75 metabolites identified
Metabolomics study requirements for GC/MS instruments
GC-HRT 1 Sensitivity √
2 Fast acquisition √
3 Robustness √
4 Reproducibility √
Unique features
1 Routine stable high resolution √
2 Routine stable high mass accuracy √
3 True peak deconvolution √
4 Elemental composition assignment √
High Resolution, High Mass Accuracy: YES or NO ID
High Resolution, High Mass Accuracy: YES or NO ID
High Resolution, High Mass Accuracy: YES or NO ID
Conclusions High Resolution GC-MS instrumentation is a must have component of mass spectrometry based modern metabolomics laboratory. High Resolution GC-MS instrumentation delivers components identification at new higher level of confidence.