Dr. Alexander MakarovThermo Fisher Scientific

September 1, 2009

18th IMSC Conference,

Bremen

Frontiers of Orbitrap Mass Spectrometry

Irkutsk

2

Zoology of ion traps

Ion trapsIon trapsThe art of confined motion

Down-side: imprisonment of innocent ionsUp-side: comparing to other MS, ions enter a world where they could live an eventful and meaningful lifeIon traps with

damping“Fishbowl”

Ion traps without damping

“Outer space”

(FAIMS traps)

• Paul traps• Linear radial• Linear axial

Magneto-static

RF Electrostatic

• FT ICR• Accelerators• Storage rings

• Multi-reflection• Multi-deflection• Orbital

• FT trap

Mass analysis:• excitation/ejection• image current• time-of-flight

Mass analysis:• excitation & ejejection

RF traps

Depends on free oscillations of narrow packets

3

Electrostatic traps: multi-reflection

Examples of Closed-type Examples of Open-type

Melzner F. US Patent 3,226,543; 1965.

Benner W.H. Anal.Chem.1997, 69, 4152-4168.

Behrisch R.; Blauth E.W.; Melzner F.; Meyer E.H. US Patent 3,174,034, 1965.

Wollnik H.; Przewloka A. Int. J. Mass Spectrom.1990, 96, 267-274.

Wollnik H., GB2,080,021, 1982

Verentchikov A.N.; Yavor M.I.; et al. Tech. Phys. 2005, 50, 73-81 and 82-86.

- image current detection- detection by excitation/ejection- time-of-flight detection

4

Electrostatic traps: multi-deflection

Examples of Closed-type Examples of Open-type

Toyoda M., Ishihara M. US7,148,473

Moller S.P. NIM A, 1997, 394, 281-286.

Matsuo T.; Toyoda M.; Sakurai T.; Ishihara M. I J. Mass Spectrom. 1997, 32, 1179-1185.

Satoh T. et al. J. Am. Soc. Mass Spectrom.2005, 16, 1969-1975.

Sakurai T.; Ito H.; Matsuo T. Anal. Chem.1994, 66, 2313-2317.

- image current detection- detection by excitation/ejection- time-of-flight detection

Bakker J.M.B. In: Advances in Mass Spectrometry. Vol. 5. 1971, 278-280.

5

Multi-reflection + Multi-deflection = Orbital

Examples of Closed-type Examples of Open-type

Makarov A. Anal. Chem. 2000, 72, 1156-1162.

Knight, R.D. Appl. Phys. Lett. 1981, 38,221-222.

Yang L.; Church D.A.; Tu S.; Jin J. Phys. Rev. A 1994, 50, 177-186.

Kingdon, K.H. A Phys. Rev. 1923, 21, 408-418.

Alimpiev S.S.; Makarov A.A. et al. SU 1,716,922, 1991.

- image current detection- detection by excitation/ejection- time-of-flight detection

6

Common features of electrostatic traps

Period of repetitive motion is T ~ (m/z)1/2

m/z-independent potential well In principle, unlimited mass rangeVery long mean free path must be provided:

• Loss of momentum widens the beam and deteriorates peak shape• Collisions take place at high energies (keVs) and could lead to prompt

or metastable ion fragmentation, thus reducing S/N Space-charge effects:

• Global mass shifts independent of m/z;• Diffusion of packet size • Synchronization of packets of different m/z (coalescence, self-

bunching)Short ion packets must be formed along the direction of m/zdispersionAccuracies of electrode shape, voltage and positioning are crucial for resolving power In closed traps, time-dependent fields are needed to introduce ions

++

++

Ø0.0001

7

What is so special about Orbitrap analyzer?

