Expand Your Research with Metabolomics and Proteomics. Christine Miller Omics Market Manager ASMS 2017

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1 Expand Your Research with Metabolomics and Proteomics Christine Miller Omics Market Manager ASMS 2017

2 New Additions to Agilent Omics Workflows Acquisition Ion Mobility Q-TOF IM All Ions MS/MS Find Features Profinder IM Browser Identify PCDL Manager Spectrum Mill Statistics GeneSpring MPP Pathway Pathway Architect Bridge DB Metabolomics dmrm Database and Method MassHunter VistaFlux IM All Ions MS/MS IM Browser B and Mass Profiler B enhancements MassHunter Profinder B GC/MS support added Spectrum Mill B support for IM and MPP 14.8 Mass Profiler Professional (MPP) 14.8

3 Agilent s Platform for Integrated Biology GeneSpring Platform LC/MS GC/MS MassHunter Qual/Quant ChemStation AMDIS Microarrays Feature Extraction Pathway Architect NGS Alignment to Reference Genome For Research Use Only. Not For Use in Diagnostic Procedures.

4 Integrated Biology Software GeneSpring Suite GeneSpring GX mrna Expression microrna Expression Variant Analysis with VCF qpcr Mass Profiler Professional Proteomics Metabolomics Pathway Architect Multi-omic Pathway Analysis NLP discovery Strand NGS DNA-Seq, RNA-Seq, ChIP-Seq, Methyl-Seq and small RNA-Seq

5 GeneSpring Suite Data Analysis and Biological Contextualization Supports multi-omics analysis Includes multivariate statistical analysis Offers correlation analysis for discovery of new biological relationships Connects meta data to biology Visualizes results directly on pathways Create pathway-directed experiments

6 MPP Proteomics Analysis Enabling Protein and Peptide Level Analysis Spectrum Mill MPP 14.8 Skyline Imports protein and peptide-level information from Spectrum Mill or Skyline Filters available for peptide-level and protein-level (abundance, frequency, PTMs) Protein/Peptide Entity Inspector for visualizing results

7 Enhanced Proteomics Workflow in MPP Filter, Analyze and Visualize at Protein and Peptide Levels Filter on peptides by PTMs Sequence Reproducibility Statistical and correlation analysis

8 Proteomics Visualizations in MPP Display Both Protein and Peptide Information

9 Increase Biological Understanding: Discovery and Targeted Approaches Discovery Metabolomics TOF or Q-TOF GC or LC Targeted Metabolomics Q-TOF QQQ GC or LC Discovery Proteomics Q-TOF LC Targeted Proteomics Q-TOF QQQ LC

10 Agilent Instrumentation For Metabolomics 7000D GC/QQQ 5977B GC/MS 7100 CE 1260 SFC Hi-DEF Q-TOF 6500 series Q-TOF 6500 series 7200B GC/Q-TOF 1290 Infinity II UHPLC QQQ 6400 Series TOF 6200 series

11 The Agilent 7250 GC/Q-TOF High Resolution and Mass Accuracy Reproducible Spectral Performance Backflush-ready Agilent 7890B GC Simultaneous High Resolution and Wide Dynamic Range Low Energy EI capability, Sensitive Detection

12 Comprehensive Metabolomics Application Software Profinder Extract features across complex data sets Qualitative flux analysis Interpret unidentified spectra VistaFlux Molecular Structure Correlator MassHunter Core Software Mass Profiler Professional Agilent METLIN & Fiehn Library Analyze data for statistically different features Annotate and identify interesting features Visualize results on pathways Pathway Architect

13 MassHunter Profinder The Power of 3D Batch Feature Finding Create Batch Untargeted MFE Targeted MFE Add all experimental data files to a project Optionally add group information Extract features from each data file in batch Compile list of all features with composite spectra Use compiled list for targeted feature extraction of each data file in batch Single software for untargeted and targeted feature extraction Fast, multi-threaded batch processing Recursive batch analysis minimizes false positives and negatives Compound centric review and manual editing

