1. Orbitrap Mass Analyser - Overview and Applications in Proteomics
Alexander Makarov, Michaela Scigelova
Thermo Electron Corporation

2. Outline Orbitrap mass analyser Linking orbitrap to linear ion trap
Flexibility of use of LTQ Orbitrap
Focus on:
High resolution
Sensitivity
Speed
Dynamic range
Conclusion

3. Principle of Trapping in the Orbitrap
The Orbitrap is an ion trap – but there are no RF or magnet fields!
Moving ions are trapped around an electrode
Electrostatic attraction is compensated by centrifugal force arising from the initial tangential velocity
Potential barriers created by end-electrodes confine the ions axially
One can control the frequencies of oscillations (especially the axial ones) by shaping the electrodes appropriately
Thus we arrive at …
Orbital traps
Kingdon (1923)

4. Orbitrap – Electrostatic Field Based Mass Analyserz φ r
Electrostatic field mass analyser
Korsunskii M.I., Basakutsa V.A. Sov. Physics-Tech. Phys. 1958; 3: 1396.
Knight R.D. Appl.Phys.Lett. 1981, 38: 221.
Gall L.N.,Golikov Y.K.,Aleksandrov M.L.,Pechalina Y.E.,Holin N.A. SU Pat , 1986.

5. Ion Motion in Orbitrap
Only an axial frequency does not depend on initial energy, angle, and position of ions, so it can be used for mass analysis
The axial oscillation frequency follows the formula
w = oscillation frequency
k = instrumental const.
m/z = …. what we want!
A.A. Makarov, Anal. Chem. 2000, 72:
A.A. Makarov et al., Anal. Chem. 2006, 78:

6. Ions of Different m/z in Orbitrap
Large ion capacity - stacking the rings
Fourier transform needed to obtain individual frequencies of ions of different m/z

7. How Big Is Orbitrap?

8. Getting Ions into the Orbitrap
The “ideal Kingdon” field has been known since 1950’s, but not used in MS. Why? There is a catch
how to get ions into it ?
Ions coming from the outside into a static electric field will zoom past, like a comet from the outer space flies through a solar system
The catch: The field must not be static when ions come in!
A potential barrier stopping the ions before they reach an electrode can be created by lowering the central electrode voltage while ions are still entering
Thus we arrive at the principle of
Electrodynamic Squeezing
A.A. Makarov, Anal. Chem. 2000, 72:
A.A. Makarov, US Pat. 5,886,346, 1999.
A.A. Makarov et al., US Pat. 6,872,938, 2005.

9. Curved Linear Trap (C-trap) for ‘Fast’ Injection
Ions are stored and cooled in the RF-only C-trap
After trapping the RF is ramped down and DC voltages are applied to the rods, creating a field across the trap that ejects along lines converging to the pole of curvature (which coincides with the orbitrap entrance). As ions enter the orbitrap, they are picked up and squeezed by its electric field
As the result, ions stay concentrated (within mm3) only for a very short time, so space charge effects do not have time to develop
Now we can interface the orbitrap to whatever we want!
Push
Trap
Pull
Lenses
Orbitrap
Gate
Deflector
A.A. Makarov et al., US Pat. 6,872,938, 2005.
A. Kholomeev et al., WO05/124821, 2005.

10. Outline Orbitrap mass analyser Linking orbitrap to linear ion trap
Flexibility of use of LTQ Orbitrap
Focus on:
High resolution
Sensitivity
Speed
Dynamic range
Conclusion

11. Linking Linear Trap with Orbitrap
Combining the features of the Finnigan LTQ…
ESI, nanospray, APCI, APPI ionsation methods
outstanding sensitivity
MSn operation
Ruggedness and ease of use It adds capabilities for the most demanding analyses
…with excellent performance of orbitrap
High resolution
Accurate mass determination
It is fast - even with high resolution/accurate mass detection

