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High-throughput capillary-flow LC-MS proteomics with maximum MS utilisation
- 1. The world leader in serving science Alexander Boychenko and Chris Pynn 2018-07-26 High-throughput capillary-flow LC-MS proteomics with maximum MS utilization
- 2. 2 Content • The Thermo Fisher LC Portfolio: The right system for every application • The need for speed in low-flow LC-MS proteomics: high-throughput low-flow LC-MS solutions • Capillary-flow LC-MS with 150 µm ID columns • High-throughput LC-MS with 75 µm ID columns • 100% MS Utilization with Tandem LC-MS • Summary
- 3. 3 The Thermo Fisher LC Portfolio: The right system for every application
- 4. 4 Column ID: 50 µm 75 µm 150 µm 300 µm 0.5 mm 1.0 mm …2.1–4.6 mm Nano: <1.5 µL/min Capillary: 1–15 µL/min Micro: 10–100 µL/min Analytical: >100 µL/min 50 µL/min–5 mL/min 100 µL/min–8 mL/min100–1000 nL/min 50 nL/min–50 µL/min Sensitivity Throughput Based on recommended flow rate Thermo Scientific™ UltiMate™ 3000 RSLCnano System Thermo Scientific™ Vanquish™ Horizon System Thermo Scientific™ UltiMate™ 3000 Systems Thermo Scientific™ Vanquish™ Flex and Vanquish™ Duo UHPLC Systems Thermo Scientific™ EASY-nLC™ 1200 System Thermo Scientific HPLC and UHPLC Systems: LC Portfolio for LC-MS Applications
- 5. 5 Why do low-flow LC-MS: what are the benefits? 0 20 40 60 80 100 120 140 160 180 200 0 100 200 300 400 500 0 10 20 30 40 50 60 0 20 40 60 80 100 CapLC-MS MicroLC-MS NanoLC-MS Analytical LC-MS Flow rate, µL/min Sensitivitygainincomparisonwith analyticalLC-MSat450µL/min Low flow LC-MS The sensitivity gains were measured as a relative peak area averaged for Cytochrome C tryptic peptides ESI-MS exhibits a mixed behavior of both concentration and mass-sensitive detectors Sensitivity gains (experimental, relative to 2.1 mm ID) microLC-MS: 2-4 capLC-MS: 4-50 nanoLC-MS: >50 sub-nanoLC-MS: >100 Reasons • Improved ionization efficiency • Smaller droplets • Increased surface to volume ratio • Increase of analyte concentration with decrease of column ID
- 6. 6 Sensitivity: why is it improved with low-flow LC? HESI Source Thermo Scientific™ EASY-Spray™ Source Significant improvements in sensitivity for LC-MS analysis using an ESI source are observed at flow rates below 5 µL/min
- 7. 7 Capillary-flow LC with the Thermo Scientific UltiMate 3000 RSLCnano system Micro flow ternary pump for fast sample loading in trap and elute experiments Flow up to 2.5 mL/min, Pressure 620 bar Near zero sample loss with unique μL pick-up injections High precision for sub-μL volume injections UHPLC binary capillary flow pump for robust separations and high retention time precision Settable flow up to 50 µL/min Two low-dispersion snap-in valves for standardized workflows and custom column switching applications ~100 nL port to port volume Thermo Scientific™ nanoViper™ Fingertight fittings for ease-of-use and leak prevention
- 8. 8 The need for speed in low-flow LC-MS proteomics: high-throughput solutions
- 9. 9 “Triangular” and “rectangular” biomarker discovery and validation pipeline Throughput Sensitivity Discovery • High number of samples • Targeted quantification • Minimal sample preparation • LC-MS vs. ELISA • Low number sample • Multiplexed labelling • Pooled samples • Fractionation for deep quantitative profiling Analytical flow LC-MS on 2.1 mm ID column Nano-flow LC- MS method on 50-75 µm ID column Sub-nano-flow LC-MS <100 nL/min Validation 1000’s samples 1000’s analytes Discovery 1000’s samples 1000’s analytes • Similar large cohort size in discovery and validation • Same sensitivity and number of targets • Same robust LC-MS technology for discovery and validation • High analytical throughput