14 m/z Profinder: GC/MS Untargeted Feature Finding MFE Created Spectrum Spectral Comparison Library Spectrum RT Co-eluting ions with similar peak shape are grouped together as a feature A library-searchable spectrum is created for each feature MFE spectra can be searched in MassHunter Qual or ID Browser

15 Profinder GC/MS Molecular Feature Extraction Compound Table EI Spectra ECC Compound Details by File

16 Profinder B SP2 Enhancements Chromatowarping Supports accurate mass CE/MS or LC/MS data Corrects retention or migration time and optionally aligns to a retention/migration time database of known standards Supports untargeted, targeted or isotopologue data processing Enhanced Add/Remove Sample Files window Add or remove multiple custom grouping columns for each sample Import or export grouping columns from a.csv file

17 MassHunter Profinder B SP2 Chromatowarping for CE/MS & LC/MS Chromatograms Aligned Feature Extraction Original chromatograms with retention time variation Warped chromatograms with aligned retention times Extracted features with compound spectra

18 PCDL Manager and ID Browser 8.0 IM and MS/MS Library Support Create & manage custom database and library Identify features using database and library

19 Metabolomics dmrm Database and Method Integrated with Agilent s Metabolomics Workflows Database covers >215 central carbon metabolites Designed for 1290 Flex pump and 6460/6470 QQQ LC/MS Systems Provides an optimized method and database with stable, robust chromatography Separate & Detect LC/QQQ Pathways QQQ Data Acquisition & Analysis Quantitation Quant Statistics Pathway Architect Mass Profiler Professional

20 Increase Biological Understanding: Discovery and Targeted Approaches Discovery Metabolomics TOF or Q-TOF GC or LC Targeted Metabolomics Q-TOF QQQ GC or LC Discovery Proteomics Q-TOF LC Targeted Proteomics Q-TOF QQQ LC

21 Agilent Instrumentation For Proteomics Nanospra y source Agilent AssayMAP Bravo Agilent Jet Stream source Nanodaptor Q-TOF 6500 series 1290 Infinity II UHPLC QQQ 6400 Series

22 Agilent 6495B LC/Triple Quadrupole System Optimal Performance for Peptide Quant Highest analytical sensitivity with Agilent ifunnel technology Higher mass range for larger m/z peptides Mass range 5 3,000 Da Lower minimum dwell time allows more MRM transitions Minimum dwell time of 0.5 ms

23 Nanoflow vs. Jet Stream Proteomics Nanoflow LC/MS Lowest detection limit when sample is limited Longer column re-equilibration times Requires cleaner samples On complex samples, may hit limit of column capacity Requires more skill to maintain and use Standard Flow LC/MS Equivalent or better detection limit when sample is not limited Faster analyses with quicker column reequilibration Higher loading and peak capacity Best run-to-run reproducibility Easiest to use and maintain Greater column selection available Can be done on the same LC/MS system as metabolomics!

24 Agilent Jet Stream Proteomics Superior to Capillary LC Jet Stream source provides 3-5x signal increase compared to ESI Agilent Jet Stream showed same signal (and LOD) for 2.1 mm ID column at 400 µl/min 0.5 mm ID column at 17 µl/min mm column 0.5 mm column Agilent Jet Stream shows massflow dependent behavior Sensitivity depends on absolute amount (mass) injected Using smaller i.d. columns with standard sources does NOT increase sensitivity! ESI AJS

25 Nanodapter Kit Converts Standard Flow LC to Nanoflow Direct injection mode Trapping mode

26 LC/Q-TOF For Discovery Omics Proteomics and Metabolomics on One System 1290 Infinity II LC with 6545 Accurate Mass Q-TOF UHPLC Reproducible, fast separations Column capacity Robustness Reproducible Variety of commercial columns MS Accurate mass Good mass resolution 5 orders of dynamic range High isotope ratio fidelity Maintains performance at high acquisition speeds MS/MS for compound identification

