Orbitrap Mass Analyser - Overview and Applications in Proteomics

Orbitrap Mass Analyser - Overview and Applications in Proteomics

Alexander Makarov, Michaela ScigelovaThermo 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

Orbital trapsKingdon (1923)

• 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 …

4

Orbitrap – Electrostatic Field Based Mass Analyser

z

φ

r

)/ln(2/2

),( 222mm RrRrz

kzrU

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. 1247973, 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

zm

k

/

w = oscillation frequencyk = instrumental const.m/z = …. what we want!

A.A. Makarov, Anal. Chem. 2000, 72: 1156-1162.A.A. Makarov et al., Anal. Chem. 2006, 78: 2113-2120.

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: 1156-1162.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

Push

Trap

Pull

Lenses

Orbitrap

Gate

Deflector

• 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 1 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!

A.A. Makarov et al., US Pat. 6,872,938, 2005.A. Kholomeev et al., WO05/124821, 2005.

10

Outline





–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 Trap2. …. are axially ejected3. …. and trapped in the C-trap4. …. they are squeezed into a small cloud and injected into the Orbitrap5. …. 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?

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



• Flexible method design for LTQ Orbitrap


–Sensitivity

–Speed

–Dynamic range

• Conclusion

16

MS/MS with precursor accurate mass only

Setup for highest MS/MS productivityCycle time 1 second

1 LTQ Orbitrap high resolution full scanand in parallel

3 low resolution ion trap MS/MS scans

SE1Full Scan

MS

SE2MS/MS

SE3MS/MS

SE4MS/MS

SE denotes a ‘scan event’

17

“All-round accurate mass” MS/MS methods

Setup for high mass accuracyCycle time 2 seconds

SE1Full Scan

MS

SE2MS/MS

SE3MS2 (or MS3)

SE4MS2 (or MS3)

1 LTQ Orbitrap high resolution full scanand sequentially

3 high resolution LTQ Orbitrap MS/MS scansExternal mass calibration

18

“All-round accurate mass” MS/MS methods

Setup for highest mass accuracyCycle time 2.2 seconds

1 LTQ Orbitrap high resolution full scanand sequentially

3 high resolution LTQ Orbitrap MS/MS scans Internal mass calibration

SE1Full Scan

MS

SE2MS/MS

SE3MS2 (or MS3)

SE4MS2 (or MS3)

19

Various combinations of MS/MS methods

Example: phosphopeptides analysisSE1Full Scan

MS

SE2MS/MS

1 Orbitrap high resolution full scanand

{ high resolution Orbitrap MS/MS scan and neutral loss triggered

Low-resolution ion trap MS3 scan }x2

External mass calibration

SE3MS3

SE4MS/MS

SE5MS3

20

Precursor phosphopeptides m/z 831: -S1 Casein 121-134; m/z 1031: -Casein 33-48

PP_28092005_10-POS #22-49 RT: 0.31-0.70 AV: 14 NL: 5.93E3F: FTMS + p NSI Full ms [ 800.00-1800.00]

800 850 900 950 1000 1050 1100 1150m/z

0

5

10

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100

Rel

ativ

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bu

nda

nce

103

1.4

2128

, +

3.3

pp

m

z=2

1042.91402z=2

830.

9031

3, +

2.5

pp

m

841.89392z=2

1050.89741z=2

1062.38000z=2

830.0 831.0 832.0 833.0 834.0 835.0m/z

830.90315

831.40519

831.90689

832.40787 1031.0 1032.0 1033.0 1034.0

m/z

1031.92296

1031.42128

1032.42430

1032.92600z=2

Samples: Dr. Martin Larsen, Prof. Ole N JensenUniversity of Southern Denmark

Orbitrap detector

21

MS/MS of m/z 1031 FQS*EEQQQTEDELQDK

Neutral lossexactly detected

976 977 978 979 980 981 982 983 984 985 986m/z

982.4320

977.43825

400 600 800 1000 1200 1400 1600 1800 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

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85

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100

Rel

ativ

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bu

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nce

982.43205

+2.7 ppm

S* denotes dehydroalanine

Orbitrap detector

22

MS3 of m/z 982 triggered upon the accurate neutral loss detection

400 600 800 1000 1200 1400 1600 1800m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

