fourier transform mass spectrometry ftms the international proteomics tutorial program hupoeupa...

Fourier Transform Mass Spectrometry FTMS

The International Proteomics Tutorial ProgramHUPOEuPA

Michaela Scigelova, Martin Hornshaw,Anastassios Giannakopulos, Alexander Makarov

(Thermo Fisher Scientific)

2

Overview

Key performance characteristics of FTMS• Mass accuracy• Resolution

Fourier Transform in mass spectrometry – FTMS Fourier Transform ion cyclotron resonance – FTICR Discussion FTICR

• Factors impacting on the resolution/accuracy of the measurement• Fragmentation techniques

Orbitrap analyzer Discussion and comparisons – FTICR and Orbitrap analyzer

3

Key Performance Characteristics of FT Mass Spectrometers

Mass Accuracy Mass Resolution

• measures of the ability to distinguish two peaks of slightly different mass-to-charge ratios Δm, in a mass spectrum

• Defined as a full width of a peak at half of the maximum peak height (FWHM).

The peaks of the measured compounds must be sufficiently well resolved in order to be able to determine their mass accurately

4

MASS ACCURACY

Accurate mass measurement can be used to determine the elemental composition of an analyte*

This acts as a powerful ‘filter’ enabling• Confirmation of target compound identification• Elimination of false positive identifications• Identification of unknowns

5

Example: Accurate Mass As a Powerful Filter

Mass measured

Tolerance [Da]

Suggestions Calc Mass

32.0 +/- 0.2 O2

CH3OH

N2H4

S

31.989832.026132.037431.9721

32.02 +/- 0.02 CH3OH

N2H4

32.026132.0374

32.0257 +/- 0.002 CH3OH 32.0261

C = 12.0000H = 1.0078N = 14.0031

O = 15.9949S = 31.9721

Accurate mass makes life easier…

6

Quercetin fragmentation spectrum interpretation

Example: Structural elucidation aided by accurate mass measurement of fragments in MS/MS (or MSn) spectra

Software used for spectrum annotation: Mass FrontierTM from HighChem

7

Example: Peptide Identification – Effect of Mass Accuracy

Peptides of human database:

Courtesy of David Fenyo, Rockefeller University

8

RESOLUTION

High resolution ensures that ions of only one exact mass contribute to a particular peak.

Implications for:• Accurate mass measurement of compounds in mixtures• Hence providing a certain confidence interval for elemental

composition/identification of measured compounds• Reliable and accurate quantitation

9

Example: Effect of mass resolution on the confidence of mass accuracy determination

Resolution used translates to a ‘confidence interval’ (tolerance) for accurate mass measurement

Knowing such a tolerance (+/- mmu) is important when used for generating elemental composition suggestions

Example of Pirimicarb m/z 239

Resolution Mass tolerance (mmu)

Number of elemental composition suggestions*

15,000 +/- 9 14

80,000 +/- 1.7 1

*Assuming CHNO elements

10

Example: Effect of mass resolution on accurate mass measurement

Accurate mass determination of Pirimicarb enabled when sufficiently high mass resolving power separates its peak from that of a co-eluting interference of nearly same mass.

Courtesy of Markus Kellmann, Thermo Fisher Scientific

11

Example: Effect of mass resolution on compound detection

The presence of an interfering compound causes a mass deviation for the compound of interest (Pirimicarb; mass deviation 6.5 ppm)

Performing a screening experiment and setting the mass tolerance to +/-5 ppm, Pirimicarb would escape detection altogether resulting in a false negative result


12

Example: a background component (‘matrix’) can not be separated from the analyte at resolution 15,000 and contributes to peak area determination (black trace).

Quantitation of the analyte is not impaired at resolution 80,000 (orange trace).

