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Velos Pro and Orbitrap Elite The Latest Evolution of Ion Trap Technology: Velos Pro and Orbitrap Elite Jae Schwartz Indianapolis Users Meeting September 25, 2011 New Instruments Velos Pro Orbitrap Elite Quadrupole Linear Ion Trap Quadrupole Linear Ion Trap + Orbitrap Hybrid Instrument 2 The Velos Pro - Motivation Feature Application Benefit • UHPLC Cycle Times • More points across a chromatographic peak gives better 1 Faster Scanning quantitative performance • More MSN Scans During a Run, Or Shorter Run • More Information/Unit Time Enhance the Robustness • Improved Robustness to Reduce Instrument Down Time with 2 of Ion Optics High sensitivity S-Lens technology • Single digit RSDs, Competitive Quantitation 3 • Up to 6 orders of linear dynamic range on Ion Traps • First Ion Trap Designed for Quantitation. Improve Dissociation • Increases flexibility for structural elucidation (both proteomics and small molecule applications) 4 No Low mass cut off/TMT • Allows Highly Sensitive TMT Based Protein Quantitation 3 Ion Trap Relationship Between Scan Rate and Peak Width 0.7 0.6 0.5 0.4 0.3 FWHM(amu) 0.2 Scan Rate Range 1 0.1 Scan Rate Range 2 Scan Rate Range 3 0 0 5000 10000 15000 20000 25000 30000 35000 Scan Rate (amu/sec) * At ~1 mtorr 4 New Mass Analysis Scan Rate: “Rapid” Scan Rate 3,500 M/∆M Rapid Ion Ion Ion Mass FWHM=.52 66 KDa/sec Injection Isolation Dissociation Analysis 5,400 M/∆M Normal FWHM=.34 33 KDa/sec n-1 1 7,000 M/∆M Enhanced FWHM=.26 10 KDa/sec 350 INTERSCAN TIME 300 OTHER OVERHEAD MASS ANALYSIS (300-2000) 10,100 M/∆M Zoom 250 ACTIVATION FWHM=.18 ISOLATION 2.2 KDa/sec 200 ION INJECTION PRESCAN 150 36,440 M/∆M 100 FWHM=.05 Ultra Zoom 50 27 Da/sec 0 LTQ XL LTQ VELOS LTQ VELOS w LTQ VELOS PRO P/AGC 5 More Real Scan Speed Improvement: C:\j.horner\...\HCD-N-N-Top20_Short_002 4/21/2011 12:17:21 PM Protein Groups Unique Peptides RT: 0.00 - 130.07 Normal Scan Rate 52.79 NL: 100 3.77E8 Base Peak 9000 8108 80 70.11 MS HCD-N-N- 8000 7477 Top20_Short 60 _002 55.33 7000 40 25.13 35.00 41.32 46.34 64.67 75.91 6000 21.86 28.64 77.28 Relative Abundance 20 4.76 99.52 5.50 82.96 103.13 118.75 5000 0 0 10 20 30 40 50 60 70 80 90 100 110 120 13047061 Scans 4000 Time (min) 3000 1974 HCD-N-N-Top20_Short_002 # 21775 RT: 54.35 AV: 1 NL: 3.95E4 1867 T: ITMS + c NSI d Full ms2 [email protected] [50.00-935.00] 2000 120.01372 1000 30000 460.27264 0 545.33057 660.43414 20000 807.47168 Multiconcensus HCD Multiconcensus HCD Intensity 260.17169 147.04297 Normal Scan Rate Rapid Scan Rate 10000 328.15677 399.20895 201.05994 480.26218 589.37354 692.42554 922.52051 0 100 200 300 400 500 600 700 800 900 C:\j.horner\...\HCDm/z -R-R-Top20_Short_007 4/24/2011 3:56:12 PM Rapid Scan Rate 52.58 69.73 NL: 100 2.60E8 Base Peak 80 MS HCD-R-R- 60 53.90 Top20_Short 40.97 56.75 24.81 64.25 _007 57.19 40 34.58 45.70 75.18 31.53 57.88 Relative Abundance 64.57 82.56 20 4.414.95 13.59 21.57 36.23 48.93 76.54 84.22 94.52 100.54 103.22 115.07118.69 123.10 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Up to 10% 52083 Scans Time (min) More Scans 814.37787 250000 961.45288 with Rapid 667.31732 200000 120.07883 150000 157.14017 574.29041 Intensity 100000 504.25012 86.09660 185.08255 50000 267.11484 375.19061 440.22287 559.83539 650.27991712.19629 797.47675 870.48254944.43738 988.43304 1074.53516 1136.89819 0 100 200 300 400 500 600 700 800 900 1000 1100 m/z 6 Velos Pro Speed : Full Ion Tree in 1 second! RT: 6.19 - 6.82 6.44 100 90 80 70 60 50 40 Relative Abundance 30 20 10 0 6.2 6.3 6.4 6.5 6.6 6.7 6.8 Time (min) 8 MSn Spectra in 1 second! 