2017 Metabolomics Seminars Pushing the Leading Edge in Protein Quantitation: Integrated, Precise, and Reproducible Protein Quantitation Workflow Solutions The world leader in serving science
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Cancer Moonshot Goal: To detect cancer at an early stage while providing additional therapies to more patients. 4
Proteins Are the Machinery, Markers, and Targets for Cancer It is the proteins that comprise most of the biomarkers that are measured to detect cancers, constitute the antigens that drive immune response and inter and intracellular communications, and it is the proteins that are the drug targets for nearly every targeted therapy that is being evaluated in cancer trials today. Conrads et al. 2016 Clinical Cancer Research 5
The Goal: Standardized, High Throughput Proteomics Large Scale Proteomics Sample Preparation Separation Data Acquisition Data Analysis 6
Versatile Workflow Solutions For Your Needs Quantitative Our Solution Precise Reproducible A new standard in quantitative, sensitivity, accuracy and precision Sound Statistical Analysis Desirable Method Standardized Methods HR DIA DDA+ Workflows Curated Methods 7
Flexible Quantitative Workflow Solutions High-Resolution DIA Workflow Unparalleled proteome coverage and dynamic range DDA+ Workflow Unsurpassed quantitative precision and reproducibility Highest depth of proteome coverage and quantitative insight Robust quantitative precision Biospecimen profiling Digital archiving Unrivaled precision in precursor quantitation Maximize complete, reproducible quantitation across samples Minimize missing values among samples Cellular signaling studies Mechanism of action studies PTM profiling 8
High-Resolution DIA Workflow High-Resolution DIA Workflow Sample Preparation Separation Data Acquisition Data Analysis QUANTITATIVE PRECISE REPRODUCIBLE STANDARDIZED 9
High-Resolution DIA: Unparalleled Proteome Coverage and Dynamic Range Workflow Thermo Scientific UHPLC Systems Thermo Scientific EASY-Spray LC Column Thermo Scientific Q Exactive HF-X Hybrid Quadrupole-Orbitrap MS Spectronaut software Thermo Scientific UltiMate 3000 RSLCnano system Direct inject or preconcentration mode Thermo Scientific Viper fittings 150 µm ID x 150 mm, Sensitivity and robustness RT stability <1% observed for 350 injections Increased acquisition speed Advanced precursor determination Same # of protein IDs half the time Designed for Speed and Coverage 10
Spectronaut Software Key Benefits Spectronaut software is specifically developed for the analysis of DIA & SWATH data sets Data analysis with retention time correction based on spiked reference peptides-hrm calibration kit or irt Kit Spectral library generation from MaxQuant and Thermo Scientific Proteome Discoverer software search results Direct visualization of qualitative and quantitative results on protein level Fast data analysis speed in less than 2 min per run Designed for high throughput DIA data analysis 11
Time (min) Balancing Efficiency Without Sacrificing Performance # of IDs # of IDs Nanoflow Greater # of proteins Greater # of peptides Greater sensitivity Longer total run times CapLC Greater Efficiency Shorter total run time ( 2X) Greater throughput Comparable protein and peptide id s Gradient (min) 400 350 300 250 200 150 100 50 0 Nano Total analysis time (min) for Triplicates Capillary Capillary 10000 8000 6000 4000 2000 0 Total Proteins Nano Capillary Capillary 80000 60000 40000 20000 0 Total Peptides Nano Capillary Capillary 12
High-Resolution DIA Workflow: Highly Precise Proteome Quantitation % of total Maximize depth of coverage 50000 Peptides 40000 Robust quantitative precision Confident in IDs Short analysis time 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Quantitation variance Proteins Peptides 0 10 20 30 40 50 %CV CapLC DIA, 4ug HeLa, 60min, 120K -> Spectronaut Analysis 30000 20000 10000 0 5000 4000 3000 2000 1000 0 90% Proteins Complete Quantification AND CV<20% AND CV<10% 74% 13
DDA+ Workflow High-Resolution DDA+ Workflow Sample Preparation Separation Data Acquisition Data Analysis QUANTITATIVE PRECISE REPRODUCIBLE STANDARDIZED 14
DDA+ Workflow: Quantitative Precision and Reproducibility Workflow UHPLC Systems EASY-Spray LC Column Q Exactive HF-X MS Thermo Scientific Proteome Discoverer 2.2 software UltiMate 3000 RSLCnano system Direct inject or preconcentration mode Viper fittings 150 µm ID x 150 mm, Sensitivity and robustness RT stability <1% observed for 350 injections Increased acquisition speed Advanced precursor determination Same # of protein IDs half the time Designed for Precision and Reproducibility 15
Proteome Discoverer 2.2 Software Key Benefits Enables large scale, multiplex proteomic studies and captures confident protein results which enables confident reproducibility Improved Label-free Quantitation Feature mapping Retention time alignment Feature linking across files Minora Feature Detector node Detects chromatographic peaks and features according to the specified quantification approach Minimizes missing data points and maximizes quantitative insights Most comprehensive data analysis platform for qualitative and quantitative proteomics research 16