R = 80,000

Example: Effect of mass resolution on compound quantitation


13

Fourier Transform in Mass Spectrometry

FTMS

14

Fourier Transform

In FTMS masses are represented by frequencies

Frequencies can be measured very accurately FTMS offers high resolution/accuracy

The signal complexity in FTMS can be considerable as illustrated by this example:

15

Fourier Transform

FT decomposes a function into a spectrum of its ‘frequency components’

#4487 IT: 19.154 ST: 1.66 uS: 1 NL: 6.06E6F: FTMS + p ESI Full m s [ 120.00-2000.00]

195.086 195.088 195.090 195.092 195.094 195.096

m /z

0

5

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Relat

ive In

tensit

y

195.0889R=201753

195.0908R=242757

195.0866R=301524

195.0862R=201486

195.0923R=224052

195.0850R=236095

195.0928R=310056

195.0949R=306152

195.0959R=197373

195.0975R=250022

Time domain Frequency domain / mass

16

Fourier Transform

17

FT: Effect of a phase shift of the time domain signal on the spectrum

FT decomposes the frequency spectrum into a complex (in the mathematical sense, i.e., containing a real and an imaginary part) spectrum.

At zero phase shift the spectrum is the absorption mode in real part (see case (a) on the figure) – the only instance of practical utility for us

At all other instances (non-zero phase shift) the data can not be used for deriving a spectrum

Ref.: James Keeler, "Understanding NMR Spectroscopy", 2nd Edition, Wiley 2009.

18

FT: ‘Magnitude’ spectrum

This operation eliminates the phase dependency at non-zero phase shift Drawback: the resolution of ‘magnitude’ spectrum is about 2x lower

19

Apodization

Figure courtesy of Robert Malek, Thermo Fisher Scientific

20

Fourier TransformIon Cyclotron Resonance

FTICR

21

FTICR - Theory

A charged particle in a magnetic fieldwith velocity vector at 90 deg to the magnetic field experiences a force normal to the plane defined by the velocity and the magnetic field.

When the vector of velocity is at any other angle then the component at 90 deg to the magnetic field (radial) will play a role in trapping the particle, while the component parallel to the field (axial) will offer no confinement, thus resulting to a helical path.

22

FTICR – Ion Trapping

Along the magnetic field lines (axial direction) ions are trapped by an applied electric potential, while on the plane perpendicular to the magnetic field lines (radial direction) ions are trapped by the magnetic field.

23

FTICR – Motion of Trapped Ions

m

qBc

B

v

qv x B

B

ωz

ω+

ω-

ωC : “unperturbed by trapping fields” angular cyclotron frequency

ω+ : “reduced” cyclotron frequencyω- : “magnetron” frequencyωz : trapping oscillatory frequencyq = z (i.e., charge)

ωc

m

qBvc 2

1

in radians/sec

in Hz

24

FTICR - Theory

2a

2

m

qVtrapz

222

22

zcc

222

22

zcc

B

ωz

ω+

ω-

The stronger the trapping potential, the greater the deviation from the unperturbed ICR frequency

a : characteristic of the trap geometrya: is the trapping electrode (end-cap) separation

23

a

210891369.13

m

qVtrapz

a: in m, m: in uz: in multiple charge elements

in SI units

a for cell geometries:

cube = 2.77373cylinder= 2.8404open = 3.8679

25

FTICR - Excitation

time domain

frequency domain

frequency sweep (chirp)

Stored Waveform Inverse Fourier Transform(SWIFT)

Stored Waveform Inverse Fourier Transform(SWIFT), excitation and ejection of part of the spectrum

“FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETRY: A PRIMER”A.G. Marshall, C.L. Hendrickson, and G.S. Jackson, Mass Spectrometry Reviews, 1998, 17, 1–35

tw

tw

tw

26

526.260 526.265 526.270 526.275m/z

0

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100

0

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40

60

80

100

Rel

ativ

e A

bund

ance

526.2606R=906700

526.2715R=1021000

526.2692R=989500526.2654

R=883500

526.2608

526.2717

526.2692

526.2654

File: 526_1200k_3us#41 scan, 3 uscans SIM at 5e5

MRFA +H +H 2 O: C 23 H 38 N 7 O 5 S 1p (gss, s /p:40) Chrg 1R: 1e +06 Res .Pwr . @FWHM

