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 k 2 2 2 Frequency of radial oscillations r U (r, z) 4z r / 2 Rm 4 ln(r / Rm ) 2 Frequency of axial oscillations z
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 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. Zeller; C. Crone; M. Mueller; E. Damoc; E. Denisov; A. Makarov; D. Nolting; T. Moehring. “Increased 23 analytical performance on a hybrid iontrap-FTMS mass spectrometer with a compact Orbitrap mass analyzer”, Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011. Protein performance
993.9770 R=110221 100 993.8706 Orbitrap Elite R=107582 994.1036 80 R=108475 0.76 sec 60 994.1885 R=109018 40 993.6367 993.0619 R=108853 993.4244 994.4433 R=109083 R=99350 R=105946 994.7200 994.9536
Relative Abundance Relative 20 R=109283 R=98949 0 994.0309 R=44604 100 993.9030 R=42104 LTQ Orbitrap Velos 80 993.7112 994.1802 R=35704 R=42804 1.52 sec 60 993.5618 994.4540 R=47304 R=52804 40 993.1357 994.6257 R=42304 R=37604 994.8610 Slide courtesy 20 R=32404 E. Damoc, M.Zeller
0 993.0 993.5 994.0 994.5 995.0 m/z Intact Yeast Enolase (46.64 kDa), 47+ ion (P<1*10-10 Torr)
Please see also: E. Damoc, E. Denisov, O. Lange, T. Moehring, A. Makarov. “Improving protein analysis in Orbitrap mass spectrometry”, Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011. Please see also: P. Compton; E. Damoc; E. Denisov; J.C. Tran; A. Wieghaus; M.W. Senko; S.R. Horning; A.Makarov; N.L. 24 Kelleher. “Top-Down Proteomics on Orbitrap-Based Mass Spectrometers” Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011. One of workflows for top-down analysis
D:\Nike_ASMS2011\7PM_low_high_high 4/1/2011 3:31:37 PM
RT: 0.00000 - 55.05627 21.34707 32.81842 NL: 631.44 8.61E8 100 848.53 32.72095 TIC MS 90 18.64681 21.63524 675.46 33.40777 7PM_low_h 1123.97 937.25 482.39 igh_high 80 33.50616 18.55103 22.79732 32.62393 35.63121 70 18.93580 526.41 584.45 41.12886 1444.73 1530.64 1139.29 309.27 780.63 42.41121 36.02652 50.36138 60 23.57190 39.73384 780.64 48.38061 916.64 47.33300 419.32 1463.26 754.62 43.13853 419.32 419.31 51.49740 50 23.86839 32.42828 TIC 526.42 419.31 419.31 40 835.40 18.35869 25.79235 32.23100 1534.62 30 1445.51 30.45841 424.36 573.39
Relative Abundance Relative 51.60215 20 419.31 17.69723 52.10238 10 1.01407 2.73260 3.74654 7.97599 9.15300 14.35969 16.30460 740.46 1445.45 391.27 371.09 371.09 371.09 391.28 391.27 564.36 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 Time (min) 7PM_low_high_high#439 RT: 21.73 AV: 1 NL:1.41E7 T: FTMS + p NSI Full ms [300.00-4000.00] 937.250 100 90 880.532 968.49668.496 857.856 1117.353 80 1001.827100 830.234 70 1037.573 Low Res Full MS 1320.304 60 1161.933 807.215 50 1210.255 40 785.442 1452.323 1262.967 1382.926 30 764.811 1333.759 1704.703 1547.289 1613.709 Relative Abundance Relative 745.272 1481.213 1662.860 20 709.007 1753.469 1808.890 1919.579 1987.083 10 572.015 660.829 0 400 500 600 700 800 900 10001 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z 7PM_low_high_high#440 RT: 21.76 AV: 1 NL:8.69E5 T: FTMS + p NSI d SIM ms [964.00-974.00] 968.46661 R=75104 100 + 1.7 ppm 90 968.40082 968.53186 High Res ddSIM 80 R=72604 R=74104 70 Carbonic Anhydrase II, 30+ ion 968.33502 60 R=73704 968.63043 (for proteins < 50kDa) R=72804 50 968.26978 968.69891 40 R=72604 967.86566 R=73704 967.96814 30 R=74104 968.76447 967.70081 R=73704 968.20258 968.93176 969.09790
