Metabolomics: Techniques and Applications ABRF

Transcription

1 Metabolomics: Techniques and Applications ABRF Sacramento, CA March 23, 2010

2 Overview Metabolomics Definitions Representative Project Sarcosine, a prostatic cancer biomarker Technical overview/ How we do it.

3 Unbiased Metabolomics Seeks to identify all chemical entities on a biological sample and comment on their relevance. All chemical entities are characterized irrespective of their identity, and indeed many are unknown. Statistical relevance does not require chemical identity Biochemical relevance does require chemical identity

4 Metabolomics DNA Genomics 25,000 Genes RNA Transcriptomics 100,000 Transcripts Protein Proteomics 1,000,000 Proteins Biochemicals (Metabolites) O OH C CH NH 2 H 2 C CH 3 CH CH 3 H OH HO HO H H H O OH OH H N N H NH 2 N N Metabolomics 1,800 Compounds

5 Chemical Ecology of a Human Cell Number of cmpds per 20 daltons Metabolome natively biosynthesized monomeric Complex metabolites polymeric (DNA, RNA, proteins, polysaccarides, etc.) Xenobiome Mass

6 Experimental design Full Platform Full Platform Full Platform

7 Results Using a combination of high throughput liquid and gas chromatography based mass spectrometry, we profiled more than 1265 metabolites across 262 clinical samples related to prostate cancer (tissue, urine, and plasma). *Funded by the Early Detection Research Network (EDRN) /NCI

8 Chemical Variation Total = 1265 compounds Total = 626 compounds (n=45) (n=12) (n=110) (n=110) (n=16) (n=14)

9 Statistical Differences

10 Principle Components Analysis (all compounds / total data set)

11 Metabolic Alterations

12 Molecular Concepts Mapping Tomlins et al., Nature Genetics 39, (2007)

13 METABOLOMIC SIGNATURE OF METASTATIC SAMPLES COMPARED TO PCa FATTY ACID INTERMEDIATES KETONE BODIES CoA PRECURSORS S-ADENOSYL METHIONINE AMINO ACIDS NUCLEIC ACID PRECURSORS

14 Sarcosine: A Methylated derivative of Glycine is elevated in Metastatic Prostate cancer Ben PCA Met Ben PCA Met CH 3 Ben PCA Met Ben PCA Met

15 Validation of Sarcosine Using an Independent GC/MS ( targeted ) Assay on the same sample set.

16 Revalidation of Sarcosine Using an Independent GC/MS ( targeted ) Assay on additional samples.

17 Characteristics Chemocentric platform Unbiased analyses Library based compound identification Quantitative Robust Reproducible CVs < 10%

18 Platform Fractions & Chemical Libraries CE MS/+ MS/ Software Data Extraction peak identification peak alignment Sample Preparation LC MS/+ MS/ peak deconvolution Chemical Identification reference databases ion spectra grouping related ions Data Set GC MS/ei compound id Quantitation Quality Control LIMS Data Reduction Preparation Analysis Informatics

19 MS Dimensionality

20 UPLC/QToF Previous Current mass > time > mass > time > time 20 sec 2 sec mass +/-0.3 amu +/ amu concentration 1x 20x Characterization Library comparison only, Formula based, may add may add fragmentation fragmentation

21 Reproducibility Unit mass resolution on ion masses, and fragmentation of molecular ions requires very high reproducibility to accurately name compounds. May 2009 August 2009 December 2009

22 Reproducibility (2) The use of LIMS, robotics & barcodes is required to achieve reproducible sample prep, and data.

23 Integration through LIMS

24 Metabolomic Library coverage Our current library has 950 compounds and is growing. Approximately 250 are shown below.

25 Variance Analyses Run periodically to test ourselves 50 aliquots from a single pooled plasma sample Processed individually with simple pooling pattern Final dataset examined statistically to identify sources of sample prep induced variance. (There are many variants to this experiment)

26 Variance Analyses Samples 1-60 Samples Pool & Realiquot Samples 1-10 Step 1 -- PPT Samples Step 1 -- PPT Samples Pool & Realiquot Samples 1-10 Step 2 -- Dry Samples Step 2 -- Dry Samples Step 2 -- Dry Samples 1-10 Step 3 Recon. Samples Step 3 Recon. Samples Step 3 Recon. Samples Pool & Realiquot Samples 1-10 Step 4 Deriv. Samples Step 4 Deriv. Samples Step 4 Deriv. Samples Step 4 Deriv. Samples Pool & Realiquot Samples 1-10 Step 5 Microwave Samples Step 5 Microwave Samples Step 5 Microwave Samples Step 5 Microwave Samples Step 5 Microwave

27 Summary It is possible to create a highly reproducible, accurate, and sensitive Metabolomics platform that can provide significant biological insight. It requires: continuous attention to detail constant repetition library of authentic standards Given the above it does not require the latest and greatest equipment

28 Thank You

29 Hierarchical Analysis Benign Localized PCa Metastatic Disease