C23H38N7O534S

C2213CH38N6

15NO5S

C2113C2H38N7O5S

C23H38N7O418OS

Experiment

Isotope Simulation

526.260 526.265 526.270 526.275m/z

0

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100

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40

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Rel

ativ

e A

bund

ance

526.2606R=906700

526.2715R=1021000

526.2692R=989500526.2654

R=883500

526.2608

526.2717

526.2692

526.2654





C23H38N7O534S

C2213CH38N6

15NO5S

C2113C2H38N7O5S

C23H38N7O418OS

Experiment

Isotope Simulation

FTICR Spectrum

Example: peptide MRFA, cluster of isotopes around m/z 526. Bottom pane: isotope simulation at a resolving power of 1,000,000.

1e

Mass of an electron

0.0005 Da

27

FTICR - Non-Ideal Conditions

FTICR requires that ions are trapped within a finite volume by the electrodes. But these electrodes produce DC and RF electric fields in the trap. This has undesirable consequences:

1. The relationship between ICR orbital frequency and m/z becomes non-linear making calibration difficult

2. ICR signal strength no longer varies linearly with rf excitation magnitude and duration

3. Coulomb forces between ions broaden (i.e., resolution suffers) and shift (i.e., mass accuracy suffers) the mass spectral peaks

4. The spatially non-uniform excitation field may eject ions axially before they can be detected (loss of signal which means shorter detection times which means lower resolution)

28

FTICR DiscussionFactors impacting on the resolution/accuracy of the

measurement

29

FTICR – Factors impacting on the resolution/accuracy of the measurement

Magnetic field strength Acquisition duration (transient) Strength and accuracy of the DC electric field used to confine the

ions axially Strength and accuracy of the RF electric field used to excite the

ions in a coherent ion cyclotron motion Homogeneity of the magnetic field Ion-ion coulomb interactions

30

FTICR – Example: Effect of Magnetic Field Strength on Resolution

Please remember that the apodized resolution will be about half the resolution displayed in this figure. It is not easy to quote directly the apodized resolution because each manufacturer will use different apodization algorithms with a different effect on the resulting resolution Note: logarithmic scale both on mass and resolution; resolution defined as FWHM

31

FTICR – Benefits of High Field Magnets

• Higher mass resolving power ( m/Δm resolving power will increase linearly with increasing magnetic field)

• Higher mass accuracy as a consequence of increased resolving power

• Data acquisition speed (time needed to acquire a time domain signal of a given mass resolving power varies as 1/B)

• Higher maximum ion kinetic energy (useful for CID, as an example at 3T an ion of 1000 Da and argon collision gas has centre of mass kinetic energy (CMCE) of 1.67eV where at 9.4T has 16.4 eV)

• Upper mass limit increases quadraticaly with magnetic field (B)

• Ion trapping duration (The length of time required for the ion magnetron radius to expand to the radius of the trap increases quadraticaly with B)

• Number of trapped ions (increases quadraticaly with B)

• Quadrupolar axialisation efficiency (the rate of conversion of magnetron to cyclotron motion increases linearly with B)

• Peak coalescence (varies as 1/B2)

32

FTICR – Benefits of High Field Magnets

14.5 T FTICR at the National High Magnetic Field Laboratory,Florida State University, USA

33

FTICR – Example: Effect of Acquisition Duration on Resolution

Note: logarithmic scale both on mass and resolution; resolution defined as FWHM

34

FTICR – Example: Increasing performance by better controlling the excitation electric field

standard open ICR cell excitation

improved excitation (Finnigan LTQ FT)

In a cell with central excitation electrodes only, all isopotential lines meet at the gap between excitation and trapping electrode. Therefore, ions are heavily exposed to axial components of the excitation field.