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bu

nda

nce

672.3

328.1 747.3 1620.7632.5965.8

1619.6632.4

876.3836.9 1332.2965.0

1619.5965.9

1818.6

1216.5836.8 1817.4966.31361.6

1087.21689.7

827.8964.8

1234.31089.3 1490.7503.3 900.4 1105.5544.81106.5584.3345.2 1702.3

1574.8390.2 1281.3 1461.51198.71070.9456.3 1817.3 1820.7

1715.4968.0 1836.3

1836.6

Linear ion trap detector

23

Interpretation of fragments from MS3 experiment

Complete y and b series are observed

24

Outline




• Focus on:–High resolution and mass accuracy

–Sensitivity

–Speed

–Dynamic range

• Conclusion

25

High Resolution &Accurate Mass

.. confident ID, PTMs, de novo sequencing, top-down

26

High Mass Resolution and Accurate Mass (in 1 second)

+ 0.7 ppm

theoretical

measured

R= 82,000

312.12181

312.13272

NOTE: All mass accuracies in this presentation are with external calibration

27

High Masses and Mass Accuracy: Apomyoglobin, charge state 10+

1695.5 1696.0 1696.5 1697.0m/z

10

20

30

40

50

60

70

80

90

100

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

1696.105601696.20570

1696.30552

1696.40580

1695.80587

1696.50579

1695.60576

1696.705971696.80571

1697.00309

1696.106511696.20677

1695.905991696.30703

1695.805721696.40729

1695.70545 1696.50755

1696.607801695.60518

1696.70805

1696.80830

1697.00881

NL:1.97E6MYO_1#245-350 RT: 4.05-7.12 AV: 104 T: FTMS + p ESI SIM ms [ 1683.50-1708.50]

NL:1.19E5

C 769 H1212 N210 O 218 S2 +H: C 769 H1222 N210 O 218 S2

p (gss, s /p:8) Chrg 10R: 60000 Res .Pwr . @FWHM

All mass accuracies < 2 ppm

theoretical

measured

28

1382.5 1383.0 1383.5m/z

0

10

20

30

40

50

60

70

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100

0

10

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lativ

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bu

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ce

1383.08684R=0

1383.23011R=0

1382.94236R=0

1383.42033R=0

1383.94076R=0

1382.79931R=0

1383.51462R=0

1382.62581R=0

1383.08962R=59809

1383.23292R=59776

1382.94634R=59798

1383.32842R=597871382.85086

R=59809

1383.42391R=597801382.75534

R=59820

1383.56712R=597621382.61205

R=59798 1383.75806R=59731

NL:5.01E3CARB_ANH_4#18-38 RT: 0.78-1.72 AV: 21 T: FTMS + p ESI Full ms2 [email protected] [ 380.00-2000.00]

NL:2.14E3

C 1312 H 2017 N 358 O 384 S 3: C 1312 H 2017 N 358 O 384 S 3p (gss, s /p:40) Chrg 21R: 60000 Res .Pwr . @FWHM

High Masses and Mass Accuracy: Carbonic Anhydrase, charge state 21+

All mass accuracies < 3 ppm

measured

theoretical

29

Long-term stability of external calibrationExternal Mass Accuracy Check

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Time [h]

RMS of 2 m/z 524.264964 m/z 1421.977862

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

De

via

tion

[pp

m]

Time, hours

Dev

iatio

n, p

pm

3 ppm

4 hours

(m/z 1422 at 100%; m/z 524 at <0.02%).

30

Internal Calibration in LTQ Orbitrap

Injection of the calibrantInjection of analyteMixing of ion populations and ejectionDetection

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: 2010-2021.

31

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

1 LTQ Orbitrap high resolution full scanand

5 fast ion trap MS/MS scans

SE1Full Scan

MS

SE2MS/MS

SE3MS/MS

SE4MS/MS

SE denotes a ‘scan event’

SE5MS/MS SE6

MS/MS

33

Complex Mixture - Selecting Ions for Fragmentation

ControlB3a #4869 RT: 41.56 AV: 1 NL: 7.39E6T: FTMS + p NSI Full ms [465.00-1600.00]

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

m/z

0

5

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20

25

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55

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lativ

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bu

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ce

600.9776

804.3450

558.7548

532.2505

649.9460

699.3472897.9816

716.0311956.8159

849.8573 974.9185 1116.5020

599.0 600.0 601.0 602.0 603.0m/z

0

50

100

Rel

ativ

e A

bund

ance

600.9776

598.6563

548 550 552 554 556 558 560 562m/z

0

50

100

Rel

ativ

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bund

ance

558.7548

547.6516

775 780 785 790 795 800 805 810m/z

0

50

100

Rel

ativ

e A

bund

ance

804.3450

777.3942

MS

/MS

MS

/MS

MS

/MS

MS

/MS

MS

/MS

34

ControlB3a #4869 RT: 41.56 AV: 1 NL: 7.39E6T: FTMS + p NSI Full ms [465.00-1600.00]