7 SLTQ-Lens Velos Ion Optics- Generation Design: I Ion LTQ Optics Velos • Dual ID Variable Spaced Stacked Lenses: • Larger ID to capture entire expansion from transfer tube • Smaller ID to focus ion beam through conductance limiting aperture • Increasing spacing = increasing field penetration to focus ion beam • Small number of electrodes (18 total in one assembly) • Stainless Steel Electrodes and Supports for Ruggedness and Easy Cleaning • Alternate Phase RF on Lenses (650KHz, 300Vpp max) • Relatively Large Exit Aperture versus Skimmer • 5-10x Transmission Improvement! 8 LTQ Velos - Generation I Ion Optics . “S-LENS” in Velos increased Sensitivity by 10 . Bent Q0 Optics Designed to Catch Neutrals . Can Improve Robustness Further 9 The “Rotated Quadrupole” with neutral beam blocker To prevent material from building up on the rods, the quadrupole has been rotated 450. This opens up a new line of sight for the neutral beam. A beam blocker is now able to capture the neutral beam out side the body of the ion optics! This allows, for the first time, neutrals and ions to be separated in a linear optics design. 10 Velos Pro Ion Optics The majority of material strikes the beam blocker outside of the ion path. 11 Velos Pro Robustness Testing • Using the new rotated Q0, the 100% Velos Pro is 2x as robust as 90% the2x LTQincrease Velos in. 80% robustness averaged 70% ions ions 60% VELOS all 50% PRO 40% 30% 20% Relative Intensity Intensity Relative 10% 0% 0 5 10 15 20 DHB Infused (mg) 12 Ion Traps for Quantitation 1.E+07 1.E+06 1.E+05 Early Ion Traps had a 1.E+04 narrow dynamic range 1.E+03 due to inherent design 1.E+02 limitations Log(SIGNAL) 1.E+01 1.E+00 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 Log(Conc) 1.E+07 1.E+06 1.E+05 • Automatic Gain Control 1.E+04 • > 1 0X More Ions 1.E+03 • 1 0 Hz Fast Scanning 1.E+02 • Isolation during loading Log(SIGNAL) High Dynamic Range 1.E+01 • Detection 1.E+00 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 Log(Conc) 13 Injection (Isolation) Waveforms With new injection=isolation waveforms, the HPC acts just like a very good single quadrupole! m/z 524 100 80 60 40 20 IsolationEfficiency 0 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 m/z at Isolation Q Velos symmetric rod stretch Isolation profiles for Velos rf field: E=0 on axis instruments during loading. Even better isolation after loading. 14 Isolation while loading is key 6 3.0x10 5 1.5x10 Select only this ion while 6 2.5x10 5 loading into 1.0x10 the trap! 6 2.0x10 4 5.0x10 6 1.5x10 0.0 710 715 720 725 730 735 740 745 750 Intensity 6 1.0x10 5 5.0x10 0.0 200 400 600 800 1000 Mass 15 Isolation while loading is key Effect of Injection Waveforms: MS/MS of m/z 735 Lactoperoxidase Digest Effect of Injection Waveforms: MS/MS of m/z 735 Off Lactoperoxidase Digest On 105 Waveform OFF Low precursor intensity produces poor quality MS/MS 4 Intensity 10 103 0 50 100 150 200 Waveform ON Scan High precursor intensity Produces high quality MS/MS •Injection waveforms allow selective ion accumulation •Injection waveforms prevent 200 400 600 800 1000 1200 1400 signal variation due to matrix Mass based space charge 16 Faster Scan Rates – Higher Detected Ion Currents 3,500 M/∆M Rapid FWHM=.52 15 usec/amu 5,400 M/∆M Normal FWHM=.34 30 usec/amu 7,000 M/∆M Enhanced FWHM=.26 100 usec/amu 10,100 M/∆M FWHM=.18 Zoom 455 