- 10. 10 How to acheive high-throughput with low-flow LCMS? UltiMate 3000 RSLCnano system Thermo Scientific™ Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ mass spectrometer HESI probe with 50 µm ID stainless steel needle EASY-Spray source Ion Max source EASY-Spray transfer line with 20 µm ID silica emitter Capillary column Q Exactive HF Capillary flow UltiMate 3000 RSLCnano Trap cartridge Recommended AlternativeThermo Scientific™ EASY-Spray™ column 150 µm x 150 mm, 2 µm – ES806 75 µm x 150 mm, 3 µm – ES800 Robust & SensitiveEasy-to-use & • Versatile platform – safe investment • Fast sample loading with integrated micro-flow loading pump • Fast injection routines • UHPLC capabilities • Optimized fluidics • Simple installation and operation • Low ID and short columns with low internal volumes • Temperature control to accommodate increased flow rates • High loading and peak capacity • Fast scan speed to detect very narrow peaks • Improved sensitivity at capillary and nano- flow rates • Advanced precursor detection and charge state recognition (APD) Solutions introduced @ASMS2017 @ASMS2018
- 11. 11 • Translational proteomics and biomarker validation • Population monitoring • Samples characterization for Biobanking • Food and beverage authentication • … Demands for high-throughput low-flow LCMS Applications Requirements 1.Robustness 2.Sensitivity 3.Throughput 4.Easy handling and setup of fluidic connections 5.Plug & play column consumable with UHPLC capability Flow rate, µL/min Throughput 75 µm ID 150 µm ID Flow rate, µL/min Sensitivity 75 µm ID 150 µm ID • Balance between sensitivity and throughput • Higher linear velocity results in higher throughput, but lower sensitivity • Ultra-fast low flow methods requires the operation of low ID columns at maximum possible linear velocity
- 12. 12 Capillary-flow LC-MS with 150 µm ID columns 150 µm x 150 mm, 2 µm ES806 Q Exactive HF-X UltiMate 3000 RSLCnano system Flow rate, µL/min 75 µm 150 µm Sensitivity
- 13. 13 Direct injection: how to get it right? 3 1.2 3 Flow rate, µL/min Sample loading Column washing and equilibration 0.0E+00 2.0E+08 4.0E+08 6.0E+08 8.0E+08 1.0E+09 1.2E+09 1.4E+09 0 20 40 60 80 100 0 10 20 30 40 50 60 Signal %B Time, min Sample loop is switched offline to reduce delay volume and is washed to avoid carryover Peptide separation
- 14. 14 Single shot capLC-MS analysis 3,507 3,813 4,392 4,060 4,301 4,826 0 1000 2000 3000 4000 5000 6000 1 ug 2 ug 5ug Proteingroups 21002 23918 2573426708 28515 30529 0 5000 10000 15000 20000 25000 30000 35000 1 ug 2 ug 5ug Peptidegroups • Deep proteome coverage with single shot analysis (>4000 protein groups in 60 min) • Comparable to nanoLC-MS performance with increased sample loading 0 20 40 60 80 Signal Time, min Direct injection 60 and 90 min cycle time 24 and 18 samples per day 60min 90min 60min 90min 60min 90min 60min 90min 60min 90min 60min 90min
- 15. 15 Quantitative reproducibility 4578 4550 4553 Column 1 Column 2 Column 3 29307 29552 30448 Column 1 Column 2 Column 3 RSD=1.8% RSD=1.9% Durable response factor 4578 4520 4209 85% 79% 87% Injection 1 Injection 350 Injection 1000 Long-term quantitative performance Protein Groups Peptide Groups Quantified, CV <20% Column to column reproducibility Protein Groups • Excellent column-to-column consistency was observed, including coverage of >4500 proteins, and ~30000 peptides all with RSD <2% between columns • Comparable proteome coverage and quantitative precision was observed throughout the 1000 injection experiment
- 16. 16 Where to find more information about capLC-MS? Link Link Link Link Link Link More information on our web-page https://www.thermofisher.com/nanoLCMS
- 17. 17 High-throughput LC-MS with 75 µm ID columns 75 µm x 150 mm, 3 µm PN ES800 Q Exactive HF-X Flow rate, µL/min Throughput 75 µm 150 µm