Relative Abundance Relative 967.03101 20 967.16669 967.43549 R=77004 R=71004 R=72104 R=73804 R=72004 R=67704 R=71504 967.33575 967.53027 969.19952 969.33167 R=75804 969.46521 969.62909 969.76202 969.92871 10 R=72204 R=69804 R=68404 R=70004 R=69204 R=69504 R=71204 R=72604 0 967.0 967.2 967.4 967.6 967.8 968.0 968.2 968.4 968.6 968.8 969.0 969.2 969.4 969.6 969.8 m/z 7PM_low_high_high#441 RT: 21.79 AV: 1 NL:2.78E5 T: FTMS + p NSI d Full ms2 [email protected] [200.00-1950.00] 1086.88208 R=74100 z=7 100
1007.41254 80 R=77300 z=7 High Res ddHCD 1175.14697 740.92303 958.47437 R=70300 60 1026.31042 R=90100 R=77900 z=6 1268.02881 z=4 z=16 R=74004 R=67200 z=? 40 539.28448 593.14032 894.56104 z=6 R=105201 R=100100 836.80676 R=79400 1122.10962 1238.67786 R=82904 z=17 1409.97607 337.18829 z=1 z=5 702.34857 R=71300 R=67900
Relative Abundance Relative 270.10934 20 R=124701 438.23633 z=? z=4 z=4 1295.20569 R=64200 1521.22876 1665.02832 1732.85364 1914.20667 R=138504 R=114001 R=91001 R=66100 z=1 z=5 R=60801 R=62704 R=54604 R=57904 z=? z=1 z=1 z=6 z=5 z=? z=? z=? 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z 25 Some records… 100 100 263.63382 197.09162 Experimental Experimental 80 R=1324004 80 R=1022504 60 197.09406 60 263.63809 197.08774 R=1328004 263.63925 40 40 R=1030104 R=1260304 197.09699 R=1089004 20 20 R=1403004 Relative Abundance Hand-selected 0 Relative Abundance 0 100 Theoretical 100 Theoretical manually-tuned 197.09190 263.63402 80 R=1398200 C8H10N4O2+H: 80 R=998200 C23H39N7O5S1 Orbitrap analyzer, 60 197.09436 60 197.08804 263.63824 40 R=1398179 263.63948 R=1398609 40 R=998692 3 sec transients 197.09728 R=998637 20 R=1398481 20 0 0 197.085 197.090 197.095 263.630 263.635 263.640 263.645 m/z m/z 100 195.08755 Experimental 100 262.63625 Experimental 80 R=1366900 80 R=1098600 60 60 263.13762 40 40 R=1106304 196.09073 263.63411 264.13567 20 197.09162 20 R=1359804 R=1104104 R=1081604 Relative Abundance R=1300904 Relative Abundance 0 0 100 195.08765 Theoretical 100 262.63612 Theoretical R=1398747 R=1099016 80 80 C H N O S C8H10N4O2+H: 23 39 7 5 1 60 60 263.13780 100 195.08739 40 40 R=1098934 196.09101
90 R=1360401 20 R=1398398 197.09190 20 263.63402 264.13570 80 R=1398200 R=1098287 R=1098095 0 m/z 0 m/z 70 195 196 197 198 262 263 264 265 60 50 262.63605 524.26495 1421.97449 40 R=1077601 R=814001 1221.98865 R=499801 1621.96021 30 R=536301 R=458001 1821.94666 Relative Abundance 20 1022.00299 R=378501 Slide courtesy 10 R=533104
0 m/z E. Damoc, E. Denisov 200 400 600 800 1000 1200 1400 1600 1800 2000 26 Research only! Q Exactive: new features relatively to Exactive
