Symposium: New Alternatives in High- Resolution Mass Spectrometry Orbitrap Mass Spectrometry: Ultra- high Resolution for Every Lab Alexander Makarov March 14, 2012 What is Orbitrap™ analyzer? Orbitrap analyzer = = Orbital trapping + Image current detection + Electrodynamic squeezing + External pulsed ion source 2 The unstable world of electrostatic trapping Earnshaw's theorem (1842): “A collection of point charges cannot be maintained in a stable stationary equilibrium configuration solely by electrostatic interaction of the charges” …but moving No promise? charges could be Orbital traps stable! Kingdon (1923) 3 TOF Adventures: Prelude to the Orbitrap analyzer k U (r, z) 4z 2 r 2 / 2 R2 4ln(r / R ) 2 m m Gall L.N., Golikov Y.K., Aleksandrov M.L., Pechalina Y.E., Holin N.A. SU Pat. 1247973, 1986. 1st implementation of quadro- logarithmic potential in mass spectrometry (1989) “A mosquito- catcher” 4 Orbital trapping “Ideal Kingdon trap”: Characteristic frequencies: Quadro-logarithmic potential # Frequency of rotation 2 k 2 2 2 # Frequency of radial oscillations 2r U (r, z) 4z r / 2 Rm 4ln(r / Rm ) 2 # Frequency of axial oscillations 2z C R S2 z D m T 1 2 E R U C R S2 D m T 2 r z E R U k z m / q Only this frequency does not depend on energy, angle, etc. “Beauty will save the world.”- F.M. Dostoevsky and is used for mass analysis 5 Detection of Ions in the Orbitrap analyzer Image current detection #All-mass detection #Noise equivalent to <10 / Frequency of axial oscillations of each ring induces an image current on split outer electrodes Multiple ions in the Orbitrap generate a complex signal with frequencies determined using a Fourier Transformation I(t) t 6 I(t) Injection of Ions into the Orbitrap analyzer A short ion packet of one m/z enters the field Increasing voltage squeezes ions “Excitation by injection” is initiated Voltage stabilises and ion trajectories are also stabilized Angular spreading forms ROTATING RINGs bouncing back and forth 7 I(t) Proof of Principle: Orbitrap MS with Laser Ion Source Field compensator Ion source 0.8 sec High voltage 8 M record length 10 Ms/s (borrowed amplifier LeCroy) 0.8 s transient f =711 kHz,f=2.39 Hz f/f 300000 M/M=½ f/f 150000 HD Technologies Ltd A.A. Makarov, Anal. Chem., v.72 (2000), No.6, From Jan. 25, 2000- within Thermo Inc. 8 p.1156-1162. (in Thermo Masslab, Manchester, UK) Reasons Why Orbitrap Would Never Work # Not possible to provide ion packets with required spatial and temporal parameters for continuous ion sources Tolerance requirements on electrodes are not realistic Injection and central slots ruin resolving power and mass accuracy Vacuum requirements are ridiculously high and can not be met Ions can not be injected with high efficiency Wide mass range can not be injected and captured Image current preamplifier will be destroyed by pick-up during injection Noise from high voltage power supply will overwhelm preamplifier Surface potentials would disturb and scatter ions Mass accuracy will be poor because of voltage drift & noise Large ion numbers cannot be properly injected or analyzed Electrodes shape, rotational and radial frequencies will cause unmanageable mass-dependent harmonics All these reasons are valid and require a lot of work! 9 Injection of Ions into the Orbitrap analyzer Ions are stored and cooled in a curved RF- C-trap only quadrupole (C- Lenses trap) RF is ramped down, radial DC is applied Deflector Ions are ejected along lines converging on the orbitrap entrance). As ions enter orbitrap, they are picked up and squeezed by its electric field All ions start simul- taneously, but light ions enter Orbitrap analyzer earlier that heavy ions 10 I(t) LTQ-Orbitrap: All Technologies Come Together 1. Ions are stored in the linear trap of LTQ 2. …are axially ejected 3. …and trapped in the C-trap and Gas <1 mtorr squeezed into a smaller cloud V 4. …then a voltage pulse across C-trap ejects ions towards the Orbitrap 5. …where they are trapped and detected x V -2.5 k V y Central Electrode Voltage -3.5 k V Transmission=30..50% 12 LTQ Orbitrap™ 2005 Major accurate-mass analyzers for Life Science FT ICR Orbitrap MS TOF MS # Excitation by injection # Motion in pre- # Ions are trapped # Motion in electro- # Detection by dominantly magnetic # Image current detection static fields (m/z- secondary electron field # Fourier transform and independent well) multiplier # Low energy injection data processing # High energy # Very high kinetic # MSn possibilities # Significant kinetic injection energy for detection # Broad-band energy during detection # High energy spread (many kV) excitation # Very long mean free upon fragmentation # Significant mean path (many km) free path (tens of m) T m / z