orbitrap mass spectrometry: ultra-high resolution for ...€¦ · orbitrap mass spectrometry:...
TRANSCRIPT
Orbitrap Mass Spectrometry: Ultra-high Resolution for Every Lab
Alexander Makarov March 14, 2012
Symposium: New Alternatives in High-Resolution Mass Spectrometry
2
What is Orbitrap™ analyzer?
Orbitrap analyzer =
= Orbital trapping
+ Image current detection
+ Electrodynamic squeezing
+ External pulsed ion source
3
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”
Orbital traps Kingdon (1923)
…but moving charges could be stable!
No promise?
4
TOF Adventures: Prelude to the Orbitrap analyzer
� �)/ln(2/2
),( 222mm RrRrzkzrU �����
Gall L.N., Golikov Y.K., Aleksandrov M.L., Pechalina Y.E., Holin N.A. SU Pat. 1247973, 1986.
“A mosquito-catcher”
1st implementation of quadro-logarithmic potential in mass
spectrometry (1989)
5
Orbital trapping
qmk
z /��
“Ideal Kingdon trap”: Quadro-logarithmic potential
22
��
� ��
RRm
zr ��
12
2
��
� ��
RRmz���
Only this frequency does not depend on energy, angle, etc. and is used for mass analysis
Characteristic frequencies: � Frequency of rotation �� � Frequency of radial oscillations �r � Frequency of axial oscillations �z
� �)/ln(2/2
),( 222mm RrRrzkzrU �����
“Beauty will save the world.”- F.M. Dostoevsky
6
Detection of Ions in the Orbitrap analyzer
I(t)
t I(t)
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
7
Injection of Ions into the Orbitrap analyzer
I(t)
� 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
8
0.8 sec
8 M record length 10 Ms/s (borrowed LeCroy) 0.8 s transient f =711 kHz,�f=2.39 Hz f/�f� 300000 M/�M=½ f/�f � 150000
A.A. Makarov, Anal. Chem., v.72 (2000), No.6, p.1156-1162.
Proof of Principle: Orbitrap MS with Laser Ion Source Field compensator Ion source
High voltage amplifier
HD Technologies Ltd From Jan. 25, 2000- within Thermo Inc. (in Thermo Masslab, Manchester, UK)
9
Reasons Why Orbitrap Would Never Work � Not possible to provide ion packets with required spatial and temporal parameters
for continuous ion sources
All these reasons are valid and require a lot of work!
� 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
10
Injection of Ions into the Orbitrap analyzer
I(t)
C-trap
Lenses
Deflector
� Ions are stored and cooled in a curved RF-only quadrupole (C-trap)
� RF is ramped down, radial DC is applied
� 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
12
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
squeezed into a smaller cloud 4. …then a voltage pulse across C-trap
ejects ions towards the Orbitrap 5. …where they are trapped and detected
-2.5 k V
-3.5 k V
Central Electrode Voltage
V
y
V
x
Gas <1 mtorr
Transmission=30..50% LTQ Orbitrap™ 2005
13
Major accurate-mass analyzers for Life Science
FT ICR Orbitrap MS TOF MS
� Motion in pre-dominantly magnetic field
� Low energy injection � MSn possibilities � Broad-band
excitation
� Ions are trapped � Image current detection � Fourier transform and
data processing � Significant kinetic
energy during detection � Very long mean free
path (many km)
� Motion in electro-static fields (m/z- independent well)
� High energy injection
� High energy spread upon fragmentation
zmT /� zmT /�
� Excitation by injection
� Detection by secondary electron multiplier
� Very high kinetic energy for detection (many kV)
� Significant mean free path (tens of m)
zmTOF /�
14
Importance of high transmission
0
1
2
3
4
5
1 10 100 1.000 10.000
RMS
Mas
s ac
cura
cy, p
pm
INJECTED ions/peak in 1 sec
High-resolution oaTOFOrbitrap
0
1
2
3
4
5
1 10 100 1.000 10.000
RMS
Mas
s ac
cura
cy, p
pm
DETECTED ions/peak in 1 sec
High-resolution oaTOF
Orbitrap
Assumptions: � oaTOF: resolving power 40,000, transmission 4% (grids x duty cycle x angular scattering on grids) � Orbitrap transmission 50%
DETECTED
INJECTED
15
2011: A new season in life of Orbitrap mass spectrometry
16
Recent developments of the Orbitrap analyzer
C-trap
Lenses
Deflector
Compact high-field trap
Enhanced FT
Parallel and multiple fills of the C-trap
x 2.Res
x 1.8.Res
17
Improvements in orbital trapping: Compact high-field analyzer
� Smaller size- 1.8x frequency at the same voltage, 2.1x at higher voltage
� New miniature lenses for focusing onto Orbitrap entrance
� Higher tolerance requirements
� Lower capacitance and new preamplifier transistors bring increased sensitivity.