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600

m/z

0

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600.9776

804.3450

558.7548

532.2505

649.9460

699.3472897.9816

716.0311956.8159

849.8573 974.9185 1116.5020

Parallel Detection in Orbitrap and Linear Ion Trap

• Total cycle is 2.4 seconds• 1 High resolution scan with

accuracies < 5 ppm• External calibration • 5 ion trap MS/MS in parallel

RT: 41.56High resolutionFull scan # 4869

High resolution full scan in Orbitrap and 5 MS/MS in linear ion trap

ControlB3a #4870 RT: 41.57 AV: 1 NL: 7.16E3T: ITMS + c NSI d Full ms2 [email protected] [150.00-1810.00]

200 400 600 800 1000 1200 1400 1600 1800

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

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Re

lativ

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bu

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an

ce

437.9462

542.7487

590.2733

983.4816

776.4982

623.5060

301.24471084.6279

1171.8290

RT: 41.57MS/MS of m/z 598.6Scan # 4870


300 400 500 600 700 800 900 1000 1100

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

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80

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lativ

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bu

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ce

1098.4486921.5529

1018.6340

480.2985

680.4445

805.3505

637.2200361.1457

952.3358

784.3491

514.2266

853.4705706.2417459.1983 588.2148871.4709

445.2212333.3748


400 600 800 1000 1200 1400 1600 1800

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

ela

tive

Ab

un

da

nce

1092.6033

1409.7291

856.3868

539.2245

1294.7877965.7724

1223.7373

654.2495 757.5266 1801.9797

1513.5245436.2499

1674.7556393.1896


200 400 600 800 1000 1200 1400 1600

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

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90

95

100

Re

lativ

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bu

nd

an

ce

535.5252

690.1100

490.3550

575.8568

450.8616361.2963

747.4839

330.2767262.1056

900.6165 1022.6853234.2242

1088.7388

RT: 41.58MS/MS of m/z 547.3Scan # 4871

RT: 41.58MS/MS of m/z 777.4Scan # 4872

RT: 41.59MS/MS of m/z 974.9Scan # 4873

RT: 41.60MS/MS of m/z 1116.5Scan # 4874


200 250 300 350 400 450 500 550 600 650 700 750

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

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90

95

100

Re

lativ

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bu

nd

an

ce

701.4880

592.5975

400.3238

729.5197

767.4117

654.3235

354.2529 683.1174371.1810

309.1429 547.4052512.5754469.5364252.0748

0.0 0.5 1.0 1.5 2.0 2..5

Time [sec]

35

Resolving Power vs Cycle Time

785.0 785.2 785.4 785.6 785.8 786.0 786.2 786.4 786.6 786.8 787.0 787.2 787.4 787.6 787.8 788.0 788.2m/z

0

20

40

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80

100

0

20

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lativ

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danc

e

0

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785.8419R=5901 786.3435

R=5900

786.8447R=5900

787.3463R=6000 787.8453

R=5800785.5934R=6200

785.8421R=23801

786.3434R=23900

786.8446R=24000 787.3457

R=24100 787.8471R=15600

785.5992R=24300

785.8419R=48101 786.3435

R=47700

786.8446R=48200 787.3458

R=47500787.8477R=42000

785.5994R=47100

785.8413R=94801 786.3428

R=95200

786.8442R=93600

787.3458R=98000785.5989

R=95800787.8477R=89200

0.9 s

1.6 s

RP 75000.2 s

RP 300000.5 s

RP 60000

RP 100000

36

Sensitivity

37

Horse Cytochrome C, Horse Myoglobin Bovine Serum Albumin, 1 fmol on column

RT: 19.01 - 42.26

20 22 24 26 28 30 32 34 36 38 40 42Time (min)

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Rel

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478.

25

637.

31

643.

13

791.

42

471.

24

863.

41

740.

40

784.

37

735.

85

582.

3258

4.81

746.

38

494.

15

879.

42

533.

60

607.

26

547.

32

480.

61

620.

36

710.

84

634.

39

861.

14

477.

37

749.

41

653.

3656

4.36

536.

16

536.

1653

6.16

536.

16

536.

16

536.

16

536.

16

485.

01

536.

16

536.

16

496.

52

536.

16

536.

16

536.

16

536.

16

536.

16

536.

16

536.

16

536.