T m / z TOF m / z 13 Importance of high transmission 5 4 3 High-resolution oaTOF 2 Orbitrap RMS Mass accuracy, ppmMass accuracy, RMS 1 0 1 10 100 1.000 10.000 DETECTED ions/peak in 1 sec 5 4 High-resolution Orbitrap oaTOF 3 2 Assumptions: oaTOF: resolving power 40,000, RMS Mass accuracy, ppmMass accuracy, RMS 1 transmission 4% (grids x duty cycle x angular scattering on grids) 0 Orbitrap transmission 50% 1 10 100 1.000 10.000 14 INJECTED ions/peak in 1 sec 2011: A new season in life of Orbitrap mass spectrometry 15 Recent developments of the Orbitrap analyzer Parallel and multiple fills C-trap of the C-trap Lenses Deflector x 1.8.Res Compact high-field trap . Enhanced FT x 2 Res 16 Improvements in orbital trapping: Compact high-field analyzer # Smaller size- 1.8x frequency at the same voltage, 2.1x at higher 12 mm 10 mm 30 mm 20 mm voltage # New miniature lenses for focusing onto Orbitrap entrance 12 526,2600 # Higher tolerance 196,0910 R=427119 Experimental 10 4 R=667444 requirements 8 526,2703 3 R=429586 6 # Lower capacitance and 196,0848 2 4 R=677597 new preamplifier 2 1 Relative Abundance Relative transistors bring 0 0 196,06 196,08 196,10 526.2608 increased sensitivity. 12 R=378620 196.0910 Theoretical 10 R=629410 4 526.2717 # Space charge shift: ca. 8 R=375454 3 70% rel. to the standard 6 2 trap at the same target 4 196.0847 Relative Abundance Relative 2 R=629316 1 m/z 0 0 196.06 196.08 196.10 526.26 526.28 17 Detection process in the Orbitrap analyzer + zero filling + apodization …. I(t) f(t ) int(m/z) ADC with n FFT + filters processing All ions are ejected from the C-trap at the same moment Coherent nature of ion packets allows for a new advanced signal processing procedure Narrow peaks in Enhanced FT 18 Enhanced FT as a tool for increasing resolving power # MS analysis of two peptides (Val5-Angiotensin II and Lys-des-Arg9-Bradikynin) differingC:\Xcalibur in\...\EDA\ massdoublets_eFTon_256m by 12.1s mmu (triply 5/9/2011charged 9:52:16 AM ions) RT: 0.00000 - 0.22545 5 9 14 17 22 26 29 34 40 42 48 NL: 0.02221 0.04009 0.06243 0.07584 0.09819 0.11606 0.12947 0.15182 0.17863 0.18757 0.21438 5.73E9 TIC MS 60 doublets_efto ff_256ms 40 3.7Hz 20 eFT off, 256ms Relative Abundance Relative 0 1 8 10 16 19 24 27 30 32 37 39 45 50 NL: 0.00372 0.03495 0.04387 0.07064 0.08402 0.10632 0.11970 0.13308 0.14201 0.16431 0.17323 0.20000 0.22230 6.07E9 TIC MS 60 doublets_eF Ton_256ms 40 3.7Hz 20 eFT on, 256ms 0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 Time (min) 344.84744 NL: 2.84E8 R=17304 doublets_eftoff_256ms# 100 38 RT: 0.17 AV: 1 T: 345.18164 FTMS + p ESI Full ms R=16800 [150.00-2000.00] 50 345.51587 R=16900 345.84967 eFT off, 256ms R=17000 Relative Abundance Relative 0 344.84711 NL: 3.78E8 R=60504 doublets_eFTon_256m 100 Val5-Angiotensin II Lys-des-Arg9-Bradykinin s#5 RT: 0.02 AV: 1 T: C H N O 345.18124 C49H71N13O12 50 75 13 11 FTMS + p ESI Full ms R=60100 [150.00-2000.00] 50 + 0.61ppm - 0.87ppm 345.51526 R=58300 345.84937 eFT on, 256ms R=56100 0 344.6 344.8 345.0 345.2 345.4 345.6 345.8 346.0 m/z Please see: O. Lange; E. Damoc; A. Wieghaus; A. Makarov. “Enhanced FT for Orbitrap Mass 19 Spectrometry”. Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011. Enhanced FT is not just a software! # Improved stabilization and filtering of voltages # Special dielectric materials # Improved preamplifier Before After 100 100 90 90 80 80 70 70 60 60 50 50 40 40 Relative Intensity Intensity Relative 30 Intensity Relative 30 20 20 10 10 t, ms t, ms 0 0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 Conclusion: detection of the first beat is very important for heavy intact proteins 20 Orbitrap Elite instrument Faster Dual trap electronics: >12 scans/sec ETDETD optionoption 2nd generation S-lens (Square quadrupole with a blocker of neutrals for improved ruggedness) Compact high-field Orbitrap analyzer+ eFT 21 Resolving power of Orbitrap Elite m/z RFWHM in 0.76s 0.76 sec acquisition 195 345,000 (std for R=60,000) Hamming apodization, R=60,000 393 248,000 T=5e5 524 215,000 1222 140,000 1822 106,000 LTQ FT equivalent: 12T 20T 32T 34T 195.0876 R=345700 100 95 90 85 80 75 70 65 60 55 50 45 1421.9780 40 R=131700 524.2652 Relative Abundance 35 1321.9849 R=215900 R=137600 1521.9713 30 262.6363 R=126500 25 R=297300 1221.9915 1621.9645 R=140900 R=122100 20 15 1721.9586 393.2248 1121.9978 R=116100 R=145600 1821.9536 10 R=248600 1022.0043 R=106500 R=152200 5 922.0103 1921.9482 R=142400 R=93500 0 m/z 200 400 600 800 1000 1200 1400 1600 1800 2000 22 Number of peptide spectrum matches for different LC gradients HCD rate >7 Hz! Orbitrap Elite LTQ Orbitrap Velos HCD rate 4 Hz Slide courtesy Sample: E.coli, HCD top 15 method M.Zeller Please see also: M.