Protein ID Maximizing Efficiency for Large Scale Proteomics Peptide ID Q Exactive HF-X MS 50% Q Exactive HF MS Maximizing protein identifications Quick screening of complex samples Quality control of complex samples Assessment of sample concentration time Q Exactive HF-X MS 40% Maximizing peptide identifications Saves time and samples in large-scale proteomics efforts Q Exactive HF MS Highest peptide coverage Deep proteome analysis Spectral library building time 17
Maintaining Sensitivity at Increased Robustness caplc with Q Exactive HF-X MS Increased sensitivity on QE HF-X Increased peptide identifications at higher robustness Higher reproducible protein identifications with reduced total run times Sensitivity Robustness 3.0E+09 2.5E+09 Q- Exactive HF-X MS Q- Exactive HF MS 4.0E+09 3.5E+09 3.0E+09 Blue= 1 µg (nanolc) cell lysate digest Red = 4 µg (caplc) cell lysate digest 2.0E+09 2.5E+09 1.5E+09 2.0E+09 1.0E+09 1.5E+09 1.0E+09 5.0E+08 5.0E+08 0.0E+00 0 10 20 30 40 50 60 1 ug HeLa + 150 um x 15 cm Easy Spray 0.0E+00 0 10 20 30 40 50 60 75 µm ID x 15 cm vs 150 µm ID x 15 cm 18
Real Benefit of Using DDA+ Workflow 97% 97% 82% DDA+ workflow compared to DDA 15% gain in completely quantified proteins 50% Quantified Proteins 47% gain in completely quantified peptides Maximizes quantitation Quantified Peptides New DDA+ Traditional DDA caplc DDA+, 4ug HeLa, 60min, 120K/7.5K, 19ms, Top 40 -> Proteome Discoverer 2.2 Label-free Quant 19
DDA+ Workflow: Protein Quantitation Rep 1 Rep 2 Rep 3 Completely quantified proteins Missing data = sparse quantitation Complete quantitation 82% quantified during MS Rep 1 Rep 2 Rep 3 caplc DDA+, 4ug HeLa, 60min, 120K/7.5K, 19ms, Top 40 -> PD 2.2 Label-free Quant Quantitation Precision Reproducibility Standardization 97% quantified from MS and Protein Discoverer 2.2 software 20
DDA+ Workflow: Near Complete Peptide Quantification Rep 1 Rep 2 Rep 3 Completely quantified peptides Missing data = sparse quantitation Complete quantitation 50% quantified during MS Rep 1 Rep 2 Rep 3 97% quantified from MS and PD 2.2 caplc DDA+, 4ug HeLa, 60min, 120K/7.5K, 19ms, Top 40 -> PD 2.2 Label-free Quant Quantitation Precision Reproducibility Standardization 21
DDA+ Enable Unrivaled Quantitative Precision % of proteins % of peptides Protein quantitation variance Peptide quantitation variance 100% 100% 80% 89% 98% 80% 81% 97% 60% 60% 40% 3329 proteins completely quantified 40% 22525 peptides completely quantified 20% 20% 0% 0 10 20 30 40 50 0% 0 10 20 30 40 50 %CV %CV Quantitation Precision Reproducibility Standardization 22
Protein groups Peptide groups DDA+ Workflow: Greater Reproducibility Between Samples Proteins Peptides 4000 3500 3000 2500 93% 30000 25000 20000 83% 2000 62% 15000 1500 1000 10000 500 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Cumulative replicates DDA+ Traditional DDA 5000 0 20% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Cumulative replicates DDA+ Traditional DDA Quantitation Precision Reproducibility Standardization 23
Inter-Site Consistency Across Different Instruments Average # of Protein Groups Instrument Standardization Test Three Locations HeLa digest metrics Protein Peptide PSMs MS/MS 60 min gradient 4500 4000 3500 3000 2500 2000 1500 1000 500 0 Germany 1 Germany 2 USA 1 Location # of Protein Groups Quantitation Precision Reproducibility Standardization 24
Multi-Center Study to Demonstrate Large-Scale Capabilities Objective: Determine analytical robustness and reliability between laboratories Comparability of measurements between laboratories and define critical parameters Ring trial participants adapt a system suitability test protocol to maintain analytical performance Determine the range of accuracy and precision that users can expect to achieve following the standardized product/assay Thermo Fisher BRIMS Center The standardization enables the transfer of measurements between laboratories Phase 1 Labs identified, resources secured, SOP established Phase 2 Study design, study setup, data collection Phase 3 Data review, report-out 25
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Median CV% Increased Throughput: Cap LC DIA Q Exactive HF-X MS vs. Q Exactive HX MS 46217 Peptide Precursors (1% FDR) ID CV% <20% CV% < 10% > 20% Increase 37367 34603 Half the time 42206 9.5 9.8 Peptide precursors per Protein Group 9.2 ID CV% <20% CV% < 10% 8.0 8.3 7.9 9.0 9.5 8.9 25990 28231 28716 21095 19772 QE HF-X 60mins QE HF 60mins QE HF-X 30mins 7.0% 8.9 % 6.9 % QE HF-X 60mins QE HF 60mins QE HF-X 30mins QE HF 60mins QE HF-X 60min QE HF-X 30min * 4ug HELA digest, 3 technical replicates, 120k @ MS1 Quantitation Precision Reproducibility Standardization 30
The Age of Multi-Omics Is Here. Are We Ready? Proteomics is being used to discover and establish the protein landscape of cancer cells or tissues Proteomic measurements complement genomics/transcriptomic measurements by reducing the vast number of potentially actionable somatic mutations and identifying genomic variances that might be actionable Proteomics Complements Genomics 31