usec/amu 36,440 M/∆M FWHM=.05 Ultra Zoom 3700 usec/amu 17 Continuous Dynode Electron Multiplier (CDEM) Versus Discrete Dynode Electron Multiplier (DDEM) Output electrons ions electrons Glass channel plate Advantages: Advantages: • Smaller . Larger Surface Area • Cost Effective . Each multiplication stage can be optimized . Larger linear Dynamic Range . Longer life times 18 Linear Dynamic Range - 195/196 Intensity Ratio Comparison of High End of Linear Range 12 Old Detection System New Detection System 10 8 Peak RatioPeak ~20 uA ~160 uA 6 4 10 100 Current (µA) 19 New Higher Dynamic Range Detection System • Discreet Dynode Electron Multipliers (ETP-SGE) • Packaged as a Direct Replacement for Previous Continuous channel (Channeltron) Electron Multipliers • Extended dynamic range • Slower aging and longer lifetimes • 24 bit High Dynamic Range Electrometer/Pre Amplifier Circuit • Novel 160 uA electrometer board designed to handle high peak currents found in fast scanning micropackets. (now 24bit) • Packaged to replace current LTQ Velos Electrometer 20 Alprazolam 6 Order Quantification on Velos Pro Pg On RSD% Column 50000 0.01 14.58 40000 30000 0.02 10.76 Area C:\j.horner\...\AlpFull-VelosPro194 4/1/2011 3:55:51 AM 20000 10000 pg on Column Velos Pro 0.04 6.63 RT: 0.00 - 0.80 SM: 7G 10000 Y = 6852.68*X R^2 = 0.9918 W: 1/X 100 0.1 4.25 0 90 0 1 2 3 4 5 6 7 80 pg on Column 0.2 3.21 70 60 0.4 3.70 50 40 Relative Abundance Relative 1 3.59 30 20 10 Alprazolam 2 3.57 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Time (min) 4 3.47 70000000 10 2.48 60000000 20 1.98 RSD 10 fg on Column: 14.58% 50000000 40 4.03 40000000 100 4.33 Area 30000000 200 2.03 400 3.40 20000000 1000 3.85 10000000 Y = 6852.68*X R^2 = 0.9918 W: 1/X 2000 1.66 0 0 2000 4000 6000 8000 10000 4000 1.27 pg on Column 10000 2.16 21 Dynamic Range Summary: 12 Compounds + 1 IS in plasma (5 min run) C:\j.horner\...\ASMS_VelosPro_Run2_088 4/9/2011 12:42:13 PM 200 pg/uL RT:0.00 - 4.40 100 90 2 Scan 80 3 Scan 4 Scan 1 Scan 1 Scan 2 Scan Events Events Events Event Event Events 70 60 50 40 30 20 IS 10 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time (min) Norcodeine 286.30 CID 4 Methampmetamine 150.24 CID 5 Propranolol 260.16 CID >5 Codeine-d3 303.37 CID 5 Methoxymethamphetamine 180.26 CID >5 Alprazolam 309.09 PQD >5 Hydrocodone 300.37 CID >5 Lidocaine 235.34 CID >5 Sulfamethazine 279.09 CID 4 Testosterone 289.43 CID 5 Terfenadine 472.32 CID 6 Methylthioamphetamine 165.22 CID IS 4.2% Bupropion 240.11 CID >5 22 Dissociation Diversity HCD dd Decision Tree ETD CID Complimentary sequence coverage Acronym Long Title Pros/Cons Mechanism CID Collision Induced Dissociation Universal, no tuning required, high Resonance excitation of precursor at low efficiency q; low energy collisions with He Low mass cutoff PQD Pulsed “q” Dissociation Selective, no low mass cutoff, low noise Short resonant excitation of precursor at high q, rapid shift to very low q, high Tuning required, low efficiency energy collisions with He HCD Higher Energy Collision High efficiency, no low mass cutoff Acceleration of ion packet through N2 cell, Induced Dissociation high energy collisions with N2 Tuning required ETD Electron Transfer Dissociation Highly selective, no low mass cutoff, very Transfer of electron from radical low noise destabilizes molecule Doesn’t work on singly charged species 23 Use Existing High Pressure Multipole Collision Cell TRAP-HCD 1.
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