Image current detection naturally yields frequencies- an “easy” way towards high resolving power and mass accuracy along with all-mass detection and high sensitivityFor a given resolving power, Orbitrap field provides the smallest dimensions and the highest possible frequencies out of all types of electrostatic traps (harmonic oscillations)As a consequence of this, additional properties appear:

• high space charge capacity• robust and maintenance-free mass

analyzerFor other types of detection, other geometries might be more appropriate…

{ })/ln(2/2

),( 222mm RrRrzkzrU ⋅+−⋅=

z

f

r

8

Injection into the Orbitrap analyzer and Formation of Ion Rings

(r,φ) (r,z)

A short ion packet of one m/z enters the field Increasing voltage squeezes ions“Excitation by injection” is initiatedVoltage stabilises and ion trajectories are also stabilizedAngular spreading forms a ROTATING RING

9

Detection of Ions in the Orbitrap

Image current detection

I(t)

tI(t)

•All-mass detection (Felgett advantage)•Noise equivalent to <10 ē/√Hz

Frequency of axial oscillations of each ring induces an image current on split outer electrodes

10

zmk/

=ω

Detection of ion rings

Multiple ions in the Orbitrap analyzer generate a complex signal whose frequencies are determined using a fast Fourier Transformation

• Lighter ions enter Orbitrap analyzer earlier therefore they are squeezed closer to the central electrode than heavier ions

11

Experiments with individual ions (myoglobin +20)

Inte

nsity

848.3 848.4 848.5 848.6 848.7 848.8 848.9m/z

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000848.70261.7 ppmR=50300

848.4498-4.5 ppm

R=38900

848.6003-2 ppm

R=46500848.7964-5.7 ppmR=22100

848.4 848.6 848.8 849.0 849.2m/z

R=46100

848.9516< 1 ppm

R=44800

0

2000000

4000000

6000000

8000000

848.70251 ppm

848.6515<1 ppm

R=45200

Inte

nsity

(1) (1) (1)

(f)

(1) (2)(1)

Velos

12

Overview of results for individual ions of +20 myoglobin

0

1

2

3

4

5

6

7

8

9

10

11

12

-5 0 5 10

Mass error, ppm

S/N

0

1

2

3

4

5

6

7

8

9

10

11

12

-5 0 5 10

Mass error, ppmS/

N

R=60,000 (0.76 sec) R=100,000 (1.52 sec)

Velos

Conclusion: Orbitrap could detect down to <10 elementary charges with mass accuracy still <3 ppm RMS

~5σ

2×

13

Importance of high transmission

0

1

2

3

4

5

1 10 100 1.000 10.000

RMS Mass accuracy, ppm

INJECTED ions/peak in 1 sec

High-resolution oaTOFOrbitrap

0

1

2

3

4

5

1 10 100 1.000 10.000

RMS Mass accuracy, ppm

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%

14

0.8 sec

8 M record length10 Ms/s (borrowed LeCroy)0.8 s transientf =711 kHz,Δf=2.39 Hzf/Δf≈ 300000M/ΔM=½ f/Δf ≈ 150000

A.A. Makarov, Anal. Chem., v.72 (2000), No.6, p.1156-1162.

First steps: Orbitrap with Laser Ion Source

Field compensator Ion source

High voltageamplifier

15

1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06

Typical number of ions per injection 1.E-09 1.E-08 1.E-07 1.E-06 1.E-05 1.E-04 1.E-03 1.E-02

Typical width of ion pulse, s

For discontinuous analyzers, continuous ion beams require external storage and pulsed extraction

Orbitrap “fast inj.”Orbitrap“slow Inj.”TOFFT ICRRF ion trap

1.E-01 1.E+00 1.E+01 1.E+02

Typical entrance area, sq.mmTypical no. of ions per injection Typical width of ion pulse, sec Typical entrance area, mm2

16

Orbitrap mass spectrometry: state of the art

Sample in solution

Ion Source

Ion Source … C-trapC-trap Orbitrap

analyzerOrbitrapanalyzer

How does it work?

17

Curved Linear Trap (C-trap) as an Injection Device

CE

OE-1

OE-2

C-trap

Lenses

Deflector

Quasi-continuous ion beam enters a gas-filled RF-only quadrupole (C-trap) Ions collide with bath gas, loose energy and get stored in the C-trapRF is ramped down, radial DC is applied across rodsIons are ejected along lines converging on the orbitrap entrance. Ions enter Orbitrap analyzer as a small packet...