In an improved version the axial components inside the trapping region are reduced by applying the excitation waveforms also to the outer electrodes, positioned adjacent to the trapping rings, and by using a grid with the excitation field applied inside the ICR cell.

35

FTICR – Example: Effect of electric field homogeneity on mass measurement accuracy

2/

BA

zm

External calibration mass accuracy is limited mainly by the variation of ion numbers in the cell

Mass assignment error given by:

wherein DB is the error of the electric field-dependent calibration parameter B

The grid cell (see previous and next slides) reduces DB by a factor of 4 Same effect on Dm could be achieved by increasing the frequency by a

factor of 2, i.e. by exchanging the 7 T magnet with a 14 T one. This is exemplified on the example below: measured mass deviations for

a population of 1e6 ions (+/- 100 ions) at m/z 1000

Improved cell7 T : 0.26 ppm

Normal cell 15 T : 0.39 ppm 7 T : 1.77 ppm

36

Grid Cell Used in LTQ FT Instrument

37

FTICR – Example: Effect of the electric field homogeneity on mass measurement accuracy

A homogenous electric field with reduced axial components of the excitation field allows use of higher excitation amplitude

This results in a significantly higher ion signal Figures show the mass deviation at m/z 524 (peptide MRFA) measured

for two different excitation amplitudes

target 1e6, excitation amplitude 0.50

-8.0

-4.0

0.0

4.0

8.0

0 20 40 60 80 100

scan number

dev

iati

on

(p

pm

)

target 1e6, excitation amplitude 0.25

-8.0

-4.0

0.0

4.0

8.0

0 20 40 60 80 100

scan number

dev

iati

on

(p

pm

)

38

FTICR – Detection, phase correction

Fourier deconvolution-based phase correction consists of a complex division of the time domain ICR signal by the spectrum of the time domain excitation waveform to yield a phased broadband response.

The critical requirement for implementing this process is that the detection event must incorporate the excitation interval, and the excitation and detection spectra must be temporarily synchronised. In practice, this simultaneous excitation and detection is very difficult due to detector saturation.

mixedmode

mixedmode

Re

Im

response FFT excitation FFT response FFTphased

“Broadband Phase Correction of FT-ICR Mass Spectra via Simultaneous Excitation and Detection” Steven C. Beu, Greg T. Blakney, John P. Quinn, Christopher L. Hendrickson, and Alan G. Marshall, Anal. Chem. 2004, 76, pp 5756-5761.

39

FTICR DiscussionFragmentation techniques

40

FTICR Fragmentation Techniques

FTICR has been used with a wide variety of fragmentation techniques

• CID• IRMPD• ECD

ECD method has some remarkable advantages:• Fragmentation not directed by peptide bond protonation• It ‘preserves’ post-translational modifications

Wide choice of applicable fragmentation techniques plus the high resolution/mass accuracy of the detected fragments make FTICR very powerful for analysis of large peptides/proteins

R.A. Zubarev, D.M. Horn, E.K. Fridriksson, N.L. Kelleher, N.A. Kruger, M.A. Lewis, B.K. Carpenter, and F.W. McLafferty, “Characterization of Multiply Charged Protein Cations”, Anal. Chem. 2000, 72, 563-573

41

FTICR – CID and ECD fragmentation spectra

A single scan ECD MS/MS spectrum of the doubly charged precursor of substance P at m/z 674.37. The spectrum exhibits intense ECD fragment ion peaks. Bearing in mind that the cyclic structure of proline does not allow formation of c- and z-type fragments, all possible N-Ca bonds are cleaved, allowing even de novo sequencing of peptides with unknown amino acid sequences.