12. LTQ Orbitrap Operation Principle
1. Ions are stored in the Linear Trap
2. …. are axially ejected
3. …. and trapped in the C-trap
4. …. they are squeezed into a small cloud and injected into the Orbitrap
5. …. where they are electrostatically trapped, while rotating around the central electrode
and performing axial oscillation
The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier
Ions of only one mass generate a sine wave signal

13. How Big Is LTQ Orbitrap?
Electrostatic field mass analyser

14. What LTQ Orbitrap Delivers
Mass resolution > 60,000 at m/z 400 at 1 sec cycle
Max. resolution over 100,000 (FWHM)
Mass accuracy < 5 ppm external calibration
Mass accuracy < 2 ppm internal calibration
Mass range 50 – 2,000; 200 – 4,000
Sensitivity sub-femtomole on column
Throughput 4 scans per second (1 high-resolution scan in the orbitrap
+ 3 MS/MS scans in the LTQ)

15. Outline Orbitrap mass analyser Linking orbitrap to linear ion trap
Flexible method design for LTQ Orbitrap
Focus on:
High resolution
Sensitivity
Speed
Dynamic range
Conclusion

16. MS/MS with precursor accurate mass only
Setup for highest MS/MS productivity
Cycle time 1 second
SE1
Full Scan MS
SE2
MS/MS
SE3
MS/MS
SE4
MS/MS
1 LTQ Orbitrap high resolution full scan
and in parallel
3 low resolution ion trap MS/MS scans
SE denotes a ‘scan event’

17. “All-round accurate mass” MS/MS methods
Setup for high mass accuracy
Cycle time 2 seconds
SE1
Full Scan MS
SE2
MS/MS
SE3
MS2 (or MS3)
SE4
MS2 (or MS3)
1 LTQ Orbitrap high resolution full scan
and sequentially
3 high resolution LTQ Orbitrap MS/MS scans
External mass calibration

18. “All-round accurate mass” MS/MS methods
Setup for highest mass accuracy
Cycle time 2.2 seconds
SE1
Full Scan MS
SE2
MS/MS
SE3
MS2 (or MS3)
SE4
MS2 (or MS3)
1 LTQ Orbitrap high resolution full scan
and sequentially
3 high resolution LTQ Orbitrap MS/MS scans
Internal mass calibration

19. Various combinations of MS/MS methods
SE1
Full Scan MS
Example: phosphopeptides analysis
SE2
MS/MS
SE4
MS/MS
SE3
MS3
SE5
MS3
1 Orbitrap high resolution full scan
and
{ high resolution Orbitrap MS/MS scan
and neutral loss triggered
Low-resolution ion trap MS3 scan } x2
External mass calibration

20. Precursor phosphopeptides m/z 831: -S1 Casein 121-134; m/z 1031: -Casein 33-48
Orbitrap detector
PP_ _10-POS #
22-49
RT:
AV: 14
NL:
5.93E3 F:
FTMS + p NSI Full ms [ ]
800
850
900
950
z=2
100 95 90
1031.0
1032.0
1033.0
1034.0
m/z 85
830.0
831.0
832.0
833.0
834.0
835.0
m/z 80
z=2 75
, ppm 70 65 60
z=2 55
Relative Abundance 50 45 40 35
, ppm 30
z=2 25 20
z=2 15 10 5
z=2
1000
1050
1100
1150
m/z
Samples: Dr. Martin Larsen, Prof. Ole N Jensen
University of Southern Denmark

21. MS/MS of m/z 1031 FQS*EEQQQTEDELQDK
Orbitrap detector
m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
Relative Abundance
Neutral loss
exactly detected
+2.7 ppm
976
977
978
979
980
981
982
983
984
985
986
m/z
400
600
800
1000
1200
1400
1600
1800
2000
S* denotes dehydroalanine