16

NL: 4.29E6Base Peak m/z= 470.00-2000.00 F: FTMS + p NSI Full ms [ 300.00-2000.00] MS BSA_CC_MYO_3fmol_each_total_01

m/z 653 (2+)theory: 653.361701measured: 653.36127 (+0.7 ppm)

BSA_CC_MYO_3fmol_each_total_01 #3588 RT: 24.90 AV: 1 NL: 7.14E3T: ITMS + c NSI d Full ms2 [email protected] [ 165.00-1320.00]

200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z

0

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Rel

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1055.55

251.21

956.52 1056.61

644.61

712.49841.44332.31 594.25

957.46465.24713.59

523.78 1168.61842.56 1046.51223.23823.48 1057.53350.23252.19 1169.71524.44 933.46

958.57819.29314.32 695.39458.63 843.59 1141.69 1170.61

dd IT MSMS on this scan (scan 3588)m/z 653

nanoLCNewObjective 75 um PicoFrit columnFlow rate: 200 nl / minFrom 98 % A (water, 0.1 % FA) to 60% B (Acetonitrile, 0.1 % FA) in 20 min

CoverageCytochrome C 67%Myoglobin 71%BSA 45%

38

RT: 19.01 - 42.26

20 22 24 26 28 30 32 34 36 38 40 42Time (min)

10

15

20

25

30

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40

45

50

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60

65

70

75

80

85

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Rel

ativ

e A

bund

ance

478.

25

637.

31

643.

13

791.

42

471.

24

863.

41

740.

40

784.

37

735.

85

582.

3258

4.81

746.

38

494.

15

879.

42

533.

60

607.

26

547.

32

480.

61

620.

36

710.

84

634.

39

861.

14

477.

37

749.

41

653.

3656

4.36

536.

16

536.

1653

6.16

536.

16

536.

16

536.

16

536.

16

485.

01

536.

16

536.

16

496.

52

536.

16

536.

16

536.

16

536.

16

536.

16

536.

16

536.

16

536.

16

NL: 4.29E6Base Peak m/z= 470.00-2000.00 F: FTMS + p NSI Full ms [ 300.00-2000.00] MS BSA_CC_MYO_3fmol_each_total_01

Protein digest mix: 1 fmol each on column

Peptide m/z 653 (2+) at RT: 24.93 min

Base Peak Chromatogram

39

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

BSA_CC_MYO_3fmol_each_total_01 #3588 RT: 24.90 AV: 1 NL: 7.14E3T: ITMS + c NSI d Full ms2 [email protected] [ 165.00-1320.00]

200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z

0

5

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25

30

35

40

45

50

55

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Rel

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1055.55

251.21

956.52 1056.61

644.61

712.49841.44332.31 594.25

957.46465.24713.59

523.78 1168.61842.56 1046.51223.23823.48 1057.53350.23252.19 1169.71524.44 933.46

958.57819.29314.32 695.39458.63 843.59 1141.69 1170.61

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Assigned Fragment Ions by SEQUEST

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Dynamic Range

..detecting minor components in complex mixtures

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Angiotensin 10 pmol/ul + Glu-fibrinogen 10 fmol/ulConcentration Difference 1000x

Angio10pmol_Glufib10fmol_Res30000 #6 RT: 0.09 AV: 1

NL: 1.18E8

T: FTMS + p ESI Full ms [ 215.00-2000.00]

400 600 1200 1400 1600 1800 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bun

danc

e

428.2281

641.8381

513.2818633.3358

1282.6699269.1610

385.7010

1305.6428

800 1000

652.8230770.3946 915.6690 1014.5159 1221.9934 1552.9739 1711.2153 1804.3352

785?

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

danc

e

785.5992 785.8419

786.3431

786.6021

786.8450787.6064

787.3463

NL: 9.35E4

784.5 785.0 785.5 786.0 786.5 787.0 787.5 788.0m/z

Measured 785.8419Calculated 785.8421m = -0.2 ppm

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#199-199 RT:5.30-5.30 NL: 6.64E3

200 300 400 500 600 700 800 900 1000 1100 1200m/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

627.3

bb 8+1

887.3bb 5

+1

515.2

480.2558

684.3457

813.3882

333.1879

942.4313

yy12+2

692.8

246.1558y2

y4

y3

y5

y6

y7

MS/MS of Glu-Fibrinogen @10 fmol/ul

Measured 246.1558Calculated 246.1561m = -1.2 ppm

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1

10

100

1000

10000

100000

100 1000 10000 100000 1000000 10000000

Target value, ions

S/B

m/z 1522

m/z 524

m/z 195

Dynamic Range in a Single Spectrum (0.75 sec Acquisition)

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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

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About the Authors

Dr. Michaela ScigelovaLC/MS application expertat Thermo Electron in UK

Dr. Alexander MakarovThe inventor of orbitrap mass analyserResearch Manager at Thermo Electron in Bremen

Orbitrap Mass Analyser - Overview and Applications in Proteomics

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