2007 GeneSpring MS GeneSpring for Metabolite BioMarker Analysis using Mass Spectrometry data Agilent Q-TOF VIP Visit Jan 16-17, 2007 Santa Clara, CA Thon de Boer GeneSpring MS Product Manager

2007 Definition of a Biomarkers Used to indicate or measure a biological process (for instance, levels of a specific protein in blood or spinal fluid, genetic mutations, or brain abnormalities observed in a PET scan or other imaging test). Detecting biomarkers specific to a disease can aid in the identification, diagnosis, and treatment of affected individuals and people who may be at risk but do not yet exhibit symptoms. Any molecular species found to provide correlation to a particular phenotype or perturbation of a biological system. Co-variant analysis of multiple biomarkers or patterns usually results in higher correlation confidence. A molecular marker associated with a biological function.

2007 GeneSpring MS – GeneSpring for Mass Spectrometry and Biomarker analysis 1.Allows the easy import, storage and visualization of LC/MS(/MS) and GC/MS data for many samples and exp. Designs 2.Identify significantly different metabolite abundances across experimental conditions without the need to be a statistical expert

2007 GeneSpring MS for Biomarker discovery Built on the Agilent GeneSpring Platform

2007 Applications of GeneSpring MS Find and monitor metabolite biomarkers for drug toxicity screening Monitor the drug production plant’s efficiency Classify unknown patient samples for disease outcome prediction based on their metabolite profiles

2007 Case study – Mayo Clinic – Poster at ASMS

2007 PCA Analysis of 347 Features from 1-way ANOVA of Pxo99 and Mock classes only in resistant & susceptible Rice Lines TP309-NT TP309-Mock TP309-Pxo99 Xa21-NT Xa21-Mock Xa21-Pxo99 Xa21_Pxo99/RaxST- Xa21 Line TP309 Line PCA before 1-way ANOVA TP309-NT & Mock TP309-NT TP309-Mock TP309-Pxo99 Xa21-NT Xa21-Mock Xa21-Pxo99 Xa21_Pxo99/RaxST- Xa21-NT Xa21- Mock & Xa21-Pxo99 (Resistant) TP309-Pxo99 & Xa21-RaxST (Infected) PCA after 1-way ANOVA ALL 1822 QC Features 347 Significant QC Features TP309TP309-Xa21

2007 GeneSpring MS GeneSpring MS is designed to serve the Metabolomics needs of our customers Metabolomics pillar of the GeneSpring Platform

2007 LC/MS Analysis Metabolomics Workflow - Overview Sample Preparation Purpose: extract and sub- fractionate metabolites from sample Typical samples: Body fluids Tissue Plants GC/MS Analysis Purpose: Separate and detect metabolites by the appropriate tool Purpose: Find meaningful differences in sample sets Statistical Analysis Purpose: Find and quantitate all metabolites Peak Finding Purpose: Correct data for retention time and response drift Data normalizati on Purpose: Understand the biological meaning of the data System Biology Analysis Metabolite Identification Needed for Understanding

2007 Feature Extraction Results in Reduced Set of Mass Features

2007 LC/MS Workflow #1: Load Agilent MHD files GeneSpring MS Identify diff. expressed Biomarkers Using statistics List of interesting mass values , pI= , pI= , pI= , pI=4..WIFF files (Q)TOF Mass Hunter Mol Feat Extr MHD Files Link to Metlin Etc. for Structure search.D files

2007 LC/MS Workflow #2: TOF-QTOF, ID before loaded in GSMS List of interesting mass values (+ RT) Create Inclusion list file Re-run Samples on MS/MS (Ion Trap?) Picking just the Interesting peaks for MS/MS MS/MS data files Spectrum Mill MS/MS Search MS/MS run (QTOF w same LC setup) Annotate peaks In GeneSpring MS GeneSpring MS Identify diff. expressed Biomarkers Using statistics.WIFF files TOF MHD Files Mass Hunter Mol Feat Extr

2007 Workflow # 4: GC/MS analysis of metabolites GeneSpring MS Identify diff. expressed Biomarkers Using statistics List of interesting mass values GC/MS AMDIS DB search (NIST) ELU/FIN files Unid/Identified masses

2007 GeneSpring MS allows you to… Load data from a variety of MS systems 1.Load data from Agilent LC/MS (Q)TOF system (MHD files) 2.Load data from Agilent GC/MS system (ELU/FIN files from AMDIS 3.Load data from Agilent QQQ (Quant Result Files) (In 1.1) 4.Load data in mzData/mzXML format from LC/MS(/MS) systems (in 1.1)

2007 GeneSpring MS allows you to… Compare mass features across different runs Each mass feature is represented as an entity with the following properties –RT; Retention time, or RI, retention index (normalized RT) –Mass; neutral mass

2007 GeneSpring MS allows you to… Compare mass features across different runs Mass features are “aligned” from one run to the next based on the similarity between the RT/mass values –For GC/MS data fragmentation spectra are used for alignment Interference Matrix Target

2007 GeneSpring MS allows you to… Find statistically significant differences in abundance Abundance of the metabolites or peptides can be compared across different runs using advanced statistics –ANOVA –T-test –Volcano plots –Correlation with meta data

2007 GeneSpring MS allows you to… Use PCA for QC and Biomarker discovery

2007 GeneSpring MS allows you to… Performs a variety of statistical analyses 1.Class prediction –K-nearest neighbors –SVM 2.Clustering –K-means –SOM –QT clustering 3.Hierarchical trees –Complete, average and single linkage (w. bootstrapping)

2007 GeneSpring MS allows you to… Visualize the MS data and analysis results in a variety of ways Variety of plots to visualize RT vs Mass plots RT vs Ratio Mass vs Ratio Graphs of abundance across conditions Scatter plots Chromatograms

2007 Continue the workflow by creating inclusion lists for LC/MS/MS run on the QTOF or the QQQ After finding potential biomarkers using the statistical techniques in GeneSpring MS, you can create Inclusion List files for the QTOF (or Ion Trap) and QQQ to determine identity of the biomarkers using the QTOF or to run large scale validation experiments with the QQQ

2007 Thon de Boer GeneSpring MS Product Manager Thank you for your attention