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.