C-trap could be used also for: de-clustering/ removal of chemical noise passing ions through (T-piece)combining multiple populations, etc.

18

Biodiversity of Orbitrap mass spectrometry: status at 2009

+ HCD (XL)(QqQ-type fragmentation)

MALDI Source(high-power paradigm)

ETD(high-intensity beams)

+++ --

-

LTQ Orbitrap familyLTQ Orbitrap family

ExactiveExactive• HCD option

LC

Velos

FAIMS(new dimension of filtering)

19

FAIMS: separating isomers

Front-end Technology Extension: FAIMS

20

New Instrument Type: ESI-Orbitrap Exactive™

First serial bench-top FTMSHighest speed FTMS (10 spectra/ second) for fast LCNo precursor mass selection High resolution, mass accuracy and sensitivity allow not only fast screening and qualitative analysis, but also quantitative analysisIon population control using a pre-scanFast polarity switching without the loss of external calibrationAccurate mass is provided for all peaks in a single shot, from very low to very high S/N (>10,000)Additional dimension of analysis: all-ions fragmentation in an HCD cell

Exactive

21

hepc_96_std_2_5_ppb02 #345-350 RT: 4.29-4.34 AV: 3 SB: 48 3.61-3.96 , 4.72-5.51 NL: 4.90E4T: FTMS {0,0} + p ESI Full ms [600.00-1000.00]

697.5 698.0 698.5 699.0 699.5 700.0 700.5 701.0 701.5 702.0 702.5m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

700.

7747

700.

5249

700.

2743

701.

0246

701.

2750

701.

5245

701.

7757

698.

0179

700.

0232

698.

2677

697.

7677

698.

5178

702.

0258

701.

3460

699.

0185

698.

7676

702.

2986

699.

7824

699.

3641

698.

6295

699.

5554

Blow-up of the

internal standard

and

analyte ions (2.5ppb)

Example: Quantitation of Hepcidin using Exactive

2.5 ppb spiked and extracted plasma sample 100k resolution

H. Li1, M. J. Rose1, B. J. Hart2, S. Sharma2, C. A. James1

1Amgen Inc, Thousand Oaks , CA; 2Thermo Scientific, San Jose, CAExactive

22

Exactive HR-AM_MS QC Plate: Quantitation

RT: 0.00000 - 9.52012 SM: 7G

0 2 4 6 8Time (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

RT: 4.34193

RT: 4.34193

RT: 6.32679

NL: 5.53E3m/z= 698.01436-698.02134+698.26425-698.27123 F: FTMS {0,0} + p ESI Full ms [600.00-1000.00] MS ICIS hepc_96_std_2_5_ppb02

NL: 1.01E5m/z= 700.52143-700.52843+700.77122-700.77822 F: FTMS {0,0} + p ESI Full ms [600.00-1000.00] MS ICIS hepc_96_std_2_5_ppb02

InternalStandard

Analyte2.5ppb

Exactive

23

Exactive HR-AM_ HCD MS QC Plate: Confirmation by HCD

RT: 0.00000 - 9.01554 SM: 7G

0 1 2 3 4 5 6 7 8 9Time (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

RT: 4.06004

RT: 4.06004

RT: 6.67110

NL: 6.08E2m/z= 501.19049-501.20051 F: FTMS {0,0} + p ESI Full ms2 1000.00@hcd39.00 [330.00-1330.00] MS ICIS qc_plate_2_5_ppb51

NL: 4.12E4m/z= 511.22024-511.23046 F: FTMS {0,0} + p ESI Full ms2 1000.00@hcd39.00 [330.00-1330.00] MS ICIS qc_plate_2_5_ppb51

InternalStandard

Analyte2.5ppb

Exactive

24

Quantitation using precursor scan: 2.5 ppb to 500ppb

Sample in spiked serum Calibration curves in triplicate 2.5 to 500 ng/mlQCs in triplicate at 4 levels (5, 15, 100 and 400 ng/ml)2/3’s of QCs must be within 25% of nominal at each level

Exactive

25

All-ion fragmentation for structure elucidation

Full scan spectra of Verapamil with and without HCD fragmentation.