The CID MS/MS spectrum of substance P. The doubly charged peptide precursor ions were subjected to CID in the linear ion trap and the fragment ions were transferred into the ICR cell and detected. The spectrum looks somewhat more complex compared to the ECD spectrum. The fragment ion peaks of this spectrum are sufficient to identify substance P in a database search, but de novo sequencing would be a challenge.

42

Peptide PKKKKYAKEAWPGKKPTPSLLI Phosphorylation on serine S(19) Diagnostic c/z fragments highlighted in the spectrum

FTICR – ECD fragmentation spectra of phosphopeptides

BD_2b_ecd_3_pS #34-103 RT: 0.61-6.22 AV: 70 NL: 1.05E6T: FTMS + p NSI Full ms2 [email protected] [email protected] [ [email protected] [email protected] ]

200 400 600 800 1000 1200 1400 1600m/z

0

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60

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70

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100

Re

lative

Ab

und

an

ce

648.12504

864.16706

499.37194

559.85648 765.45219 989.66334243.18168 371.27679 1296.251341174.66047

900.48418

1471.78633324.06304

848.83281

495.33516 1601.83381

666.40792

P K K K K Y A K E A W P G K K P T P S L L I

c2 c3 c4 c5 c6 c7 c9c8 c10 c12 c13 c14 c16 c19c20c21

z8z9z10z12z13z14z15z16z17z18z19z20z21[M+4H]4+

[M+4H]3+•

z3

c3c2

c4 c5

c8

c9

z13

c142+

c132+

c162+ c19

2+

c202+

c212+z9

z10z10

z14

Courtesy of Etienne Waelkens, University of Leuven, Belgium, and Martin Zeller, Thermo Fisher Scientific

43

Peptide PKKKKYAKEAWPGKKPTPSLLI Phosphorylation on theronine T(17) Diagnostic c/z fragments highlighted in the spectrum


BD_2b_ecd_3_pS #34-103 RT: 0.61-6.22 AV: 70 NL: 1.05E6T: FTMS + p NSI Full ms2 [email protected] [email protected] [ [email protected] [email protected] ]

200 400 600 800 1000 1200 1400 1600m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

und

ance

648.12504

864.16706

499.37194

559.85648 765.45219 989.66334243.18168 371.27679 1296.251341174.66047

900.48418

1471.78633324.06304

848.83281

495.33516 1601.83381

666.40792


c2 c3 c4 c5 c6 c7 c9c8 c10 c12 c13 c14 c16 c19

z8z9z10z12z13z14z15z16z17z18z19z20z21[M+4H]4+

[M+4H]3+•

z3

c2 c3

c4

c5

c8

c162+

c192+

z8

z9

z10

z12z13


44

Peptide PKKKKYAKEAWPGKKPTPSLLI Phosphorylation on tyrosine Y(6) Diagnostic c/z fragments highlighted in the spectrum


BD_2a_ecd_2_pY #1-77 RT: 0.01-4.16 AV: 77 NL: 1.41E6T: FTMS + p NSI Full ms2 [email protected] [email protected] [ [email protected] [email protected] ]

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z

0

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30

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Re

lative

Ab

un

da

nce

648.13

648.38

648.63

864.17

864.84

750.08 869.98826.14499.37

649.13

243.18 1198.67371.28 627.47 696.41 1069.63

788.11

1296.25870.48324.06 941.53500.38432.08


c2 c3 c4 c5 c6 c7 c9c8 c10 c12 c13 c14 c16 c18c19c20c21

z6z8z9z10z12z13z14z15z16z17z18z19z20z21

c5

[M+4H]4+

[M+4H]3+•

c6z16

2+

z172+

c2 c3

c4

c7c8

c9c10


45

FTICR – Intact protein measurement

1071.0 1071.5 1072.0 1072.5m/z

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

Inte

nsity

1071.45190R=545300

z=81071.57703R=537804

z=81071.20178R=528104

z=8

1071.70227R=495804

z=81071.07678R=553704

z=8 1071.82764R=467004

z=8

1072.07813R=480004

z=81070.95154R=600804

z=8 1072.32959R=351604

z=8

1072.58118R=700304

z=8

Corresponds to resolution 1,000,000 at m/z 400

Spectra courtesy of E. Damoc, Thermo Fisher Scientific

Analysis of intact proteins benefits from ultra-high resolution Ubiquitin (MW 8560), a detail of charge state 8+