- 18. 18 How to maximize low-flow LC-MS throughput? Delay volume is around 1.0 µL 100nL 70nL 300nL 70nL 100nL 150nL 400nL Back Flush 100nL Gradient delay volume is below 300 nL Total internal volume ~ 1 µL ES800 75 µm x 150 mm, 3 µm Separation pump + Column compartment + Micro-flow loading pump Autosampler • Low gradient delay volume • Low total internal volume • High linear velocity – low delay time • Fast and precise gradient formation Nano-flow standard Pre-concentration kit
- 19. 19 High-throughput low-flow LC method explained Fast injection routine (< 1 min) Fast sample loading onto the column with micro-flow loading pump (6 sec @150 µL/min) Parallel sample elution and fluidic washing steps Parallel column washing/equilibration and sampler injection routine
- 20. 20 Typical BPC and TIC chromatograms Wide peptide elution window Extremely low carryover Zoom in
- 21. 21 Robustness of High-throughput LC-MS Method 0 2000 4000 6000 8000 10000 12000 0 30 60 90 120 150 180 Injection No. Protein groups PSMs MS/MS Peptide groups 0 1 2 3 4 5 6 0 30 60 90 120 150 180 Retentiontime,min Injection No. 0 1 2 3 4 0 30 60 90 120 150 180 PWHM,sec Injection No. [K].LTDcVVMR.[D] [K].SLTNDWEDHLAVK.[H] [K].STELLIR.[K] [K].TVTAMDVVYALK.[R] [K].VDNDENEHQLSLR.[T] [K].VNQIGSVTESIQAcK.[L] [R].EDSQRPGAHLTVK.[K] [R].TVSLGAGAK.[D] Protein groups Peptide groups PSMs MS/MS 0.0E+00 1.0E+07 2.0E+07 3.0E+07 4.0E+07 5.0E+07 0.E+00 2.E+06 4.E+06 6.E+06 8.E+06 1.E+07 0 30 60 90 120 150 180 Area Area Injection No. Retention time Peak area PWHM SD < 0.1 min PWHM < 3 sec RSD < 10% Maximum speed TOP 40 MS2 7.5K IT MS2 14 sec
- 22. 22 How do we outperform competitors? 14.4 minutes 8 minutes Vendor “E” UltiMate 3000 RSLCnano 180 samples/day MS utilization – 75% per run 100 samples/day MS utilization – 42% per run SignalSignal 0 71 62 53 4 Previous injection Next injection Previous injection Next injection 1 min 3 min UltiMate 3000 RSLCnano system compared to Vendor “E”: Almost double the throughput Higher peak capacity More equal peptide distribution
- 23. 23 ASMS2018 poster High-throughput LC-MS: where to find more information? Post 1 Post 2 Post 3 Tweet 3 Tweet 2 Tweet 1 Link In review
- 24. 24 2 x 75 µm x 150 mm, 3 µm; PN 164568 Q Exactive HF-XTandem Pre-concentration setup with UltiMate 3000 RSLCnano system Flow rate, µL/min Throughput 75 µm 150 µm 100% MS Utilization with Tandem LC-MSTandem low-flow LCMS for maximum MS utilization
- 25. 25 Enhanced Productivity with Tandem low-flow LC • Tandem nano LC is applied to increase throughput. • Analyte separation is carried out on two columns in an alternating fashion • While one column is used for separation the other is washed and re-equilibrated for the next run • Tandem nano LC employed for long gradient / deep dive proteomics applications run on long columns, enables multiple washing steps to be carried out on the offline column. Tandem operation – Separation matches washing and equilibration Cleaning operation -Long gradient for separation on Column A is combined with consecutive short gradients for washing column B 0 20 40 60 80 100 1 11 21 31 41 51 61 71 81 91 101 111 121 Column A Column B 0 20 40 60 80 100 1 11 21 31 41 51 61 71 81 91 101 111 121 Column A Column B %,B %,B
- 26. 26 High throughput low-flow Tandem LC setup to ESI MS waste waste Separation pump Equilibration pump Separation pump + Column compartment + Micro-flow loading pump Equilibration pump Autosampler 3 pumps 2 nano columns 2 trap cartridges 2 switching valves One LC-MS method