Identification of precursors and fragments is facilitated by very high resolution and mass accuracy

Exactive

1.3 ppmO

O

N

O

O

1.4 ppm

1.2 ppm

N

N

O

O

2.1 ppmMH+

150 200 250 300 350 400 450m/z

0

20

40

60

80

1000

20

40

60

80

100 455.2890

167.0126

239.1614 311.2189

165.0912

303.2071

260.1642 455.2895

1.3 ppmO

O

N

O

O

1.4 ppm

1.2 ppm

N

N

O

O

2.1 ppmMH+

HCD off

HCD on

150 200 250 300 350 400 450m/z

0

20

40

60

80

1000

20

40

60

80

100 455.2890

167.0126

239.1614 311.2189

165.0912

303.2071

260.1642 455.2895

Quantitation

Confirmation

Replaces MRMs

26

New hybrid for proteomics: LTQ Orbitrap Velos

Main emphasis: • higher speed of MS/MS not on paper (e.g. for calibration mixtures)

but for real-life samples • higher quality and choice of MS/MS methods

Tuesday 3:00 – 3:20Eduard Denisov,, E. Damoc, J. Griep-Raming, O. Lange, A. Makarov, H. Kuipers, P. Remes, J. Schwartz, D. Taylor, T. Moehring and V. Zabrouskov:"A New Instrument for High-speed Proteomics: Orbitrap Mass Analyzer Interfaced to a Dual Linear Trap"

Velos

27

What to expect from Orbitrap mass spectrometry?

Faster and more powerful Orbitraps• High-field Orbitraps (dimensions, voltage)• Higher-performance (resolving power, mass accuracy) Orbitraps

Higher space charge capacity C-trap

“Intelligence and speed are better than haste”: multiple fills allow to improve quality of analysis

• Stepped collision energy in 1 scan• Multiple SIM windows in 1 scan • Multiple MS/MS in 1 scan• ....

28

Example 1: High-field Orbitrap

Smaller gap and higher voltage- 1.8x frequencySpace charge shifts: <1 ppm over the whole dynamic rangeMost “best” parameters could be demonstrated, but tuning ranges much narrower; more research is needed.

12 m

m

18 m

m

Research only!

30 m

m

526.26 526.28m/z

0

1

2

3

4

0

1

2

3

4

Rel

ativ

e A

bund

ance 526.2614

R=377733

526.2722R=375264

526.2756R=354997

526.2608R=378620

526.2717R=375454

526.2747R=359061

196.06 196.08 196.1002

4

6

8

10

12

02

4

68

10

12196.0913R=630118

196.0850R=598468

196.0910R=629410

196.0847R=629316R

elat

ive

Abu

ndan

ce

Experimental

Theoretical

RFWHM ≈630,000 RFWHM ≈370,000

A. Makarov et al. JASMS, 2009

A. Makarov, E. Denisov, O. Lange J. Am. Soc. Mass Spectrom. 2009, 20, 1391–1396.

29

Example 2: Overtone Orbitrap

Each of outer electrodes is split in two, all outer electrodes sustained at virtual ground

Research only!

30 m

m

30

SIM from overtone Orbitrap

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

Rel

ativ

e In

tens

ity

157.9970

3rd harmonics: 41%R≈107,400

1422.9772

56.8789

355.4934

2nd harmonics: 1%R≈71,100

5th harmonics: 4%R≈177,300

1st harmonics: 54%R≈35,500

•Use of 3rd harmonics allows to drastically increase resolving power in short acquisitions-though at the expense of sensitivity (↓ x2.5)•Extensive filtering is required to remove undesired harmonics

31

Example 3: Compact Orbitrap

Smaller size- 1.5x frequency at the same voltage, 1.8x at higher voltage Higher tolerance requirementsNew lenses for focusing onto Orbitrap entranceLower capacitance

12 m

m

20 m

m

Research only!