46

Ubi_12_ECD #1-71 RT: 0.01-18.48 AV: 71 NL: 1.81E5T: FTMS + p NSI Full ms2 [email protected] [email protected] [email protected] [ 195.00-1900.00]

200 400 600 800 1000 1200 1400 1600 1800m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

714.7266

1136.6500

390.2168961.0637277.1327

852.9706

640.3762636.3536

1347.2296537.2853 978.5806

1264.7448

1579.9248

1693.79291478.8775

x10 x10

Spectra courtesy of E. Damoc, Thermo Fisher Scientific

ECD fragmentation spectrum of Ubiquitin (12+)

FTICR – Fragmentation of Intact Protein

47Courtesy of M. Zeller, Thermo Fisher Scientific

Sequence coverage within ECD fragmentation spectrum of Ubiquitin (12+) 71 out of 72) possible bonds were cleaved obtaining 147 fragment ions

FTICR – Fragmentation of Intact Protein

48

Orbitrap FTMS Analyzer

49

Orbitrap Analyzer – Electrostatic Field

Copyright: Thermo Fisher Scientific

50

Orbitrap Analyzer – Trapping Ions

Orbital trapsKingdon (1923)

The Orbitrap analyzer is an ion trap 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 …

51

Ion Injection and Formation of Ion Rings

(r,φ) (r,z)

A short ion packet of a particular m/z enters the field Increasing the voltage on the central electrode squeezes ions to a curved

trajectory around the central electrode Voltage stabilizes and ion trajectories are also stabilized Angular spreading forms a ROTATING RING High charge capacity can be achieved due to the shielding effect of the central

electrode (e.g., can not see the ions on the other side of the electrode)

52

Orbitrap Analyzer – Detection

zm

k

/

Electrostatic Axially Harmonic Orbital Trapping: A High-Performance Technique of Mass Analysis, Alexander Makarov, Anal. Chem. 2000, 72, 1156-1162

Image current detected on outer electrodes Frequency dependence on ions’ m/z Frequencies pertaining to ion populations of a particular m/z

obtained using Fourier Transform

53

Intact Protein Analysis – Depth of Information

Myoglobin infusionOrbitrap detectionRP 100,000

54

enolase_5ms_resolved_1300_avg #1 RT: 90.84 AV: 1 NL: 6.10E3T: FTMS + p ESI Full ms [700.00-1200.00]

1061.0 1061.5 1062.0 1062.5 1063.0m/z

0

5

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15

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70

75

80

85

90

95

100R

ela

tive

Ab

un

da

nce

1061.67R=61504

1061.65R=64804

1061.72R=61404

1061.60R=69104 1061.76

R=59304

1061.78R=47204

1061.83R=513041061.58

R=45204

1061.54R=67604

Intact Protein Enolase ~46 kDa

DeconvolvedMonoisotopic Mass

1.0 ppm

enolase_5ms_resolved_1300_avg #1 RT: 90.84 AV: 1 NL: 6.10E3T: FTMS + p ESI Full ms [700.00-1200.00]

950 1000 1050 1100 1150 1200m/z

0

5

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Rela

tive

Abund

ance

1061.67R=615041038.12

R=53604994.01

R=66800 1086.34R=604041015.58

R=51700 1112.25R=54900 1139.31

R=617041167.74R=64904

973.30R=55800

1197.73R=54804

953.44R=57600

934.41R=50000

LTQ Orbitrap XLRP 100,000 at 400 m/z

55

enolase_5ms_MSMS_2300avg #1 RT: 75.78 AV: 1 NL: 3.41E3T: FTMS + p ESI Full ms2 [email protected] [275.00-2000.00]