22. MS3 of m/z 982 triggered upon the accurate neutral loss detection
Linear ion trap detector
672.3
100 95 90
328.1 85
632.5
747.3
1620.7 80
965.8 75
632.4
1619.6 70
876.3 65
836.9
965.0
1332.2
1619.5 60
965.9
1818.6 55 50
836.8
1216.5
Relative Abundance
966.3
1817.4
1361.6 45
1087.2
1689.7
827.8 40
964.8
1089.3
1234.3 35
503.3
544.8
900.4
1490.7
1105.5 30
345.2
584.3
1106.5
1702.3 25
390.2
1281.3
1574.8
456.3
1070.9
1198.7
1461.5 20
1817.3
1820.7 15
968.0
1715.4
1836.3 10 5
1836.6
400
600
800
1000
1200
1400
1600
1800
m/z

23. Interpretation of fragments from MS3 experiment
Complete y and b series are observed

24. Outline Orbitrap mass analyser Linking orbitrap to linear ion trap
Flexibility of use of LTQ Orbitrap
Focus on:
High resolution and mass accuracy
Sensitivity
Speed
Dynamic range
Conclusion

25. .. confident ID, PTMs, de novo sequencing, top-down
High Resolution &
Accurate Mass
.. confident ID, PTMs, de novo sequencing, top-down

26. High Mass Resolution and Accurate Mass (in 1 second)
NOTE: All mass accuracies in this presentation are with external calibration
theoretical
R= 82,000
+ 0.7 ppm
measured

27. High Masses and Mass Accuracy: Apomyoglobin, charge state 10+
measured
All mass accuracies < 2 ppm
theoretical

28. High Masses and Mass Accuracy: Carbonic Anhydrase, charge state 21+
measured
All mass accuracies < 3 ppm
theoretical

29. Long-term stability of external calibration
Deviation, ppm
3 ppm
4 hours
Time, hours
(m/z 1422 at 100%; m/z 524 at <0.02%).

30. Internal Calibration in LTQ Orbitrap
Detection
Mixing of ion populations and ejection
Injection of analyte
Injection of the calibrant
Olsen, J.V.; de Godoy, L.M.; Li, G.; Macek, B.; Mortensen, P.; Pesch, R.; Makarov, A.A.; Lange, O.; Horning, S.; Mann, M.
“Parts per million mass accuracy on an orbitrap mass spectrometer via lock-mass injection into a C-trap.”
Mol. Cell. Proteomics 2005, 4:

31. ..while delivering accurate mass
Speed
..while delivering accurate mass
in MS, MS/MS and MSn

32. Complex Protein Digests: ‘Big 5’ Experiment
Digging deep into the baseline for low abundant co-eluting peptides
Total time 2.4 seconds
SE1
Full Scan MS
SE2
MS/MS
SE3
MS/MS
SE4
MS/MS
SE5
MS/MS
SE6
MS/MS
1 LTQ Orbitrap high resolution full scan
and
5 fast ion trap MS/MS scans
SE denotes a ‘scan event’

33. Complex Mixture - Selecting Ions for Fragmentation
599.0
600.0
601.0
602.0
603.0
m/z 50
100
Relative Abundance
548
550
552
554
556
558
560
562
m/z 50
100
Relative Abundance
775
780
785
790
795
800
805
810
m/z 50
100
Relative Abundance
MS/MS
MS/MS
MS/MS
MS/MS
MS/MS

34. Parallel Detection in Orbitrap and Linear Ion Trap
RT: 41.57
MS/MS of m/z 598.6
Scan # 4870
RT: 41.58
MS/MS of m/z 547.3
Scan # 4871
MS/MS of m/z 777.4
Scan # 4872
RT: 41.59
MS/MS of m/z 974.9
Scan # 4873
RT: 41.60
MS/MS of m/z
Scan # 4874
RT: 41.56
High resolution
Full scan # 4869
High resolution full scan in Orbitrap and 5 MS/MS in linear ion trap
Time
[sec]
Total cycle is 2.4 seconds
1 High resolution scan with accuracies < 5 ppm
External calibration
5 ion trap MS/MS in parallel

