Skyline & Panorama: Key Tools for Establishing a Targeted LC/MS Workflow

Transcription

1 Skyline & Panorama: Key Tools for Establishing a Targeted LC/MS Workflow Kristin Wildsmith Scientist, Biomarker Development LabKey User Conference October 6, 2016

2 Biomarkers enable drug development Drug development Research Pre-IND Phase I Phase II Biomarker development Biomarker Discovery Phase III (Pivotal) Clinical Practice Clinical Practice 2 Clinical validation Preclinical validation Technical development Pharmacodynamic Diagnostic Therapeutic Response

3 Mass spectrometry is a quantitative tool for biomarker development and Labkey server is used for data management LTQ-Orbitrap (High resolution) Biomarker Discovery Clinical validation Clinical Practice Discovery (unbiased) Multiplex panel Targeted-discovery Single analyte quantitation Triple Quad (High sensitivity) Labkey is a data management solution for MS data Development of customized folders accelerated Targeted MS assay development

4 New model for developing protein clinical biomarkers: Hypothesis driven multiplexing with mass spectrometry Target Specific Pathway Specific Disease Specific Proteomics Discovery (Literature) Candidate Biomarker List (1000s-10 0s) Targeted- MS assay Candidate Biomarker List (100-10s) Targeted-MS assay or immunoassay Clinical Marker(s) Exploratory Studies 100 s s of Candidates hypothesis-based focusing 10 s-100s of Biomarkers MS can provide a faster and more successful translation of protein biomarkers to the clinic

5 Multiplexed MRM assay developed for candidates Multiplex Candidate proteins in CSF Protein protease digestion Peptide 1 Peptide 2 Light (endogenous) Signature peptides +internal standards (heavy peptides) Peptide 3 Quantitation Light (endogenous) Heavy (std) Peptide 4 Heavy (std) LC-MS/MS

6 Skyline supports targeted MS techniques Skyline is an open-source software from MacCoss lab at University of Washington Vendor neutral Supports method development and data analysis: Targeted MS/MS Selected reaction monitoring (SRM, aka MRM) Parallel reaction monitoring (PRM) Data-independent-acquisition (DIA) / SWATH Targeted discovery with MS 1 quantitative methods Discovery proteomics with MS 2 methods Open-source software available from MacCoss lab at University of Washington:

7 Multiplexing enabled by targeted-proteomics software Peptide List Quick visual check of quantitation Check peak shape Light Retention time consistency Heavy istd Open-source software available from MacCoss lab at University of Washington:

8 Old workflow for multiplexed MRM creates data management challenges 8 Target Proteins Select Peptides & build MRM method Generate Data Peak integration and calibration curve generation QC sample data Iterative method development Complex, MANUAL data analysis Large data files from multiple platforms Statistical analysis and data visualizations

9 Improving the targeted proteomic workflow Data management simplification with Panorama Panorama is a repository server application supporting Skyline Module within LabKey server Open-source bioinformatics data management platform Searchable database storing Skyline data files 9 ASMS 2013 Panorama

10 Panorama Public Panorama increases rate of method development * MacCoss-lab hosted instance (includes CPTAC, ABRF shared libraries) Shares read-only data to support publications Workflow for sharing data with reviewers and then general public ASMS 2015 Panorama

11 Panorama Public: CPTAC Enabling data sharing 11

12 Panorama Partnership enables use of Panorama in secure environment and expand capabilities Collaborate directly with MacCoss lab and LabKey teams Installed Labkey server on-site Guide Skyline & Panorama software development priorities All partners contribute to common development fund All partners give input on priorities and design decisions Genentech/Roche partners since Oct 2013 (1 of 4) Field strongly supporting Skyline and Panorama development Academic/Government: 2 R01s 1 P41 1 U01 Industry: h"ps://skyline.gs.washington.edu/labkey/wiki/home/so7ware/skyline/page.view?name=funding

13 Sharing data internally Accelerating method building: Chromatogram libraries 13

14 Key areas of focus for Panorama Partnership Vision: Data analysis directly in Panorama 14 Tracking method development (versioning) Implementing data analysis tools directly in Panorama Quality Control folder AutoQC Statistics Adaptation of CPTAC Response Curve R script by Laura Harris on Roche Panorama test server

15 Panorama Partners progress Tracking method development: Version Chaining

16 Panorama Partners QC folder Development of QC folder to monitor instrument performance 16 View retention time, peak area, H/L ratio, etc. Add notes Zoom to details

17 Panorama Partners QC folder Define reference regions for QC statistics QC Levey-Jennings Plots

18 Panorama Partners QC folder Evaluate performance of multiple peptides Retention Time Run Date

19 Panorama Partners QC folder Track instrument performance

20 Panorama Partners QC folder Determine which feature drives variability in method Retention time variable

21 Panorama Partners QC folder Automatically upload QC runs to Panorama Vagisha Sharma, Skyline team, MacCoss Lab, UW

22 Panorama Partners QC folder Monitor active instruments using QC summary view 22

23 Skyline and Panorama expansion in 2016 Small molecule support 23 ASMS 2016

24 Panorama Partners vision: Simplifying the workflow Connecting Labkey s Panorama to data analysis tools 24 Data Storage and Analysis Data Transformation Method build and refinement Data Summarization and Visualization

25 Data analysis within Panorama: QuaSAR Accelerating method development using automated tools 25 QuaSAR processes SRM/ MRM-MS data calculating: limits of detection and quantification (LOD and LOQ), Mean and Coefficient of variation (CV) for all transitions of each peptide in a set of samples Peptide analyte concentration in unknown samples Runs the AuDIT algorithm to evaluate the transition quality and detect interference Shadi Eshghi Modified from QuaSAR script at

26 Panorama Partners Progress: Data Summarization Connecting LabKey s Panorama to Spotfire for visualization

27 Labkey helping to accelerate biomarker development through support of MS data management LTQ-Orbitrap (High resolution) Biomarker Discovery Clinical validation Discovery (unbiased) Multiplex panel Targeted-discovery Single analyte quantitation Triple Quad (High sensitivity) Developing customized features within Labkey to simplify method development and data analysis Clinical Practice 27 Data Storage and Analysis Data Transformation Method build and refinement Data Summarization And Visualization

28 Acknowledgements Genentech OMNI Biomarker Development Paul Auger Veronica Anania Shadi Eshghi Rod Mathews Lee Honigberg gred IT Jon Sasano Spotfire Laura Harris Joshua Haznedar Panorama Partners Josh Eckels (Labkey) Brendan MacLean (UW) Vagisha Sharma (UW) Tom Dunkley (Roche) Michel Petrovic (Roche) UW Targeted Proteomics Course 2014

29 Appendix

30 Large data files from multiple platforms creates data management challenges 30 Method and data generation Data QC and analysis Types of files generated: Skyline method and data visualization files Instrument method file Raw MS data Quantitation file Excel spreadsheets for import to Spotfire Spotfire QC file Spotfire biomarker result file Additional (externally generated) biostats *.csv files and R plots

31 Peptide levels by diagnostic group Spotfire visualization of multiplex results Joshua Haznedar PKPD

32 Panorama Partners QC folder Development of QC folder to monitor instrument performance 32 Retention Time Peak Area Light/Heavy Ratio

33 Panorama Partners Progress 2014 Custom Reporting

34 Panorama Partners Progress 2014 Point-and-Click Visualizations

35 35