1151 1152 1153 1154 1155 1156 1157 1158m/z

0

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Rela

tive A

bundance

1153.84R=79000

z=21

1153.79R=81304

z=211153.88R=79504

z=21

1153.69R=75204

z=21 1154.03R=78104

z=21

1153.65R=80604

z=21

1154.08R=84604

z=211153.60R=75104

z=21 1157.22R=79200

z=211154.17R=76904

z=21

1152.98R=79904

z=21

1157.08R=77704

z=21

1157.36R=83104

z=211152.41R=85204

z=21

1156.31R=80704

z=21

1155.36R=78604

z=211157.51R=73604

z=21

1151.34R=78504

z=? 1158.25R=62404

z=21

Fragmentation of intact protein - Enolase

enolase_5ms_MSMS_2300avg #1 RT: 75.78 AV: 1 NL: 5.48E3T: FTMS + p ESI Full ms2 [email protected] [275.00-2000.00]

400 600 800 1000 1200 1400 1600m/z

0

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Re

lative

Abu

nd

an

ce

1038.10R=75504

z=?

1044.10R=86500

z=15

1153.84R=79000

z=21686.33

R=106501z=2

1211.43R=80200

z=20

1275.19R=78300

z=19921.38R=91600

z=17

670.35R=107801

z=21345.97R=75100

z=18870.25R=93000

z=18

788.91R=99104

z=?

1425.09R=73300

z=17

994.04R=72800

z=1

472.26R=125001

z=1

577.27R=116501

z=2

1514.10R=71400

z=16390.15R=133901

z=11614.97R=68900

z=15

y221

0.63 ppm (mono) y222

0.95 ppm (mono)

24 kDa fragments

LTQ Orbitrap XLRP 100,000 at 400 m/z

56

High Masses: IgG (~147,000 Da) analyzed by LC/MS with the Orbitrap detection

P.Bondarenko et al., Mass measurement and top-down HPLC/MS anakysis of intact monoclonal antibodieson a hybrid linear quadrupole ion trap-orbitrap mass spectrometer JASMS 2009, 20, 1415-1424.

57

Orbitrap analyzer – Fragmentation techniques

As implemented within a hybrid linear ion trap–Orbitrap instrument, the Orbitrap device is used solely as a mass analyzer

Fragmentation of peptides is carried out in an ion trap or a multipole, i.e., outside the Orbitrap analyzer

CID used for a vast majority of experiments aiming at peptide identification/quantitation

ETD (similar to ECD on FTICR) applied to PTM and large peptide/protein analysis*

CID and/or ETD can be engaged based on the analyzed peptide characteristics. Decisions are taken automatically by the instrument on-the-fly**

*McAlister, G.C., Phanstiel, D., Good, D.M., Berggren, W.T. and Coon, J.J. Implementation of electron-transfer dissociation on a hybrid linear ion trap/orbitrap mass spectrometer. Anal. Chem. 79, 3525–3534 (2007).

Danielle L. Swaney, Graeme C. McAlister and Joshua J. Coon.Decision tree–driven tandem mass spectrometry for shotgun proteomics. Nature Methods 5, 959-964 (2008).

58

c142+ / c

142+

z213+ / z

213+

z61+

Orbitrap ETD fragmentation: Top-down Analysis of Proteins

http://upload.wikimedia.org/wikipedia/en/b/b6/PBB_Protein_DEFA5_image.jpg

alpha_Defensin-5_ETD_4+_245-4000 #1 RT: 243.51 AV: 1 NL: 4.39E5T: FTMS + p ESI sa Full ms2 [email protected] [245.00-4000.00]

500 1000 1500 2000 2500 3000 3500m/z

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

Re

lativ

e A

bu

nd

an

ce 1435.68079z=2

713.28473z=1

1893.86353z=2

2384.15430z=1

633.29504z=1 1997.96533

z=1 2871.36401z=1 3396.53052

z=1

942.41772z=1

*

Example:• α-Defensin 5 with

intact disulfide links • ETD on 4+ precursor

ions with Orbitrap detection

793 794 795 796 797

59

Orbitrap Analyzer – Detection, Phase Correction

All ions are ejected at moment t=0 from the C-trap along lines converging on the Orbitrap entrance.