- 27. 27 Tandem low-flow LC-MS method explained Peptide elution window: ~ 6.5 min Column 1, cycle time: 7 min Method duration and raw file length: 7 min Prepare for next injection routine Sample loop strong solvent wash Trap 2 strong solvent wash & equilibration Done in parallel with loading pump, syringe, 2nd nano pump Sampler wash between reinjections Sample load onto trap 2 Sample transfer to 2nd column Column 2 washing Column 2 equilibration Trap 2 washing Next gradient on column 2 Previous gradient on column 2 Look ahead injections: sample is loaded onto the second trap cartridge in parallel with current run Trap cartridge is washed with strong wash solvent using micro- flow pump Injection fluidics is washed in parallel with peptide separation The delay in peptide elution is removed by intelligent use of post column flow diversion Method can be fully adapted according to the required gradient length The same pump is used for separation on column 1 and column 2
- 28. 28 High throughput, Tandem low-flow LC: what are the benefits? RT: 0.00-7.01 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time(min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 3.63 3.65 3.71 4.50 3.13 1.80 2.401.82 5.74 3.43 5.77 1.78 2.82 5.80 3.78 4.17 2.42 1.84 2.55 0.820.80 1.04 1.13 4.94 3.95 4.971.470.490.34 1.97 5.38 5.854.600.30 5.15 6.345.99 6.856.87 6.46 NL: 9.46E8 BasePeakF: FTMS+p NSIFull ms [350.0000-2000.0000] MS 7min_HeLa_200ng_MS1_ column1_2018042022021 4 RT: 0.00-7.01 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time(min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 RelativeAbundance 3.62 3.60 3.68 4.543.70 4.52 4.56 3.06 3.05 5.96 3.42 5.93 2.24 4.16 1.60 1.59 2.71 1.62 4.212.26 2.98 2.280.15 3.28 5.571.65 0.66 5.020.16 5.031.41 6.003.950.81 4.99 0.99 5.590.34 6.025.234.742.021.02 6.09 6.481.99 6.50 6.60 NL: 7.91E8 BasePeakF: FTMS+p NSIFull ms [350.0000-2000.0000] MS 7min_HeLa_200ng_MS1_ column2_2018042022092 5 Column 1 Column 2 7 min 7 min 972 5374 6129 10399 1175 5587 6689 12948 0 2000 4000 6000 8000 10000 12000 14000 Pre-concentration Tandem Protein groups Peptide groups PSMs 0 200 400 600 800 Count 0.500 1.000 1.500 2.000 2.500 3.000 3.500 4.000 4.500 5.000 5.500 6.000 6.500 7.000 PSMs-RT[min] PSMs > 200 samples per 24 hours ~100% MS utilization Good separation performance Reproducible peptide separation profiles on column 1 and column 2 Maximum MS speed utilization
- 29. 29 Summary
- 30. 30 Conclusions • Time is Money! • There is an increasing demand for speed, sensitivity and MS utility in LC-MS applications in industrial and translational proteomics and beyond • The UltiMate solution for all your low-flow needs • The UltiMate 3000 RSLCnano is uniquely capable of delivering ultra high throughput, low-flow applications due to its: • Integrated micro-flow pump for high speed online sample loading and desalting and trap and loop washing • Wide pressure / flow footprint of 50 nL/min to 50 µL/min at up to 900 bar column pressure • Robust pressure stable tool free industry leading nanoViper connections • Offering high throughput AND sensitivity • High through-put low-flow LC-MS methods using nano columns and capillary flow rates yield uncompromising levels of speed and sensitivity whilst delivering as yet unseen levels of MS utility • Make the most of your MS with Tandem low-flow LCMS • Tandem LC-MS is possible through the simple addition of a low-flow pump and column set • Intelligent method control combined with look ahead injections enable continuous back-to-back MS data generation for the first time in the history of low-flow LC-MS
- 31. 31 • Complete with schematics, method parameters and example data Last but not least…. The ALL NEW • Comprehensive and easy to follow • Detailed information from instrument setup to best practices • Available for free download on the RSLCnano webpage • https://www.thermofisher.com/order/catalog/product/ULTIM3000RSLCNANO • And as a hard copy shipped with each application kit • UltiMate 3000 RSLCnano Standard Applications Manual (V 3.0)
- 32. 32 Thank you!