IMPLEMENTATION OF PANORAMA INTO DAILY WORKFLOWS AND QUANTITATIVE PROTEIN PK ANALYSIS IN THE PHARMACEUTICAL SETTING Kristin Geddes Associate Principal Scientist Pharmacokinetics, Pharmacodynamics and Drug Metabolism Discovery Bioanalytics, West Point, PA Skyline Users Group Meeting, San Diego, CA June 3, 2018
Outline 1. Overview of workflows and instrumentation 2. Skyline and data processing 3. Panorama AutoQC Standard Curves PK Calculations 2
RT: 0.32-69.93 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 2.74 21.59 27.21 30.10 31.54 33.00 33.34 21.00 48.88 38.40 58.16 17.97 59.92 57.83 3.82 8.94 9.17 17.35 61.62 67.98 5 10 15 20 25 30 35 40 45 50 55 60 65 Time (min) 39.70 42.65 45.35 52.63 52.74 52.86 53.04 54.04 1963053 LC-MS Protein PK Assay Development NL: 1.58E7 Base Peak MS Relative Abundance 45.27 45.14 54.20 22.62 45.77 33.78 54.32 48.78 59.04 34.21 58.30 51.34 68.24 24.92 12.12 16.49 64.86 Nanoacquity Orbi Velos Mapping Peptide selection CE Opt Matrix Check 10 min Methods Agilent 6495 QQQ Target Assay Development/Refinement Sample Prep: ImmunoCapture In-solution Digestion Tissue Homogenization Digestion Optimization Standard curves Automation Sample Preparation 3
Thermo Orbitrap Velos Foundational Analytical Platforms Thermo QE-HF Waters Xevo-TQS QQQ Discovery and mapping Agilent 6495 QQQ Discovery, mapping and PRM experiments Thermo TSQ Vantage QQQ High throughput peptide/protein quantitation AB Sciex 6500 Q-Trap High throughput peptide/protein quantitation Quantitation of biomarkers and endogenous proteins High throughput protein/peptide quantitation 4
Skyline: The Foundational Software Tool With the wide variety of instrumentation in our laboratories, data analysis using Skyline is critical. Method building and refinement CE Optimization Spectral Libraries Calibration Curves Peak Areas Retention times 5
From Skyline Data to Reports for Collaborators SKYLINE Excel PKLIMS reporting tool Watson LIMS 6
How do we efficiently go from Skyline to a PK report? 7
Panorama and P3 Panorama is a freely-available, open-source repository server application for targeted mass spectrometry assays that integrates into a Skyline mass spec workflow. It has been implemented as a module within LabKey Server, an open-source bioinformatics data management platform with extensive support for proteomics and small molecule data and a security model rich enough to support clinical studies AutoQC uploader Organize projects across the group Allows for multiple projects to be set up on the server 8
Tracking Instrument and Assay Performance in Panorama: AutoQC We use instrument QC s for things such as instrument life-cycle management and knowing the instrument status before starting a run. AutoQC is great because it automatically sweeps system suitability files up to Panorama and adds the replicates to a Skyline document Allows for quick and easy view of the history of each instrument s system suitability (process control) Customizable views for date range, metric type, plot type, etc. 9
Assay History and Document Summary in Panorama Gives an overview of the file: Version of Skyline, Proteins, Peptides, precursors, transitions, replicates, calibration curves Under the Calibration curve sub divider, you can see the peptides in the run, navigate to the Figures of merit (FOM) and perform pharmacokinetic (PK) calculations. Gather information about the calibration curve 10
Interacting with Quantitative Data in Panorama: Details By selecting the details of the peptide of interest, you are able to drill down to see the individual peptide data 11
Interacting with Quantitative Data in Panorama: Calibration Curves Panorama reads the information from the Skyline file Summarizes by peptide Ability to export a PDF or image of the standard curve 12
Interacting with Quantitative Data in Panorama : Calibration Curves Set up calibration curves by changing the sample type in Skyline and assigning the analyte concentration Under peptide settings, chose regression fit, normalization method, regression weighting, MS level, units, and newly added in Skyline Daily Figures of merit 13
Interacting with Quantitative Data in Panorama : Figures of Merit Quickly view figures of merit by peptide for the entire document Easily obtained from the document summary 14
Interacting with Quantitative Data in Panorama : Figures of Merit The Figures of Merit report provides the following calculations: Mean - The mean of all replicates for a given sample. CV(%) - Coefficient of Variation for all replicates for a given sample (the standard deviation divided by the mean). Bias(%) Difference between the average values of the replicates for a given sample and the expected value Export Function n - The Number of samples used for above calculations. LOQ - The limit of quantitation, i.e., lowest concentration at which the %CV is less than 30%. ULOQ - The upper limit of quantitation, i.e., the highest concentration for standards at which the %CV is less than 30% SD - Standard deviation of all replicates for a given sample. 15
Interacting with Quantitative Data in Panorama : PK Calculations Requires some up-front input into Skyline Annotate Dose, time point, Dose Group, Subject ID, Dose Units 16
Interacting with Quantitative Data in Panorama : PK Calculations LabKey server 18.1 Currently supports Single Dose IV non-iv workflows are still in development Chose both C0 and Terminal time points Both Linear and Log-Linear graphs 17
Automated and Integrated Data Analysis Chorus Discovery Proteomics, Cloud Based, Qualitative and Quantitative Data Analysis and Storage Skyline Keystone Software for Targeted Protein Quantitation Assay Development and Data Analysis Panorama Server Software for Aggregating and Publishing Targeted Protein Quantitation Data Licensed by PPDM in 2015 (Merck is a Panorama Partner) Study Request Seamless Integration of Discovery Data Analysis, Targeted Method Development, Statistical Analysis, Method Sharing, and Report Generation Electronic notebook 18
Acknowledgements Merck PPDM Bioanalytical Group Dan Spellman Lisa O Callaghan BaoJen Shyong Kevin Bateman James Marr II LabKey Josh Eckels Marty Pradere Ron Dashwood Cory Nathe Skyline Brendan MacLean Nick Shulman Vagisha Sharma Michael MacCoss Panorama Partners Program 19
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