Quadrupole mass filter Enhanced Fourier transform (eFT™) for Orbitrap data processing Predictive automatic gain control (pAGC) and parallel filling & detection Possibility of multiple fills for spectrum multiplexing S-lens for higher transmission (like in LTQ Orbitrap Velos) with rugged optics C-trap directly interfaced to HCD (like in LTQ Orbitrap Velos)
Michalski, A; Damoc, E; Hauschild, JP; Lange, O; Wieghaus, A; Makarov, A; Nagaraj, N; Cox, J; Mann, M; Horning, S 27 “Mass Spectrometry-based Proteomics Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass Spectrometer”. Mol. Cell Proteomics 10, (2011) Resolving power and mass accuracy of the Orbitrap analyzer
Calmix_140kRes_1.8Hz Res Transient Max. scan # 1 RT: 0.01 AV: 1 NL: 2.73E8
195.0877 100 R=156902 setting @ length, speed, 95 90 85 m/z 200 ms Hz 80 75 524.2650 R=97002 70 65 140,000 512 1.5 60 55 50 45 1421.9777 70,000 256 3 40 1321.9840 Relative AbundanceRelative R=58702 R=60402 35 1521.9713 R=56502 30 1221.9905 R=63302 1621.9652 25 393.2244 R=54702 R=111702 20 35,000 128 7 1121.9971 1721.9592 15 R=66202 R=52902 10 271.1222 1022.0035 1821.9531 R=133902 R=50502 1921.9470 5 R=68302 R=48402 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 17,500 64 12 m/z
Calmix_17.5kRes_13.2Hz 140 # 82 RT:0.10 AV: 1 NL: 3.01E8 nanoLC of 100 195.0876 R=20302 95 120 90 E.coli: 85 80 524.2648 Sample of 75 R=12302 100 70 7775 points 65 60 80 r.m.s.1.5 ppm 55 50 1421.9779 45 R=7502 1321.9841 1521.9711 40 Relative AbundanceRelative R=7702 R=7202 60
35 1621.9658 Ion Score R=7002 30 1221.9904 25 393.2243 R=8002 R=14102 20 1721.9590 40 R=6702 1121.9962 15 R=8402 1821.9518 10 271.1220 R=6502 R=17002 1022.0034 1921.9468 5 R=8602 R=6202 20 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z
0 -10-8-6-4-20246810 28 Mass Error, ppm Long-term mass accuracy with external calibration
5
4
3
2
1 m/z 391 0 m/z 445 -1
Deviation [ppm] Deviation -2
-3
-4
-5 Time [h] 0 102030405060 2 Realistic conditions of an 1 average lab Air cooling only! 0 3㼻C/5.5㼻 Experiment was carried -1 F out with temperature variations up to 3°C peak- -2 to-peak, up to 1°C/hour Temperature Change [K] Change Temperature -3 29 Time [h] 0 102030405060 Mass accuracy with polarity switching and external calibration
1 positive + 1 negative scan in 1 second
30 Spectral acquisition speed with predictive AGC and parallel filling/detection
Scan speed does 14 90% not change until ion fill time 80% 12 reaches 50 ms 70%
10 60%
8 50% Scan speed 6 40% Duty cycle 30%
Scans per second,Scans Hz 4
20% of ion % cycle filling, Duty Up to 65% of the 2 total time could be 10% spent meanwhile on 0 0% accumulating ions! 0 20406080100 Ion fill time, ms Resolving power setting: 17,500 (min.) At higher resolving powers, ion fill times and
31 duty cycles are even longer! HCD performance
m/z 524 (MRFA peptide) m/z 1522 (Ultramark)
20110427_HCD_524 #142 RT: 1.27 AV: 1 NL: 6.84E7 20110427_HCD_1522 #150 RT: 1.35 AV: 1 NL: 1.63E7 T: FTMS + p ESI Full ms2 [email protected] [70.00-1400.00] T: FTMS + p ESI Full ms2 [email protected] [120.00-2000.00] 524.2648 1521.9714 100 100
95 95
90 90
85 85
80 80
75 75
70 TIC=8.3e7 70 TIC=1.9e7
65 65 60 60 55 55 50 50 45 45 Relative Abundance
40 Relative Abundance 40 35 35 30 30 25 25 20 20 15 15 10 10 5 5 0 100 150 200 250 300 350 400 450 500 550 600 650 700 0 m/z 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z
20110427_HCD_524 #229 RT: 2.11 AV: 1 NL: 1.14E7 20110427_HCD_1522 #244 RT: 2.28 AV: 1 NL: 1.39E6 T: FTMS + p ESI Full ms2 [email protected] [70.00-1400.00] T: FTMS + p ESI Full ms2 [email protected] [120.00-2000.00] 271.1221 247.9327 100 100
95 95
90 90
85 85
80 80
75 75
70 70 65 TIC=6.7e7 65 TIC=1.3e7 60 60
55 55 50 104.0533 NCE=35 50 NCE=40
45 45 Relative Abundance Relative Abundance 1521.9728 40 40
35 120.0811 35 1077.9513
30 30 288.1486 1289.9592 453.2276 524.2648 25 25 1189.9652 237.1234 306.1590 1389.9537 20 376.1977 20 965.9371 229.1006 259.1554 321.9291 865.9434 1037.9609 653.9364 15 15 753.9297 541.9229 185.9492 441.9289 10 10 421.9226 138.0662 185.1034 5 393.2241 5 95.0610 174.0696 217.1335 322.1870 418.1899 0 0 100 150 200 250 300 350 400 450 500 550 600 650 700 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z m/z
Fill times are similar to those in LTQ Orbitrap Velos instrument Direct interfacing of the C-trap to HCD cell minimizes losses during fragmentation
32 Q Exactive LC-MS/MS in action: E.coli digest
12000 10543 Unique peptides 1800 Unique proteins 1600 10000 1701 8430 <1% FDR 1400 E. Coli 1701 8000 1200 1329 5804 digest 1000 6000 1329 1039 2h gradient, 800 4000 Proxeon 600 1039 NanoUPLC 3 search 400 2000 engines 200 0 0 5ug 100ng 20ng 5ug 100ng 20ng
70,000 17,500 256 ms 64 ms
HCD HCD HCD HCD HCD HCD HCD HCD HCD HCD Full MS 1 2 3 4 5 6 7 8 9 10 ddTOP10 HCD acquisition Fill method times
0 0.2 0.4 0.6 0.8 1 sec 33 Data dependent acquisition cycle time [sec] Q Exactive LC-MS/MS in action: HeLa digest
Unique Peptide IDs at <1% FDR Protein IDs 25000 4500
4000 4202 4233 22584 22479 20000 3500 3000 15000 3028 3068 2500 run I 14255 14266 2000 10000 run II 1500 1000 5000 500 0 0 2ug HeLa 5ug HeLa 2ug HeLa 5ug HeLa 1h grad 2h grad 1h grad 2h grad Sample Total: 17142 27324 3344 4621 courtesy >3000 pph K. Mechtler Please see also: C. Paschke; Y. Xuan; E. Damoc; T. Ueckert; U. Comberg; H. Grensemann; M. Kellmann; B. Delanghe. “Breaking the 2000 proteins barrier in a standard LC run using a new benchtop Orbitrap instrument and multiple search engines.”. Proc. 59th 34Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011. What do we gain by selected ion monitoring?
195.0876 N=248402.81 NL: 1.94E8 100 [150.00-2000.00] 80 In Full MS, total C-trap charge Full MS 60 Lowest signal capacity is shared between 40 S/N = 745 IT= 0.245 ms 250330 multiple signals of different 20 0
intensity 195.0877 N=20741.58 100 NL: 1.12E8 SIM (10amu) [190.10-200.10] Signal-to-noise ratio becomes 80 dependent on the ratio of 60 For the same target: Lowest signal 40 S/N = 5400 IT= 1.321 ms compound of interest to other 20 28240 analytes- much less so in SIM! 0 In Orbitrap instruments, SIM Gain in sensitivity (7x) could become MRM without any 6000 additional overhead! 5000 Caffeine 4000
3000
2000
Sensitivity gain 5 – 10 x with SIM mode (spectrum) S/N
The gain will be higher in more 1000 complex matrices 0 195.082 195.084 195.086 195.088 195.09 195.092 195.094 35 S/N (FMS) S/N (SIM10) Alprazolam, Full-Scan Experiment
Alprazolam Y = 6366.31+514.015*X R^2 = 0.9967 W: 1/X
5500000 50 ppt – 10 ppb 5000000 250 fg oc - 50 pg oc 4500000 4000000 3500000 3000000 200000 Zoom in 50 ppt- 100ppt 180000
Area 2500000 160000 140000 2000000 120000
100000Area 1500000 80000 60000 1000000 40000 20000 0 500000 0 50 100 150 200 250 300 fg/uL 0 0 2000 4000 6000 8000 10000 fg/uL
36 Alprazolam SIM Experiment
Alprazolam Y = -3135.8+552.216*X R^2 = 0.9982 W: 1/X
6000000 10 ppt – 10 ppb 5000000 50 fg oc - 50 pg oc
4000000
120000 Zoom 10 ppt- 100ppt 110000 3000000 100000
Area 90000 80000 70000
2000000 Area 60000 50000 40000 30000 1000000 20000 10000 0 0 20 40 60 80 100 120 0 fg/uL 0 2000 4000 6000 8000 10000 fg/uL See also: X. He; M. Kozak. “Evaluation of quantitative performance for testosterone analysis in plasma on a novel quadrupole 37Orbitrap mass spectrometer”. Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011, Poster WP077. Spectrum multiplexing: principle of operation
Standard operation mode
vs
Spectrum multiplexing
5.0x108
4.5x108
4.0x108
3.5x108
8 3.0x10 C-Trap storage: No ion loss 8 2.5x10 over a broad range of 2.0x108 m/z195.08770 m/z262.60000 storage times! Ion count (arb.) 1.5x108 m/z393.20000 m/z524.26500 (St.Steel, fused silica, ceramics) 1.0x108 m/z1622.00000
5.0x107 0.0 1 10 100 1000 38 Inject time [ms] Spectrum multiplexing: example of 4-plex SIM
Experimental Setup: 1 Cycle 12 Hz Acquisition Rate 48 Precursors / second
1s 4-plex injection
Collecting 4 isolation windows 64ms detection
1 5 9 13 17 21 25 29 33 37 41 45 2 6 10 14 18 22 26 30 34 38 42 46 3 7 11 15 19 23 27 31 35 39 43 47 4 8 12 16 20 24 28 32 36 40 44 48
Please see: O. Lange; J.-P. Hauschild; A. Makarov; U. Fröhlich; C. Crone; Y. Xuan; M. Kellmann; A. Wieghaus. “Multiple C- Trap Fills as a Tool for Massive Parallelization of Orbitrap Mass Spectrometry- a new Concept for Targeted Mass Analysis”.
39 Example Fluoxastrobin, [M+H]+ calc. 459.0866
RT: 2.1989 - 2.4164 Iso3 NL: 8.83E8 100 All 4-plex TIC MS 459.0859 1.5 ppm (ext.).) 80 acquisitions 100 110418_tSIM_48pest_4plex_1min_30ppb _01 C21H16ClFN4O5 90 60 80 40 70 20 60 Relative Abundance 0 459.0860 50 NL: 8.49E7 100 m/z= 459.0847-459.0893 F: FTMS + ESI Following just one 40 80 459.0861 SIM msx ms [393.15-397.15, 404.07-408.07, 457.09-461.09, Iso4 target out of that 4-plex Abundance Relative 30 Iso1 Iso2 60 493.20-497.20] MS 459.0859 20 110418_tSIM_48pest_4plex_1min_30ppb _01 40 10
20 459.0860 459.0859 0
Relative Abundance 459.0859 459.0860 459.0863 380 400 420 440 460 480 500 520 540 0 m/z 2.20 2.22 2.24 2.26 2.28 2.30 2.32 2.34 2.36 2.38 2.40 Iso – Isolation window Time (min) zoom
RT: 2.2543 - 2.2815 NL:N 4.01E84.01E8 100 All 4-plex If we were doing this by TICT MMSS 80 acquisitions 110418_tSIM_48pest_4plex_1min_30ppb1110418_tSIM_48pest_4plex_1min_30ppb _01_ conventional SIM, we would 60 have had 4 times less data 40 2.2759 2.2594 points across the peak! 20 Relative Abundance 0 459.0860 NL:N 8.49E78.49E7 100 Following just one m/z=mm/z= 459.0847-459.0893459.0847-459.0893 F: FTMSFTMS + ESIESI SIMS msxAt msm shigh [393.15-397.15,[393.15-39 resolution7.15, and mass 80 459.0861 target out of that 4-plex 404.07-408.07,4404.07-408.07, 457.09-461.09,457.09-461.09, 60 493.20-497.20]4493accuracy,.20-497.20] MS fragmentation is 110418_tSIM_48pest_4plex_1min_30ppb1110418_tSIM_48pest_4plex_1min_30ppb 40 0.99s _01_ frequently not needed! 20 Relative Abundance 0 2.255 2.260 2.265 2.270 2.275 2.280 Time (min)
40 Boosting dynamic range by multiple fills
20110509 JPH JG DEP2 DynRangeTest Sol2_2_70k #520 RT: 2.48 AV: 1 NL: 1.44E9 T: FTMS + p ESI Full ms [210.00-600.00] 477.2303 Research only! R=51306 N=881374.69 Loperamide C 29 H 34 O 2 N 2 Cl -0.0343 ppm 100 80 60
40 499.8216 266.1534 313.1679 361.2800 411.2662 R=25200 565.2441 20 20110509 JPH JG DEP2 DynRangeTestR=65306 Sol2_2_70kR=60400 #520 RT:R=553062.48 AV: 1 NL:R=475001.44E9 N=903012.81 R=31100 Relative Abundance Relative N=712762.00 N=770267.69 N=855751.44 N=854691.25 N=904773.75 T: FTMS0 + p ESI Full ms [210.00-600.00] 250 300 350 400 450 500 [m/z550 200..600]@600 0.1ms & 500.7180 m/z R=37800 266.1534 N=903871.50 zoomR=65306 [m/z 374..378]@ 250ms 361.2800 N=712762.00 313.1679 R=55306 R=60400 N=855751.44 1.0 N=770267.69 411.2662 R=47500 0.5 500.9751 565.2441 N=854691.25 R=30300 R=31100 N=904117.69 N=904773.75 Relative Abundance Relative 0.0 250 300 350 400 450 500 550 600 zoom m/z 20110509 JPH JG DEP2 DynRangeTest Sol2_2_70k #520 RT: 2.48 AV: 1 NL: 1.44E9 T: FTMS + p ESI Full ms [210.00-600.00] 376.1476 (A+1) Isotope: 377.1513 R=56802 R=53502 N=169.29 N=169.13 Haloperidol C 21 H 24 O 2 NClF 0.4562 ppm S/N=25 0.0015 rel.abundance = 0.00031% 377.1971 DR>320.000 374.0984 R=55302 0.0010 R=49906 375.0941 N=169.12 N=6879.50 R=52602 376.3792 375.6917 R=40500 0.0005 N=169.45 R=53400 N=169.25 N=169.36 Relative Abundance Relative 0.0000 41 373.5 374.0 374.5 375.0 375.5 376.0 376.5 377.0 377.5 378.0 m/z Conclusion
Ultra-high resolution Quantitation MS/MS sensitivity Throughput
The Orbitrap Mass Analyzer is a new type of mass analyzers with its own unique combination of analytical parameters Orbitraps are still evolving… Higher speed Higher resolving power and mass accuracy Higher sensitivity More routine applications Exciting new applications continue to emerge 42 Acknowledgements
S. Horning My Family: I. Mylchreest M. Antonczak T. Moehring A.Guiller E. Hemenway E. Denisov Anna Makarova E. Schroeder M. Senko A. Kholomeev Dasha Makarova R.A.Purrmann J. Syka A. Wieghaus Nikita Makarov R. Pesch J. Schwartz W. Balschun My parents: J. Srega V. Zabrouskov O. Lange Alla & Alexei Makarov I. Jardine T. Second O. Hengelbrock T. Ziberna K. Strupat T. Second S. Moehring K.Scheffler J. Griep-Raming F. Paffen U. Froehlich B. Rose D. Nolting A. Boegehold F. Czemper J. Grote R. Malek W. Huels A. Kuehn A. Schumbera T. Rietpietsch S. Simmel R. Malek M. Zeller M. Kellmann M. Biel C. Henrich M. Mueller A.Venckus 7th European Framework Program: Health-F4-2008- 43 F. Grosse-Coosmann 201648/PROSPECTS
Thank you ! vv
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