� Space charge shift: ca. 70% rel. to the standard trap at the same target
12 m
m
20 m
m
30 m
m
2
3
4 526,2600 R=427119
526,2703 R=429586
0
1
526.26 526.28 m/z
0
1
2
3
4
R=378620
526.2717 R=375454
196.06 196.08 196.10 0 2
4
6
8
10
12 196.0910 R=629410
196.0847 R=629316 R
elat
ive
Abu
ndan
ce
Theoretical
Experimental
196,06 196,08 196,10 0
2
4
6
8
10
12
Rel
ativ
e A
bund
ance
196,0910 R=667444
196,0848 R=677597
10 m
m
526.2608
18
All ions are ejected from the C-trap at the same moment
Detection process in the Orbitrap analyzer
I(t) ADC with
filters
f(tn) FFT + processing
int(m/z)
+ zero filling + apodization ….
Narrow peaks in Enhanced FT
Coherent nature of ion packets allows for a new advanced signal processing procedure
19
Enhanced FT as a tool for increasing resolving power
Please see: O. Lange; E. Damoc; A. Wieghaus; A. Makarov. “Enhanced FT for Orbitrap Mass Spectrometry”. Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011.
C:\Xcalibur\...\EDA\doublets_eFTon_256ms 5/9/2011 9:52:16 AM
RT: 0.00000 - 0.22545
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22Time (min)
0
20
40
60
0
20
40
60
Rel
ativ
e A
bund
ance
140.06243
90.04009
420.18757
170.07584
290.12947
220.09819
260.11606
340.15182
50.02221
480.21438
400.17863
10.00372
190.08402
80.03495
100.04387
300.13308
320.14201
370.16431
160.07064
390.17323
270.11970
240.10632
450.20000
500.22230
NL:5.73E9TIC MS doublets_eftoff_256ms
NL:6.07E9TIC MS doublets_eFTon_256ms
344.6 344.8 345.0 345.2 345.4 345.6 345.8 346.0m/z
0
50
100
0
50
100
Rel
ativ
e A
bund
ance
344.84744R=17304
345.18164R=16800
345.51587R=16900 345.84967
R=17000344.84711R=60504
345.18124R=60100
345.51526R=58300 345.84937
R=56100
NL: 2.84E8doublets_eftoff_256ms#38 RT: 0.17 AV: 1 T: FTMS + p ESI Full ms [150.00-2000.00]
NL: 3.78E8doublets_eFTon_256ms#5 RT: 0.02 AV: 1 T: FTMS + p ESI Full ms [150.00-2000.00]
3.7Hz
3.7Hz
eFT off, 256ms
eFT on, 256ms
eFT off, 256ms
eFT on, 256ms
Val5-Angiotensin II C49H71N13O12
Lys-des-Arg9-Bradykinin C50H75N13O11
+ 0.61ppm - 0.87ppm
� MS analysis of two peptides (Val5-Angiotensin II and Lys-des-Arg9-Bradikynin) differing in mass by 12.1 mmu (triply charged ions)
20
Enhanced FT is not just a software!
� Improved stabilization and filtering of voltages � Special dielectric materials � Improved preamplifier
0 1 2 3 4 5 6 7 8 9 10 0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e In
tens
ity
t, ms
0 1 2 3 4 5 6 7 8 9 0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e In
tens
ity
t, ms
Before After
Conclusion: detection of the first beat is very important for heavy intact proteins
21
ETD option ETD option
Orbitrap Elite instrument
2nd generation S-lens (Square quadrupole with a blocker of neutrals for improved ruggedness)
Compact high-field Orbitrap analyzer+ eFT
Faster Dual trap electronics:
>12 scans/sec
22
Resolving power of Orbitrap Elite
0.76 sec acquisition (std for R=60,000) Hamming apodization, T=5e5
200 400 600 800 1000 1200 1400 1600 1800 2000 m/z 0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
195.0876 R=345700
1421.9780 R=131700
1321.9849 R=137600 1521.9713
R=126500
524.2652 R=215900
262.6363 R=297300 1621.9645
R=122100 1221.9915 R=140900
1721.9586 R=116100 1121.9978
R=145600 393.2248 R=248600 1821.9536
R=106500 1022.0043 R=152200 1921.9482
R=93500 922.0103 R=142400
LTQ FT equivalent: 12T 20T 32T 34T
m/z RFWHM in 0.76s 195 345,000
393 248,000
524 215,000
1222 140,000
1822 106,000
R=60,000
23
Number of peptide spectrum matches for different LC gradients
Orbitrap Elite LTQ Orbitrap Velos
Sample: E.coli, HCD top 15 method
HCD rate >7 Hz!
HCD rate 4 Hz
Slide courtesy M.Zeller
Please see also: M. Zeller; C. Crone; M. Mueller; E. Damoc; E. Denisov; A. Makarov; D. Nolting; T. Moehring. “Increased 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.
24
Protein performance
993.0 993.5 994.0 994.5 995.0m/z
0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
993.9770R=110221
994.1036R=108475
993.8706R=107582
994.1885R=109018993.6367
R=108853993.0619R=109083 994.4433
R=105946993.4244R=99350 994.7200
R=109283994.9536R=98949
994.0309R=44604
993.9030R=42104
994.1802R=42804
994.4540R=52804
993.5618R=47304
993.7112R=35704
994.6257R=37604
993.1357R=42304 994.8610
R=32404
Orbitrap Elite 0.76 sec LTQ Orbitrap Velos 1.52 sec
Intact Yeast Enolase (46.64 kDa), 47+ ion (P<1*10-10 Torr)
Slide courtesy E. Damoc, M.Zeller
Please see also: P. Compton; E. Damoc; E. Denisov; J.C. Tran; A. Wieghaus; M.W. Senko; S.R. Horning; A.Makarov; N.L. Kelleher. “Top-Down Proteomics on Orbitrap-Based Mass Spectrometers” Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011.
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.
25
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
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 54Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
32.81842631.4421.34707
848.5332.72095
675.46 33.40777482.39
21.63524937.25
18.646811123.97
33.50616584.45
32.62393526.41
35.63121309.27
22.797321139.29
18.551031444.73
18.935801530.64
41.12886780.63 42.41121
780.6436.02652916.64
50.36138419.32
23.571901463.26
48.38061419.31
47.33300419.32
39.73384754.62 51.49740
419.3143.13853
419.3132.42828
526.4223.86839835.40
25.792351534.6218.35869
1445.5132.23100
424.3630.45841573.39 51.60215
419.3117.69723
740.4652.102381445.45
16.30460564.36
1.01407391.27
2.73260371.09
3.74654371.09
7.97599371.09
9.15300391.28
14.35969391.27
NL:8.61E8TIC MS 7PM_low_high_high
7PM_low_high_high#439 RT: 21.73 AV: 1 NL: 1.41E7T: FTMS + p NSI Full ms [300.00-4000.00]
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
937.250
968.496880.5321117.3531001.827830.234
1037.573
1320.3041161.933807.215
1210.255785.442 1452.3231262.967 1382.926
764.811 1704.7031547.289 1613.709745.272 1481.213 1662.8601333.759
1753.469709.007 1919.5791808.890 1987.083
857.856
660.829572.015
7PM_low_high_high#440 RT: 21.76 AV: 1 NL: 8.69E5T: FTMS + p NSI d SIM ms [964.00-974.00]
967.0 967.2 967.4 967.6 967.8 968.0 968.2 968.4 968.6 968.8 969.0 969.2 969.4 969.6 969.8m/z
0
1020
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
968.46661R=75104
968.40082R=72604
968.53186R=74104
968.33502R=73704 968.63043
R=72804968.69891R=72604
968.26978R=73704967.86566
R=74104 967.96814R=73704 968.76447
R=72104967.70081R=77004
969.09790R=72004
968.93176R=73804
968.20258R=71004967.03101
R=67704967.16669R=71504
967.43549R=75804 967.53027
R=69804969.19952R=68404
967.33575R=72204
969.33167R=70004
969.46521R=69204
969.62909R=69504
969.76202R=71204
969.92871R=72604
7PM_low_high_high#441 RT: 21.79 AV: 1 NL: 2.78E5T: FTMS + p NSI d Full ms2 [email protected] [200.00-1950.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z
0
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
1086.88208R=74100
z=7
1007.41254R=77300
z=71175.14697
R=70300z=6
740.92303R=90100
z=4
958.47437R=77900
z=161268.02881
R=67200z=6894.56104
R=79400z=17
593.14032R=100100
z=5
539.28448R=105201
z=1836.80676R=82904
z=?
1122.10962R=71300
z=4
1238.67786R=67900
z=41409.97607
R=64200z=5
337.18829R=124701
z=1
702.34857R=91001
z=1
1026.31042R=74004
z=?
270.10934R=138504
z=?
1295.20569R=66100
z=6
438.23633R=114001
z=1
1914.20667R=57904
z=?
1521.22876R=60801
z=5
1665.02832R=62704
z=?
1732.85364R=54604
z=?
+ 1.7 ppm
TIC
Low Res Full MS
High Res ddSIM (for proteins < 50kDa)
High Res ddHCD
Carbonic Anhydrase II, 30+ ion
1
68.496
100
26
Some records…
200 400 600 800 1000 1200 1400 1600 1800 2000 0
10 20 30 40 50 60 70 80 90
100
Rel
ativ
e Ab
unda
nce
262.63605 R=1077601
524.26495 R=814001
1421.97449 R=499801 1221.98865
R=536301 1621.96021 R=458001 1821.94666
R=378501 1022.00299 R=533104
m/z 200
195.08739 R=1360401
195 196 197 198m/z0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance 195.08755
R=1366900
196.09073R=1359804 197.09162
R=1300904
195.08765R=1398747
196.09101R=1398398 197.09190
R=1398200
C8H10N4O2+H:
262 263 264 2650
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance 262.63625
R=1098600
263.13762R=1106304
263.63411R=1104104
264.13567R=1081604
262.63612R=1099016
263.13780R=1098934
263.63402R=1098287
264.13570R=1098095
C23H39N7O5S1
m/z
Experimental
Theoretical
Experimental
Theoretical
197.085 197.090 197.095m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e A
bund
ance 197.09162
R=1324004
197.09406R=1328004197.08774
R=1260304 197.09699R=1403004
197.09190R=1398200
197.09436R=1398179197.08804
R=1398609197.09728R=1398481
263.630 263.635 263.640 263.645m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e A
bund
ance 263.63382
R=1022504
263.63925R=1089004
263.63809R=1030104
263.63402R=998200
263.63948R=998637
263.63824R=998692
C8H10N4O2+H: C23H39N7O5S1
Experimental
Theoretical
Experimental
Theoretical
Slide courtesy E. Damoc, E. Denisov
Hand-selected manually-tuned Orbitrap analyzer, 3 sec transients
Research only!
27
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 “Mass Spectrometry-based Proteomics Using Q Exactive, a High-performance Benchtop Quadrupole Orbitrap Mass Spectrometer”. Mol. Cell Proteomics 10, (2011)
28
0
20
40
60
80
100
120
140
-10 -8 -6 -4 -2 0 2 4 6 8 10
Ion
Scor
e
Mass Error, ppm
Resolving power and mass accuracy of the Orbitrap analyzer
Res setting @ m/z 200
Transient length,
ms
Max. scan speed,
Hz
140,000 512 1.5
70,000 256 3
35,000 128 7
17,500 64 12
Calmix_140kRes_1.8Hz
# 1 RT: 0.01 AV: 1 NL: 2.73E8
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
195.0877 R=156902
524.2650 R=97002
1421.9777 R=58702 1321.9840
R=60402 1521.9713 R=56502 1221.9905
R=63302 1621.9652 R=54702
393.2244 R=111702
1121.9971 R=66202
1721.9592 R=52902
271.1222 R=133902
1821.9531 R=50502
1022.0035 R=68302 1921.9470
R=48402
Calmix_17.5kRes_13.2Hz
# 82 RT: 0.10 AV: 1 NL: 3.01E8
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
195.0876 R=20302
524.2648 R=12302
1421.9779 R=7502
1521.9711 R=7202
1321.9841 R=7702
1621.9658 R=7002
1221.9904 R=8002 393.2243
R=14102 1721.9590 R=6702 1121.9962
R=8402 1821.9518 R=6502 271.1220
R=17002 1022.0034 R=8602
1921.9468 R=6202
nanoLC of E.coli: Sample of 7775 points r.m.s.1.5 ppm
29 -3
-2
-1
0
1
2
0 10 20 30 40 50 60
Tem
pera
ture
Cha
nge [
K]
Time [h]
Long-term mass accuracy with external calibration
-5
-4
-3
-2
-1
0
1
2
3
4
5
0 10 20 30 40 50 60
Dev
iatio
n [p
pm]
Time [h]
m/z 391m/z 445
3 C/5.5F
� Realistic conditions of an average lab � Air cooling only! � Experiment was carried out with temperature variations up to 3°C peak-to-peak, up to 1°C/hour
30
Mass accuracy with polarity switching and external calibration
1 positive + 1 negative scan in 1 second
31
Spectral acquisition speed with predictive AGC and parallel filling/detection
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
0
2
4
6
8
10
12
14
0 20 40 60 80 100
Duty
cycl
e of
ion
fillin
g, %
Scan
s per
seco
nd, H
z
Ion fill time, ms
Scan speedDuty cycle
Scan speed does not change until ion fill time reaches 50 ms
Up to 65% of the total time could be spent meanwhile on accumulating ions!
Resolving power setting: 17,500 (min.) At higher resolving powers, ion fill times and
duty cycles are even longer!
32
HCD performance
� Fill times are similar to those in LTQ Orbitrap Velos instrument � Direct interfacing of the C-trap to HCD cell minimizes losses during fragmentation
20110427_HCD_524 #142 RT: 1.27 AV: 1 NL: 6.84E7T: FTMS + p ESI Full ms2 [email protected] [70.00-1400.00]
100 150 200 250 300 350 400 450 500 550 600 650 700m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
524.2648
TIC=8.3e7
20110427_HCD_524 #229 RT: 2.11 AV: 1 NL: 1.14E7T: FTMS + p ESI Full ms2 [email protected] [70.00-1400.00]
100 150 200 250 300 350 400 450 500 550 600 650 700m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
271.1221
104.0533
120.0811
288.1486524.2648453.2276
237.1234 306.1590 376.1977229.1006 259.1554
138.0662 185.1034393.2241
217.1335 418.189995.0610 174.0696 322.1870
TIC=6.7e7 NCE=35
20110427_HCD_1522 #150 RT: 1.35 AV: 1 NL: 1.63E7T: FTMS + p ESI Full ms2 [email protected] [120.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1521.9714
TIC=1.9e7
20110427_HCD_1522 #244 RT: 2.28 AV: 1 NL: 1.39E6T: FTMS + p ESI Full ms2 [email protected] [120.00-2000.00]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
247.9327
1521.9728
1077.9513
1289.9592
1189.96521389.9537
965.9371321.9291 865.9434653.9364
753.9297541.9229441.9289185.9492
421.9226
1037.9609
TIC=1.3e7 NCE=40
m/z 524 (MRFA peptide) m/z 1522 (Ultramark)
33
1701
1329
1039
0 200 400 600 800
1000 1200 1400 1600 1800
5ug 100ng 20ng
Full MS HCD 1
HCD 2
HCD 3
HCD 4
HCD 5
HCD 6
HCD 7
HCD 8
HCD 9
HCD 10
Data dependent acquisition cycle time [sec]
Fill times
1 sec
70,000 256 ms
17,500 64 ms
0.2 0.4 0.6 0.8
ddTOP10 HCD acquisition method
1701
1329
1039
0
2000
4000
6000
8000
10000
12000
5ug 100ng 20ng
10543
8430
5804
Unique peptides Unique proteins <1% FDR
E. Coli digest
2h gradient,
Proxeon NanoUPLC
3 search engines
0
Q Exactive LC-MS/MS in action: E.coli digest
34
0
500
1000
1500
2000
2500
3000 3500
4000
4500
2ug HeLa 1h grad
5ug HeLa 2h grad
run I run II
Protein IDs
3028 3068
4202 4233
0
5000
10000
15000
20000
25000
2ug HeLa 1h grad
5ug HeLa 2h grad
Unique Peptide IDs at <1% FDR
14255 14266
22584 22479
Q Exactive LC-MS/MS in action: HeLa digest
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 Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011.
Total: 17142 27324 3344 4621 Sample courtesy K. Mechtler >3000 pph
35
� Sensitivity gain 5 – 10 x with SIM mode � The gain will be higher in more complex matrices
What do we gain by selected ion monitoring?
� In Full MS, total C-trap charge capacity is shared between multiple signals of different intensity
� Signal-to-noise ratio becomes dependent on the ratio of compound of interest to other analytes- much less so in SIM!
� In Orbitrap instruments, SIM could become MRM without any additional overhead!
0
20
40
60
80
100
0
20
40
60
80
100
195.0876 N=248402.81
195.0877 N=20741.58
NL: 1.94E8 [150.00-2000.00]
NL: 1.12E8 [190.10-200.10]
Full MS
SIM (10amu)
S/N = 745 IT= 0.245 ms
For the same target: S/N = 5400 IT= 1.321 ms
Lowest signal 250330
Lowest signal 28240
0
1000
2000
3000
4000
5000
6000
195.082 195.084 195.086 195.088 195.09 195.092 195.094
S/N
(spe
ctru
m)
S/N (FMS) S/N (SIM10)
Gain in sensitivity (7x)
Caffeine
36
Alprazolam Y = 6366.31+514.015*X R^2 = 0.9967 W: 1/X
0 2000 4000 6000 8000 10000 fg/uL
0 500000
1000000 1500000
2000000
2500000 3000000
3500000
4000000 4500000
5000000
5500000
Are
a Alprazolam, Full-Scan Experiment
0 50 100 150 200 250 300 fg/uL
0 20000 40000 60000 80000
100000 120000 140000 160000 180000 200000
Are
a
50 ppt – 10 ppb 250 fg oc - 50 pg oc
Zoom in 50 ppt- 100ppt
37
Alprazolam Y = -3135.8+552.216*X R^2 = 0.9982 W: 1/X
10 ppt – 10 ppb 50 fg oc - 50 pg oc
0 2000 4000 6000 8000 10000 fg/uL
0
1000000
2000000
3000000
4000000
5000000
6000000
Are
a Alprazolam SIM Experiment
Zoom 10 ppt- 100ppt
0 20 40 60 80 100 120 fg/uL
0 10000 20000 30000 40000 50000 60000 70000 80000 90000
100000 110000 120000
Are
a
See also: X. He; M. Kozak. “Evaluation of quantitative performance for testosterone analysis in plasma on a novel quadrupole Orbitrap mass spectrometer”. Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011, Poster WP077.
38 1 10 100 1000
0.0
5.0x107
1.0x108
1.5x108
2.0x108
2.5x108
3.0x108
3.5x108
4.0x108
4.5x108
5.0x108
Ion
coun
t (ar
b.)
Inject time [ms]
m/z195.08770 m/z262.60000 m/z393.20000 m/z524.26500 m/z1622.00000
C-Trap storage: No ion loss over a broad range of
storage times! (St.Steel, fused silica, ceramics)
Standard operation mode
vs
Spectrum multiplexing
Spectrum multiplexing: principle of operation
39
Spectrum multiplexing: example of 4-plex SIM
64ms detection
4-plex injection
Collecting 4 isolation windows
1s
12 Hz Acquisition Rate 48 Precursors / second
Experimental Setup: 1 Cycle
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”.
40
0.99s
RT: 2.2543 - 2.2815
2.255 2.260 2.265 2.270 2.275 2.280 Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
2.2759 2.2594
459.0860
459.0861
NL: 4.01E8 TIC MS 110418_tSIM_48pest_4plex_1min_30ppb _01
NL: 8.49E7 m/z= 459.0847-459.0893 F: FTMS + ESI SIM msx ms [393.15-397.15, 404.07-408.07, 457.09-461.09, 493.20-497.20] MS 110418_tSIM_48pest_4plex_1min_30ppb _01
NNNNNNL:NN 4.01E8TTTTTTTICTTT MS1111110418_tSIM_48pest_4plex_1min_30ppb______01__
NNNNNNL:NN 8.49E7mmmmm/zmm = 459.0847-459.0893 F: FTMS + ESISSSSSSIMSS msx ms [393.15-397.15, 4444440444 .07-408.07, 457.09-461.09, 44444493444 .20-497.20] MS 1111111011 418_tSIM_48pest_4plex_1min_30ppb_____01___
Example Fluoxastrobin, [M+H]+ calc. 459.0866 RT: 2.1989 - 2.4164
2.20 2.22 2.24 2.26 2.28 2.30 2.32 2.34 2.36 2.38 2.40 Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
459.0860
459.0861
459.0859
459.0859 459.0860 459.0859 459.0860 459.0863
NL: 8.83E8 TIC MS 110418_tSIM_48pest_4plex_1min_30ppb _01
NL: 8.49E7 m/z= 459.0847-459.0893 F: FTMS + ESI SIM msx ms [393.15-397.15, 404.07-408.07, 457.09-461.09, 493.20-497.20] MS 110418_tSIM_48pest_4plex_1min_30ppb _01
zoom
380 400 420 440 460 480 500 520 540m/z
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abun
danc
e
459.0859
Iso1 Iso2
Iso3
Iso4
Iso – Isolation window
1.5 ppm (ext.)C21H16ClFN4O5
.)5
Following just one target out of that 4-plex
� If we were doing this by conventional SIM, we would have had 4 times less data points across the peak! � At high resolution and mass accuracy, fragmentation is frequently not needed!
All 4-plex acquisitions
Following just one target out of that 4-plex
All 4-plex acquisitions
41
20110509 JPH JG DEP2 DynRangeTest Sol2_2_70k #520 RT: 2.48 AV: 1 NL: 1.44E9T: FTMS + p ESI Full ms [210.00-600.00]
373.5 374.0 374.5 375.0 375.5 376.0 376.5 377.0 377.5 378.0m/z
0.0000
0.0005
0.0010
0.0015
Rel
ativ
e A
bund
ance
376.1476R=56802N=169.29
C 21 H 24 O 2 N Cl F0.4562 ppm
377.1971R=55302N=169.12
374.0984R=49906
N=6879.50375.0941R=52602N=169.45 375.6917
R=53400N=169.36
376.3792R=40500N=169.25
Boosting dynamic range by multiple fills
20110509 JPH JG DEP2 DynRangeTest Sol2_2_70k #520 RT: 2.48 AV: 1 NL: 1.44E9T: FTMS + p ESI Full ms [210.00-600.00]
250 300 350 400 450 500 550 600m/z
0.0
0.5
1.0
Rel
ativ
e A
bund
ance
266.1534R=65306
N=712762.00 361.2800R=55306
N=855751.44
411.2662R=47500
N=854691.25
313.1679R=60400
N=770267.69
565.2441R=31100
N=904773.75
500.9751R=30300
N=904117.69
500.7180R=37800
N=903871.50
20110509 JPH JG DEP2 DynRangeTest Sol2_2_70k #520 RT: 2.48 AV: 1 NL: 1.44E9T: FTMS + p ESI Full ms [210.00-600.00]
250 300 350 400 450 500 550 600m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
477.2303R=51306
N=881374.69C 29 H 34 O 2 N 2 Cl
-0.0343 ppm
266.1534R=65306
N=712762.00
361.2800R=55306
N=855751.44
411.2662R=47500
N=854691.25
313.1679R=60400
N=770267.69
499.8216R=25200
N=903012.81565.2441R=31100
N=904773.75
zoom
zoom
Loperamide
[m/z 200..600]@ 0.1ms & [m/z 374..378]@ 250ms
(A+1) Isotope: 377.1513 R=53502 N=169.13 S/N=25
rel.abundance = 0.00031% Haloperidol
DR>320.000
Research only!
42
Conclusion
� Ultra-high resolution � MS/MS sensitivity
� Quantitation � 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
43
Acknowledgements S. Horning T. Moehring E. Denisov A. Kholomeev A. Wieghaus W. Balschun O. Lange O. Hengelbrock K. Strupat S. Moehring J. Griep-Raming U. Froehlich D. Nolting F. Czemper R. Malek A. Kuehn T. Rietpietsch R. Malek M. Kellmann M. Biel C. Henrich M. Mueller A.Venckus F. Grosse-Coosmann
M. Antonczak E. Hemenway M. Senko J. Syka J. Schwartz V. Zabrouskov T. Second T. Ziberna T. Second K.Scheffler F. Paffen B. Rose A. Boegehold J. Grote W. Huels A. Schumbera S. Simmel M. Zeller
My Family: Anna Makarova Dasha Makarova Nikita Makarov My parents: Alla & Alexei Makarov
I. Mylchreest A.Guiller E. Schroeder R.A.Purrmann R. Pesch J. Srega I. Jardine
7th European Framework Program: Health-F4-2008-201648/PROSPECTS
44
vv
Thank you !