30 m

m

2

3

4526,2600R=427119

526,2703R=429586

0

1

526.26 526.28m/z

0

1

2

3

4

R=378620

526.2717R=375454

526.2747R=359061

196.06 196.08 196.1002

4

6

8

10

12196.0910R=629410

196.0847R=629316R

elat

ive

Abu

ndan

ce Theoretical

Experimental

196,06 196,08 196,100

2

4

6

8

10

12

Rel

ativ

e A

bund

ance

196,0910R=667444

196,0848R=677597

32

0

1

2

3

4

5

6

7

8

9

10

‐6 ‐4 ‐2 0 2 4 6

S/N

Mass error, ppm

Lower capacitance improves sensitivity: detection of individual ions of +10 Ubiquittin

~5σ

R=120,000 (0.76 sec)

S/N≈6

S/N≈3

Conclusion: Combination of compact Orbitrap analyzer with a customized preamplifier allows increasing sensitivity by a factor of x1.7

33

Multiple fills: Stepped collision energy in 1 scan

500 1000 1500m/z

0

50

1000

50

1000

50

1000

50

1001421.9792

1189.9635977.9568653.9355247.9324

321.9290 977.9571 1421.9774765.9498

247.9324

321.9291 725.9576 937.9651 1318.06881421.9767

247.9322

1189.9630977.9565321.9289

a) CE=99 eV (NCE=35)

b) CE=121 eV (NCE=45)

c) CE=148 eV (NCE=55)

d) CE=99…148 eV (NCE=35…55)

Perfluoroalkylphosphazine, C28H19O6N3P3F44, m/z 1421.97842

Σ3 different MS/MS

spectra in 1 Orbitrap scan

34

MPX(432.9_3_530.8_2_379.2_2)_100k_01 NL: 3.30E5T: FTMS + p ESI Full ms [100.00-1980.00]

200 300 400 500 600 700 800 900 1000m/z0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

Multiple fills: multiple MS/MS

Bradikinin 1-7Angiotensin I (human)Bradikinin

y2

y2y6

y8y7b8

b5,b5

MH2+

b1,b1

y1

b62+,b62+

a8

a6a5

b6 b8

b6

a6

b4,b4b72+

Data acquired by Dr. M.Kellmann

35

Conclusion

Orbitrap mass analyzer offers a unique and very valuable combination of analytical parameters

New analytical methods are made possible by Orbitrap technology

Orbitraps are still evolving …..• Higher speed• Higher resolving power and mass

accuracy• Higher sensitivity• Mass production

Exciting new applications continue to emerge

36

Acknowledgements

My Family:Anna, Dasha, Nikita MakarovMy parents: Alla & Alexei Makarov

Manchester, UK:S. DavisA. HoffmannR. LawtherM. HardmanN. DemetriadesJ. HughesB. McKnightK. WorthingtonS. Smirnov

Bremen, DE:E. DenisovA. KholomeevW. BalschunO. LangeK. StrupatS. HorningR. PeschJ. SregaG. JungW. Huels F. CzemperO. HengelbrockA. Wieghaus

San Jose, CA:E. HemenwayM. Antonczak M. SenkoJ. SykaJ. SchwartzV. ZabrouskovT. SecondI. JardineT. ZibernaJ-J. DunyachE. WoutersM. SplendoreP. RemesD. TaylorB. TehlirianV. KovtounN. Izgarian

Development teams:LTQLTQ FTMALDIETDExactive

J. Griep-RamingE. SchroederU. FroehlichD. NoltingR. MalekT. MoehringH. MuensterM. KellmannM. ZellerS. StrubeS. MoehringR. SeedorfS. Kanngiesser

Colleagues:P. FongJ. GabelS. FenskeP. CardenasM. KonicekR. HermezianW. DeweyB. SiebertP. AthertonM. AhrensJ. SklenarS. ZanonW. WangJ. PhillipsJ. HornerJ. BlethrowH. Bui

37

Thank you !