27 Comprehensive Proteomics Application Software Spectrum Mill Identify proteins from discovery data Extract peptides features and MS/MS spectra from IM All Ions data IM 4D Feature Extraction MassHunter Core Software Skyline Targeted peptide analysis Extract peptides across complex data sets Profinder Mass Profiler Professional Analyze data for statistically different features Visualize results on pathways Pathway Architect

28 Agilent Spectrum Mill Proteomics Data Analysis Toolbox Improved search performance 2-5x faster IM Q-TOF support Spectral matching for IM All Ions MS/MS spectra CCS and drift time information included in exported spectral libraries for Skyline Integration with Mass Profiler Professional (MPP) workflows Export protein-level discovery results to MPP Export protein results to MPP with detailed peptide information Predicting peptides from Pathway Architect generated target protein list Concatenate databases tool for proteogenomics studies and host cell protein method development

29 Instrumental Details Hyphenating IM with Q-TOF Low drift field ion mobility ~60 Resolving Power (z = 1) Optics Cell Drift Tube Rear Funnel Q Detector Ion Source Agilent s design has the mobility separation before the collision cell

30 IM Separation Intensity IM Q-TOF Provides 4 Dimensions of Information Q-TOF Data 3 Dimensions of Information Chromatographic Time Intensity (color coded) IM-Q-TOF Data Adding a 4th Dimension of Information Chromatographic Time

31 Visualizing IM Q-TOF Data Agilent IM Browser MS Spectrum X = m/z Y = abundance Drift Separation X = drift time Y = abundance Selected drift frame Heat Map X = m/z Y = drift time Color = abundance

32 Agilent IM All Ions MS/MS Drift separation instead of quadrupole isolation All ions entering the collision cell are subjected to fragmentation voltage Fragment ions will be in the same drift frame as the precursor Better duty cycle compared to wide-band quadrupole isolation with equivalent resolution power!

33 IM All Ions MS/MS Acquisition Alternates Between No Collision Energy and Collision Energy No collision energy (MS only) With collision energy (MS/MS)

34 New 4D Feature Extraction for IM All Ions MS/MS Features are defined by retention time, accurate mass, abundance and drift time 4D feature finding can be performed in IM Browser B (1 file at a time) Mass Profiler B (batch of files) No collision energy (precursor) and collision energy (fragment ions) data are associated during 4D feature finding 4D MFE searchable spectra can be exported from IM Browser

35 Ion Mobility Proteomics Workflows Discovery and Targeted Analysis Mass Profiler Skyline Spectrum Mill MS/MS Library Viewer MPP Discovery Pathway Architect Targeted

36 IM All Ions MS/MS Proteomics Article in Rapid Communications in Mass Spectrometry This paper uses Jet Stream proteomics on the 6560 IM Q-TOF IM All Ions MS/MS acquisition 4D feature extraction Database searching of extracted MS/MS spectra

37 IM All Ions MS/MS for Proteomics Reproducible MS/MS Results No precursor isolation so product ions always produced Fast cycle time compared to wide-band isolation More points across chromatographic peak yields better reproducibility MS/MS Library Product Ion EIC areas for Replicate Analysis

38 Enhance Your Metabolomics and Proteomics Results Accelerate Your Research Integrated biology is a powerful approach to better understand biology Agilent continues to expand it s integrated biology offering ensuring that new technology and software integrates with existing multi-omic workflows Advance GeneSpring and MPP software Improve GC/Q-TOF performance for metabolomics Expand Profinder capabilities Introduced IM All Ions MS/MS workflows for proteomics

39 Latest Information From Agilent Omics eseminar Series LinkedIn Agilent Integrated Biology page agilent-integratedbiology?trk=rr_brands_carousel_lo go

40 Dublin st September Early Registration Deadline: 14 th June Visit: