Alexander MAKAROV - IMP

Late Summer Proteomics
Seminar at IMP Vienna
Orbitrap Mass Spectrometry
for Quantitative Analysis
A.Makarov
August 27, 2012

There are different views on this world…
Life
Genomics
…-omics
Proteomics
C.Flammarion (1842—1925)
Instrumentomics
2
http://commons.wikimedia.org/wiki/File:THE_TURTLE_AND_ELEPHANT.jpg

Mass spectrometry for discovery proteomics
Magnetic
sectors
Res. power
Transmission
Mass accuracy
Dynamic range
Speed
Quantitation
All-ion detection
Simplicity
Time-of-flight (oa)
FT ICR
Ion traps
Res. power
Transmission
Mass accuracy
Dynamic range
Speed
Quantitation
All-ion detection
Simplicity
Quadrupoles
3
Res. power
Transmission
Mass accuracy
Dynamic range
Speed
Quantitation
All-ion detection
Simplicity
Trapping
Use of shaped electrodes
Image current detection
Pulsed injection
Electrostatic fields

What is Orbitrap analyzer?
Orbitrap analyzer =
= Orbital
trapping
+ Image current
detection
+ Electrodynamic
squeezing
+ External pulsed
ion source
4

Orbital trapping
“Ideal Kingdon trap”:
Quadro-logarithmic potential
U ( r,
z)
=
k
⋅
2
2 2 2
{ z − r / 2 + R ⋅ln(
r / )}
m
R m
Characteristic frequencies:
• Frequency of rotation ω φ
• Frequency of radial oscillations ω r
• Frequency of axial oscillations ω z
ω ϕ
=
ω z
2
⎛
⎜
⎝
R m
R
2
⎞
⎟
⎠
−1
ω r
= ω z
ω
z
=
⎛
⎜
⎝
R m
R
k
m / q
2
⎞
⎟
⎠
−
2
“Beauty will save the world.”- F.M. Dostoevsky
5
Trapping and quantitation?
Only this frequency does not
depend on energy, angle, etc.
and is used for mass analysis

Detection of Ions in the Orbitrap analyzer
• 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
Image current detection
•All-mass detection
•Noise equivalent to

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)
“Built-in” excitation is
independent of trapping,
hence no limit on linearity

Proof of Principle: Orbitrap with Laser Ion Source
Field compensator
Ion source
0.8 sec
High voltage
amplifier
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
8
A.A. Makarov, Anal. Chem., v.72 (2000), No.6, p.1156-1162.

Injection of Ions into the Orbitrap analyzer
Lenses
Deflector
C-trap
• Ions are stored and
cooled in a curved RFonly
quadrupole (Ctrap)
• 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 simultaneously,
but light
ions enter Orbitrap
analyzer earlier that
heavy ions
9
I(t)
Double trappingand
quantitation?

Could a trapping device be used for quantitation?
Quantitation requires: What a trap needs: Missing:
•High linearity
(signal/input)
•Precise control over
input-dependent effects
(i.e. space-charge effects)
•Linear detection system
Automatic gain
control (AGC),
corrections
•High discriminating
power
•High dynamic range
•High resolution
•High mass accuracy
•High ion capacity
•Concept of “charge budget”
AGC
Intelligent filling
•Low limits of
quantitation (LOQ)
•High transmission
•High sensitivity of
detection
•(UHP)LC-compatible
speed of analysis
10
Help is needed!
•Fast/ultra-fast acquisition
Detectionindep.
filling

Instrumentomics in action: Orbitrap hybrid No. 1
Detect
speed
“Instrument
polymerase”
Speed
of
isolation
Thermo Scientific LTQ Orbitrap LC-MS n
11
Image from:
http://commons.wikimedia.org/w/index.php?title=File:DNA_Repair.jpg&oldid=45779464

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
V
Gas

LTQ Orbitrap for Absolute SILAC experiments
Experiment requires:
Resolving power &
Mass accuracy &
Dynamic Range &
Parallel MS/MS &
Scan Speed &
Sensitivity
Linearity of Quantitation of a Protein
A
Pure Form
B
Background of
Total Cell Lysate
S. Hanke, H. Besir, D. Oesterhelt, M. Mann
J. Proteome Res. 2008, 7 (3), 1118–1130.
0.5 fmol
150 fmol
45,000 fmol
13

Orbitrap Elite instrument
Dual trap
up to 12
scans/sec
“Freedom of
fragmentation”:
CID, HCD, ETD
ETD option
Top-of-the-range
RF-lens interface
Compact high-field
Orbitrap analyzer+ eFT:
up to 8 scans/sec
14

Reporter-based quantitation: MS n for gas-phase purification
MS 1
MS 2
Full MS scan
Precursor ion isolation at m/z p (z>1)
CID in LT
PTR in LT
Isolation at m/z f >m/z p
MS 3 Ting L, Rad R, Gygi SP, Haas W.
HCD of m/z f
(+ CID of precursor)
Reporter ion quantitation
15
Nat Methods. 8 (11) (2011), p. 937-940.
QuantMode
Wenger CD, Lee MV, Hebert AS, McAlister
GC, Phanstiel DH, Westphall MS, Coon JJ.
Nat Methods. 8 (11) (2011), p. 933-935.

Instrumentomics in action:
Orbitrap hybrid No.2
Detect
speed
“Instrument
polymerase”
Speed
of
isolation
16
Thermo Scientific Q Exactive LC-MS/MS
Image from:
http://commons.wikimedia.org/w/index.php?title=File:DNA_Repair.jpg&oldid=45779464

Q Exactive: ultra-high resolution hybrid on bench-top
• Quadrupole mass filter interfaced to a standard Orbitrap analyzer
• Enhanced Fourier transform (eFT) for Orbitrap data processing
• Parallel filling & detection
• Possibility of multiple fills for spectrum multiplexing
• Common with Orbitrap Velos Pro/Elite:
• Predictive automatic gain control (pAGC: MS/MS on the basis of full MS scan)
• S-lens for higher transmission (like in LTQ) with rugged optic
• C-trap directly interfaced to HCD (like in LTQ Orbitrap Velos)
17
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)

Automatic gain control (AGC) in Orbitrap hybrids
• Linear trap hybrids
• Orbitrap Series: e.g. Elite, Velos Pro, ...
• Two independent detectors (Linear Ion Trap + Orbitrap)
• AGC for the Orbitrap is done via complementary detector
• Inject time is accurately determined in all cases
• Bench-Top systems
• Exactive Series: Exactive, Q Exactive
• Single detector (Orbitrap)
• AGC is done via a prescan with reduced acquisition time
(typ. 20ms)
• Inject time is accurately determined in almost all cases
An exception: The AGC-prescan contains mainly highly
charged species and many peaks below the noise threshold
18
Poster: Th623
Improved analysis of biopharmaceutical samples using an MS-only Orbitrap mass spectrometer
O. Scheibner; E. Damoc; E. Denisov; J.-P. Hauschild; O. Lange; F. Czemper; A. Kholomeev, A. Makarov;
A. Wieghaus; M. Bromirski

Example: HeLa run with incompletely digested proteins
HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #18610 RT: 61.67 AV: 1 NL: 4.62E4
T: FTMS + p NSI Full ms [350.00-2000.00]
Relative Abundance
Relative Abundance
100
80
60
40
20
19
0
100
80
60
40
20
0
Relative Abundance
100
80
60
40
20
0
497.00040
R=36500
z=?
10 15 20 25 30 35 40 45 50 55 60 65 70
Time (min)
636.95868
R=41204
z=3
19.43
722.46362
R=33202
z=?
19.47
29.90
29.59
25.98 29.96
28.32 31.79
18.23
22.59
15.67 32.74
13.23 37.11
40.93
34.07 42.77
44.39 50.03
Suspiciously empty end of elution…
827.94812
R=33700
z=?
921.32239
R=31906
z=6 1125.25989
998.30304
R=27506
z=4
R=26906
z=6
1294.64734
R=23002
z=?
1350.11121
R=25406
z=5
1381.47974
R=24406
z=4
1549.78955
R=21506
z=6
1697.28662
R=14800
z=?
54.10 57.65 59.30 60.86 64.90 71.68
55 60 65 70
1824.30591
R=16600
z=?
1977.32275
R=19600
z=?
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #19027-19394 RT: 65.00-68.00 AV: 293 NL: 4.71E3
T: FTMS + p NSI Full ms [350.00-2000.00]
946.55941
R=33317
z=3
445.12314
R=51912
z=?
615.96752
R=32939
z=1
713.40758
R=37751
z=?
912.85357
R=34397
z=3
1034.52732
R=31884
z=3
1101.56294
R=30690
z=5
1318.66850
R=19466
z=1
1481.74376
R=16917
z=1
1 spectrum
1542.77453
R=26371
z=2
1693.91682
R=20607
z=?
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
54.33
55.27
57.65
57.69
1866.41480
R=24006
z=6
59.56
60.30
60.86
1976.16243
R=14206
z=1
x25
64.90 67.76
71.68
71.78
NL: 1.82E9
Base Peak F: ms
MS
HeLa_LysC_noEM
_1e6_1e5_mz350_
2000_fm150HCD_
1
Σ

C-Trap Charge Detection (CTCD)
TIC CTCD : every 5-10 sec
TIC CTCD /TIC Orbitrap is used to
adjust the inject time, if needed
TIC Orbitrap
20
I(t)

Removal of signal suppression by C-Trap Charge Detection
Relative Abundance
4
3
2
1
0
Relative Abundance
100
80
60
40
20
0
p [ ]
497.00040
R=36500
z=?
636.95868
R=41204
z=3
722.46362
R=33202
z=?
827.94812
R=33700
z=?
921.32239
R=31906
z=6 1125.25989
998.30304
R=27506
z=4
R=26906
z=6
1294.64734
R=23002
z=?
1350.11121
R=25406
z=5
1381.47974
R=24406
z=4
1549.78955
R=21506
z=6
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
50.93
54.33
10 15 20 25 30 35 40 45 50 55 60 65 70
55.27
1697.28662
R=14800
z=?
57.65
57.69
59.56
NL=4.6e4
1824.30591
R=16600
z=?
60.30
60.86
1977.32275
R=19600
z=?
64.90 67.76
71.68
CTCD
OFF
NL: 1.82E9
Base Peak F: ms
MS
HeLa_LysC_noEM
_1e6_1e5_mz350_
2000_fm150HCD_
1
Relative Abundance
4
3
2
1
53.96
58.94
54.38
59.36
62.14
70.36
70.33
62.28 66.97
64.10
NL: 1.53E9
Base Peak F: ms
MS
hela_lysc_wem_3e
6_1e5_mz350_20
00_fm150hcd_1
0
21
10 15 20 25 30 35 40 45 50 55 60 65 70
445.11856 536.16394
Time (min)
R=52407 R=47707
z=1 z=1
Relative Abundance
100
80
60
40
20
0
674.55774
R=39206
z=5
825.46289
R=36104
z=3
950.84613
R=32206
z=3
1056.53552
R=31307
z=2
1191.24426
R=28006
z=3 1322.64648 1478.22644 1577.42529 1694.47510 1826.72253
R=22204 R=20002 R=19902 R=18700 R=20702
z=2
z=? z=? z=? z=?
NL=2.7e6
1972.63916
R=16902
z=?
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
CTCD
ON

Spectral acquisition speed with predictive AGC and
parallel filling/detection
AGC
Orbitrap acquisition
Orbitrap acquisition
Orbitrap acquisition
Inject to C-trap
Inject to C-trap
Inject to C-trap
14
12
90%
80%
Scan speed does
not change until fill
time reaches 50 ms
Resolving power
setting: 17,500
(fastest rate)
At higher R, ion
fill times and
duty cycles
increase further!
Scans per second, Hz
10
8
6
4
2
0
70%
60%
50%
40%
30%
20%
10%
0%
Duty cycle of ion filling, %
Scan speed
Duty cycle
Up to 65% of the
total time could be
spent meanwhile on
accumulating ions!
0 20 40 60 80 100
22
Ion fill time, ms

Concept of “charge budget” in ion trap world
• Any trapping device has a limit on the
number of stored ions- “charge budget”
• Typically, this budget is spent as a
“blanket investment”, all peaks large and
small accumulated for the same time. This
limits dynamic range of analysis.
• But the presence of a beam-type mass
filter allows to break this rule and
accumulate intelligently:
• Peaks of interest only- in any desired
combination
• Enhancing certain m/z ranges by “focused
investment”
Q
23

„Charge budget“ concept in targeted analysis
• Sensitivity gain 5 – 10x with SIM
mode
• The gain will be higher in more
complex matrices
100
80
60
40
20
0
100
80
60
40
20
195.0876
N=248402.81
195.0877
N=20741.58
Full MS
S/N = 745
IT= 0.245 ms
SIM (10amu)
For the same target:
S/N = 5400
IT= 1.321 ms
NL: 1.94E8
[150.00-2000.00]
Lowest signal
250330
NL: 1.12E8
[190.10-200.10]
Lowest signal
28240
0
Gain in sensitivity (7x)
6000
In Orbitrap instruments, SIM could
become MRM without any
noticeable time overhead!
S/N (spectrum)
5000
4000
3000
2000
1000
Caffeine
24
0
195.082 195.084 195.086 195.088 195.09 195.092 195.094
S/N (FMS) S/N (SIM10)

Alprazolam quantitation experiment
50 ppt – 10 ppb
250 fg oc - 50 pg oc
Full MS in matrix
6000000
5000000
10 ppt – 10 ppb
50 fg oc - 50 pg oc
SIM
Area
4000000
3000000
2000000
1000000
0
0 2000 4000 6000 8000 10000
fg/uL
Orbitrap 25 mass spectrometer”. Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 2011.
Zoom 10 ppt- 100ppt
120000
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
0 20 40 60 80
fg/uL
100 120
See also: X. He; M. Kozak. “Evaluation of quantitative performance for testosterone analysis in plasma on a novel quadrupole
Area

48 Protein Experiment on Q Exactive
• 204 identical peptides were targeted by three approaches:
• Full scan MS,
• Targeted SIM (t-SIM),
• Targeted MS/MS (t-MS2) = t-SIM+ HCD (practically no time overhead)
• Number of peptides quantifiable at respective spike concentration
Total peptides quantified
250
#peptides quantified
200
150
100
50
0
1
197 201 204 204 204
164
148
137
123
106
99
35
4 0
0.5 5 12.5 25 >25
Amount of spiked peptides [fmol/column]
Full MS
t-SIM
t-MS2
26
t-MS2 method performs best

Minimum total number of ions needed for quantitation
3000
Min. number of ions for CV=10%
2500
2000
1500
1000
500
Qq-Q
Qq-Orbi
Qq-TOF
.
.
.
1
2
3
N
27
0
1 2 3 4 5 6 7 8 9 10
Number of transitions in MRM
CV=10% on the sum of fragment ion
intensities
Assumptions:
• The same Qq for all
• Transmission: 50% for Q3,
30% for Orbitrap, 4% for TOF
• Equal intensities of fragments
• No interferences in QqQ…

Utilization of “Charge budget concept” using spectrum
multiplexing
Standard
operation mode
vs
Spectrum
multiplexing
5.0x10 8
4.5x10 8
4.0x10 8
3.5x10 8
Ion count (arb.)
3.0x10 8
2.5x10 8
2.0x10 8
1.5x10 8
1.0x10 8
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)
5.0x10 7
28
0.0
1 10 100 1000
Inject time [ms]

Low Attomole LOD, 4 Orders of Linear Dynamic Range
Heavy-labeled peptides of eicosanoid pathway enzymes in 250 ng CSF digest.
1E+09
1E+09
Peak Area
100000000
160000000
10000000
140000000 1000000
100000
120000000
10000
100000000
1000
1 10 100 1000 10000 100000
80000000
60000000
40000000
y = 1372.1x + 278774
R² = 0.9998
VAHTVAYLGK
Peak area
450000000
400000000
350000000
300000000
250000000
200000000
150000000
100000000
100000000
10000000
1000000
100000
10000
1000
1 10 100 1000 10000 100000
y = 3820.1x + 578349
R² = 0.9999
YFQGYAR
20000000
50000000
0
0 20000 40000 60000 80000 100000 120000
Sample amount (amole)
0
0 20000 40000 60000 80000 100000 120000
Sample amount (amole)
Protein Peptide LOQ (amole) LOD (amole)
PTGDS GPGEDFR 25 8
PTGS2 QFQYQNR 25 8
PTGS1 LVLTVR 10 3
HPGDS STLPFGK 25 8
PTGES VAHTVAYLGK 30 10
PTGIS FLNPDGSEK 50 17
29
TBXA1 SVADSVLFLR 100 33
ALOX15 YTLEINVR 250 83
ALOX12 LWEIIAR 500 167
LTCS4 YFQGYAR 10 3
Data courtesy Y. Xuan, M. Scigelova, ThermoFisher Scientific- Workshop 5

Data Independent Acquisition: why multiplexing?
Classical data-independent acquisition (DIA )
30
• Lacks the specificity of DDA
• 40 scans in this example to cover 400 amu

Data Independent Acquisition: multiplexing improves
signal-to- noise
Multiplexed DIA (msx-DIA )
12 Hz Acquisition Rate
60 Precursors / second
Broad
isolation
5-plex injection
Adding 5 x 4 amu
isolation windows
1s
64ms detection
1 6 11 16 21 26 31 36 41 46 51 56 …
2 7 12 17 22 27 32 37 42 47 52 57 …
3 8 13 18 23 28 33 38 43 48 53 58 …
4 9 14 19 24 29 34 39 44 49 54 59 …
5 10 15 20 25 30 35 40 45 50 55 60 …
• 20 scans in this example to cover 400 amu
msx-SIM:
“great
equalizer”
m/z
m/z
31
ASMS Poster ThP571: „Multiplexed Data Independent Acquisition for Comparative Proteomics.“
Jarrett D. Egertson 1 ; Andreas Kuehn 2 ; Gennifer Merrihew 1 ; Nicholas Bateman 3 ; Brendan MacLean 1 ; Ying S.
Ting 1 ; Jesse D. Canterbury 4 ; Markus Kellmann 2 ; Vlad Zabrouskov 4 ; Christine Wu 3 ; Michael J. MacCoss 1
1
University of Washington, Seattle, WA; 2 Thermo Fisher Scientific, Bremen, Germany; 3 University of
Pittsburgh, Pittsburgh PA; 4 Thermo Fisher Scientific, San Jose, CA

Current and future uses of spectrum multiplexing
Multiplexed SIMs/MRMs in a single Orbitrap
spectrum
Multiple fragmentation conditions in a single
Orbitrap spectrum (e.g. HCD energy scan)
• Selection of multiple charge states of the same
precursor, each fragmented at optimum
conditions, will increase sequence coverage
• Combining several fragmentation techniques
for the same precursor
• Segmenting mass range of full MS (inc. datadependent)
• ……
De-couple
LC from MS,
better LOQs
↑ quality of
spectra and
ID rate
without
increase of
data files
↑ dynamic
range
32

Conclusion
• Ultra-high resolution
• MS/MS sensitivity
• Quantitation
• Throughput
33
• Advances in technology continue to make quantitative Orbitrap mass spectrometry
increasingly more sensitive and accurate
• For peptide quantitation, Orbitrap mass spectrometry is the best technical solution
• Further improvements of quantitation will be directed along directions of:
• Better selectivity of precursors
• Improvements of ion population control and of corresponding corrections
• Higher spectral acquisition speed
• Further development of spectra multiplexing

Acknowledgements
34
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
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
7 th European Framework Program: Health-F4-2008-
201648/PROSPECTS
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

vv
Thank you !
35