Ions enter Orbitrap analyzer as a short packet at the maximum Z

The moment of entry is

Injection at the maximum Z automatically initiates axial oscillations detected as image current at frequency

CE

OE-1

OE-2

C-trap

Lenses

Deflector

L eff

Z

zmeV

Lzmt eff

inj

/2

/

zm

ekzm

//

phase correction is much simpler in Orbitrap analyzer since there is no excitation step and the t=0 is the ejection from the c-trap

60Figures courtesy of E. Damoc, Thermo Fisher Scientific

Phase correction OFF

Phase correction ON

Orbitrap Analyzer – Implementing Phase Correction

Resolving power improvement: 1.6-1.7

61

DiscussionFTICR and Orbitrap Analyzers

62

Use of FTICR as part of a hybrid instrument

63

Use of Orbitrap analyzer as part of a hybrid instrument

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 Orbitrap analyzer5. …. 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 halves of the Orbitrap outer electrode, which can be detected using a differential amplifier

Ions of only one mass generate a sine wave signal

64

Use of FTICR or Orbitrap analyzer as part of a hybrid instrument

Parallel acquisition delivers accurate mass on the precursor ion together with ion trap MS/MS spectra of selected precursor ions

1 High resolution full scan detected in the FTMS

3 Unit resolution MS/MS scans detected in the LTQ ion trap

(up to 10 MS/MS spectra detected in the LTQ Velos ion trap)

Full Scan MS

MS/MSIon 1

MS/MSIon 2

MS/MSIon 3

65

Up to 10 MS/MS spectra

Use of FTICR or Orbitrap analyzer as part of a hybrid instrument

66

Combination of various fragmentation and detection modesExample: phosphopeptide analysis

MS/MS

MS3

MS/MS

MS3

Full Scan MS

High resolution full scan detected in the FTICR / Orbitrap

High resolution MS/MS scans detected in the FTICR / Orbitrap

Unit resolution MS3 detected in the ion trap

These two scans provide information on possible neutral loss of phosphate group from the precursor

Provides information about the phosphate location within the peptide

Use of FTICR or Orbitrap Analyzer as part of a hybrid instrument

67

Resolution vs Mass Dependence Comparison of FTICR and Orbitrap Analyzer

Note: logarithmic scale both on mass and resolution; resolution defined as FWHM

68

Relative Instrument Sizes

FTICR Cell

Superconducting Magnet

Benchtop Orbitrap MS

69

Relevant topics on videos

Mass spectrometry basics• http://www.youtube.com/watch?v=rBymrFzcaPM&NR=1&feature=fvwp• http://www.youtube.com/watch?v=J-wao0O0_qM

FTICR• http://www.youtube.com/watch?v=7EHngA4S3Ws&feature=related

Fourier Transform• http://www.youtube.com/watch?v=gZNm7L96pfY&feature=related

Recommended reading:

August issue of JASMS 2009 dedicated to Orbitrap analyzer and its applications

http://www.youtube.com/watch?v=rBymrFzcaPM&NR=1&feature=fvwp

http://www.youtube.com/watch?v=J-wao0O0_qM

http://www.youtube.com/watch?v=7EHngA4S3Ws&feature=related

http://www.youtube.com/watch?v=gZNm7L96pfY&feature=related

fourier transform mass spectrometry ftms the international proteomics tutorial program hupoeupa...

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