35. Resolving Power vs Cycle Time
785.0
785.2
785.4
785.6
785.8
786.0
786.2
786.4
786.6
786.8
787.0
787.2
787.4
787.6
787.8
788.0
788.2
m/z 20 40 60 80
100
Relative Abundance
R=5901
R=5900
R=6000
R=5800
R=6200
R=23801
R=23900
R=24000
R=24100
R=15600
R=24300
R=48101
R=47700
R=48200
R=47500
R=42000
R=47100
R=94801
R=95200
R=93600
R=98000
R=95800
R=89200
RP 7500
0.2 s
RP 30000
0.5 s
RP 60000
0.9 s RP
1.6 s

36. Sensitivity

37. Horse Cytochrome C, Horse Myoglobin Bovine Serum Albumin, 1 fmol on column
m/z 653 (2+)
theory: measured: (+0.7 ppm)
dd IT MSMS on this scan (scan 3588)
m/z 653
nanoLC
NewObjective 75 um PicoFrit column
Flow rate: 200 nl / min
From 98 % A (water, 0.1 % FA) to 60% B (Acetonitrile, 0.1 % FA) in 20 min
Coverage
Cytochrome C 67%
Myoglobin 71%
BSA 45%

38. Protein digest mix: 1 fmol each on column
Peptide m/z 653 (2+) at RT: min
Base Peak Chromatogram
NanoLC run with
75 um PicoFrit column, flow rate: 200 nl / min
Fast gradient (20 min)
1 LTQ Orbitrap Full Scan resolution 60,000
6 DD LTQ MS/MS Scan
Cycle time 2.6 seconds

39. Data Dependent MS/MS of Peptide m/z 653 (2+)

40. Assigned Fragment Ions by SEQUEST

41. ..detecting minor components in complex mixtures
Dynamic Range
..detecting minor components in complex mixtures

42. Angiotensin 10 pmol/ul + Glu-fibrinogen 10 fmol/ul Concentration Difference 1000x
Angio10pmol_Glufib10fmol_Res30000 # 6
RT:
0.09
AV: 1 T:
FTMS + p ESI Full ms [ ]
100
NL:
1.18E8 95 90 85 10 20 30 40 50 60 70 80 90
100
Relative Abundance
NL:
9.35E4
784.5
785.0
785.5
786.0
786.5
787.0
787.5
788.0
m/z
Measured
Calculated
Dm = -0.2 ppm 80 75 70 65 60 55
Relative Abundance 50 45 40 35 30 25 20
785 ? 15 10 5
800
1000
400
600
1200
1400
1600
1800
2000
m/z

43. MS/MS of Glu-Fibrinogen @10 fmol/ul y 12 +2
692.8 y2 y4 y3 y5 y6 y7
# RT: NL: 6.64E3
200
300
400
500
600
700
800
900
1000
1100
1200
m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
Relative Abundance
Measured
Calculated
Dm = -1.2 ppm
627.3 b 8 +1
887.3 5
515.2

44. Dynamic Range in a Single Spectrum (0.75 sec Acquisition)

45. Conclusion
The orbitrap mass analyzer is first fundamentally new mass analyzer introduced commercially in over 20 years
The last novel mass spectrometer introduction was the RF Ion Trap (Finnigan MAT) in the early1980’s
The main advantages of the orbitrap mass analyzer are:
Unsurpassed dynamic range of mass accuracy
High resolution
High sensitivity
High stability
Compact package
Maintenance-free
The LTQ Orbitrap is the first implementation of the orbitrap analyzer in a hybrid instrument
Isolation, fragmentation and MSn is provided mainly by the linear trap
The C-trap supports multiple ion fills, CID and future expansion
The orbitrap is and will be used as a detector

46. About the Authors Dr. Alexander Makarov Dr. Michaela Scigelova
The inventor of orbitrap mass analyser
Research Manager at Thermo Electron in Bremen
Dr. Michaela Scigelova
LC/MS application expert
at Thermo Electron in UK