7 th Czech Mass Spectrometry Conference April 11th, 2018 Next Generation Technology for Reproducible and Precise Proteome Profiling Lars Kristensen, Ph.D. Application and training specialist The world leader in serving science
Agenda 1) Introduction and Q Exactive HF-X 2) High Resolution DIA Workflow 3) DDA+ Workflow (Label free quantification) 2
The Goal: Standardized, High Throughput Proteomics Large Scale Proteomics Sample Preparation Separation Data Acquisition Data Analysis 3
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 4
Q Exactive HF-X New architecture Optimized Scan Matrix with accelerated HCD: 40 Hz MS/MS Advanced DDA for bottom-up and top-down: Advanced Peak Determination HyperQuad Mass Filter with Advanced Quadrupole Technology Advanced Active Beam Guide Electrodynamic Ion Funnel C-Trap HCD Cell Ultra-High Field Orbitrap Mass Analyzer Dedicated transient for TMT 10plex: 50 K res High Capacity Transfer Tube Brighter ion beam: 2-3x More sensitive Improved S/N for intact proteins: 7.5 K res 5
Q Exactive HF-X Basis for increased speed and sensitivity Q Exactive HF MS/MS 18-22 Hz 32 ms maxit Q Exactive HF-X 28 Hz 32 ms maxit 32 ms Longer fill time Reduced scan overhead ahcd 32 ms Brighter ion beam (ion funnel), reduced scan overhead, and accelerated HCD (ahcd) is boosting acquisition speed Advantage for both MS and MS/MS mode Fast and high quality MS/MS acquisition up the 40 Hz with new 16 msec transient (7,500 resolution setting) 40 Hz 16 ms 16 ms maxit Comparable fill time Reduced scan overhead ahdc Orbitrap detection Maximum fill time with precursor ions Inter and intra scan overheads 6
High-Resolution DIA Workflow High-Resolution DIA Workflow Sample Preparation Separation Data Acquisition Data Analysis QUANTITATIVE PRECISE REPRODUCIBLE STANDARDIZED 7
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 Pulsar 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 using Cap flow ( 1.2 µl/min) 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 8
Spectronaut Pulsar Software Key Benefits Spectronaut Pulsar is specifically developed for the analysis of DIA & SWATH data sets Data analysis with retention time correction based on spiked reference peptides using irt Kit Spectral library generation from MaxQuant, Protein Pilot, and Thermo Scientific Proteome Discoverer Direct visualization of qualitative and quantitative results on protein level including FDR and P-values Processing of very large experiments (1000s of runs) Fast data analysis speed in less than 2 min per run DirectDIA library generation from DIA data (no need to create a DDA-based library) Designed for high throughput DIA data analysis 9
Balancing Efficiency Without Sacrificing Performance Nanoflow Greater # of proteins Greater # of peptides Greater sensitivity Longer total run times CapLC Greater Efficiency Shorter total run time ( 2X) Greater throughput More robust Less protein and peptide id s Gradient (min) Time (min) 400 350 300 250 200 150 100 50 0 Total analysis time (min) for Triplicates 60 min 60 min 30 min Nano Capillary Capillary # of IDs 10000 8000 6000 4000 2000 0 Total Proteins 60 min 60 min 30 min Nano Capillary Capillary # of IDs 80000 60000 40000 20000 0 Total Peptides 60 min 60 min 30 min Nano Capillary Capillary 10
High-Resolution DIA Workflow: Highly Precise Proteome Quantitation 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% % of total Quantitation variance 0 10 20 30 40 50 %CV Proteins Peptides CapLC DIA, 4ug HeLa, 60min, 120K -> Spectronaut Analysis 30000 20000 10000 0 5000 4000 3000 2000 1000 0 90% Proteins 83% Complete Quantification AND CV<20% AND CV<10% 74% 68% 11
DDA+ Workflow High-Resolution DDA+ Workflow Sample Preparation Separation Data Acquisition Data Analysis QUANTITATIVE PRECISE REPRODUCIBLE STANDARDIZED 12
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 using Cap flow ( 1.2 µl/min) 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 13
Proteome Discoverer 2.2 Software Key Benefits Enables large scale, multiplex proteomic studies (TMT 11-plex) 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 14
Maximizing Efficiency for Large Scale Proteomics Protein 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 Same # of protein ID in half the time Peptide ID time Q Exactive HF-X MS 40% Q Exactive HF MS Maximizing peptide identifications Highest peptide coverage Deep proteome analysis Spectral library building Saves time and samples in large-scale proteomics efforts time 15
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 16
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 17
DDA+ Enable Unrivaled Quantitative Precision Protein quantitation variance Peptide quantitation variance 100% 100% % of proteins 80% 60% 40% 20% 89% 98% 3329 proteins completely quantified % of peptides 80% 60% 40% 20% 81% 97% 22525 peptides completely quantified 0% 0 10 20 30 40 50 0% 0 10 20 30 40 50 %CV %CV Quantitation Precision Reproducibility Standardization 18
DDA+ Workflow: Greater Reproducibility Between Samples Protein groups 4000 3500 3000 2500 2000 1500 1000 500 0 Proteins 93% 62% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Cumulative replicates DDA+ Traditional DDA Peptide groups 30000 25000 20000 15000 10000 5000 0 Peptides 83% 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 19
Inter-Site Consistency Across Different Instruments Instrument Standardization Test Three Locations HeLa digest metrics Protein Peptide PSMs MS/MS 60 min gradient Average # of Protein Groups 4500 4000 3500 3000 2500 2000 1500 1000 500 # of Protein Groups 0 Germany 1 Germany 2 USA 1 Location Quantitation Precision Reproducibility Standardization 20
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