MALDI-TOF mass spectrometry: Theory and principles

MALDI-TOF mass spectrometry: Theory and principles

Peter Roepstorff

Protein Research GroupDepartment of

Biochemistry and Molecular BiologyUniversity of Southern Denmark

[email protected]

Rio, Ferbruary 6, 2006

Matrix-assisted Laser Desorption/Ionization (MALDI)Time-Of-Flight (TOF)mass spectrometer

ion source(MALDI)

mass analyser(TOF)

detector

t (m/z)

Computer

MALDI”solid solution” deposit of analyte

doped matrix crystals

matrix molecules

analyte molecules

•Strong absorption at the laser wavelength

•Homogeneous solid-state mixing with the analyte

•Ability to undergo photochemical reaction leading to proton transfer to or from analyte

Important properties of the MALDI matrix

1. Dilute and isolate analyte molecules.

-Prevents their interaction.

2. Absorption of energy from the laser.

-Minimizes sample damage -Efficient energy transfer to the analyte

The matrix serves two major functions

-+

MASS ANALYSER

MATRIX IONSAND NEUTRALS

MATRIXMOLECULES

ANALYTE IONS

ANALYTEMOLECULES

+

-LASER

Explosive phase transition.Gas jet of matrix and analyte molecules


ion source(MALDI)

mass analyser(TOF)

detector

t (m/z)

Computer

Linear time-of-flight mass spectrometer.

detectorflight tube(10-8 – 10-9 torr)ion source

t (m/z)mass: > >

20 kV

Ion source Continuous extraction

• All ions obtain the same kinetic energy• Their final velocity is proportional to (m/q)

20 kV 0 kV

++

t0: Ek=0 mv2

2ta: Ek= q U =


mass: > >

20 kVd

mv2

2 Ek= q U = ze U =

dv

t =

d2

2Ue t2 = m/z


detectorflight tube

ion source

same mass, but higherinitial kinetic energy than

t (m/z)

-poor resolution !-Poor mass accuracy 0.5-1 Da

20 kV

m

m

resolution= mm

h

½ h

m=FWHM

Resolution is limited by:

• The initial time distribution.– The ion production time.

• The initial spatial distribution.– Size of the volume where the

ions are formed.

• The initial energy distribution.– Variation of the initial kinetic

energy.

Delayed extraction

Reflectron

Mass resolution is affected by factors creating a distribution in flight times among ions with the same m/z ratio.

Delayed ion extraction (DE)

U (kV) : 20 0

TOF-MS

20

laser pulse

t = t0


U (kV) : 20

020

m/z : = ;V0 ( ) > V0 ( )

t tif

Tif = time of ion formation


U (kV) : 20

01819.7

19.2U = 0.5 kV

t = td

Td = delayed extraction


U (kV) : 20

018

V ( ) > V ( )

+ 0.5 kV


U (kV) : 20

018

V ( ) = V ( )

+ 0.2 kV


U (kV) : 20

018

V ( ) < V ( )

Time focus plane(ideal for ion detection)

Delayed vs. continuous extraction.

Additional benefits of DE

• Lower level of cheminal noise arising from fragmentation in the ion source.

• Reduction of matrix background.

• Significant reduction of the dependence of ion flight times on laser intensity.

Resolution is limited by:

• The initial time distribution.– The ion production time.

• The initial spatial distribution.– Size of the volume where the

ions are formed.

• The initial energy distribution.– Variation of the initial kinetic

energy.

Delayed extraction

Reflectron

Mass resolution is affected by factors creating a distribution in flight times among ions with the same m/z ratio.

Reflector time-of-flight mass spectrometry

Ion source reflector

flight tube

detector

same m/z, but higherinitial kinetic energy than

20 kV 23 kV

t (m/z)

Good resolution !Good mass accuracy 0.1-0.3 Da

The reflectron

• Consists of a series of grids and ring electrodes creating a retarding field that acts as an ion mirror.

• Corrects the energy dispersion of the ions leaving the source with the same m/z ratio.

• Increases the mass resolution at the expense of sensitivity and introduces a mass range limitation.

DE-MALDI-rTOF-MS

Uvar ,t1

Uacc

t (m/z)

High resolution!

UReftime focus plane(virtual source)

Mass accuracy ± 10-50 ppm

Sensitivity (low femto-mole)

Improvement of resolution.MS of (Glu)-fibrinopeptide B

1560 1566 1572 1578 1584 1590

Mass (m/z)

1616.7

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 Linear Spec #1 MC=>SM3=>MC=>MC[BP = 1570.7, 1617]

Linear mode

+ Delayed extraction

÷ Reflector

Resolution = 3,500

1560 1566 1572 1578 1584 1590

Mass (m/z)

2191.4

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

4700 Linear Spec #1 MC=>SM3=>MC=>MC[BP = 1571.8, 2191]

Continuous extraction

÷ Delayed extraction

÷ Reflector

Resolution = 500

1560 1566 1572 1578 1584 1590

Mass (m/z)

2642.4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

4700 Reflector Spec #1 MC=>MC[BP = 1570.7, 2642]

Resolution = 10,000

Reflector mode

÷ Delayed extraction

+ Reflector

Reflector mode

+ Delayed extraction

+ Reflector1560 1566 1572 1578 1584 1590

Mass (m/z)

1751.0

0

10

20

30

40

50

60

70

80

90

100

% I

nten

sity

4700 Reflector Spec #1 MC=>MC=>MC[BP = 1570.7, 1751]m/z = 1570.68

Resolution = 20,000


ion source(MALDI)

mass analyser(TOF)

detector

t (m/z)

Computer

Ion detection.

Secondary Electron Multipliers (SEM)Micro-channel plates (MCP)

Ions Electrons Amplification Oscilloscope

Computer

Dual microchannel plate detector

Array of leadglass tubes

oscilloscope

0kV -1.6 -0.9-0.8 0

e-

-0.1

e-

Advantages of MALDI TOF MS

•Theoretical unlimited mass range

•TOF MS is fast and sensitive

•Compatible with pulsed ion sources

•The high resolution of modern instruments results in a mass accuracy of 10-100 ppm

•Applicable to a broad range of biopolymers and complex mixture.

•Structural information can be obtained by MS/MS analysis

MS/MS with TOF instruments.

• MALDI TOF/TOF • MALDI Q/TOF

Mass analyser 1

Mass analyser 2

CID DetectorSource

Precursor selection

- TOF

- Quadrupole

Fragment determination

-TOF

MALDI Q-TOF from Micromass.

4700 TOF/TOF from Applied Biosystems

V2V1

Laser

Source 1Source 2

Reflector Detector Reflector

CID Cell

Sample Plate

TOF 1 TOF 2

MS/MS Voltages

V 1V 2

6.7 kV

8 kV

16.1 kV

10.4 kV

6 kV

7 kV

14 kV

Vo

ltag

e

Reg ion

Timed ion selector operation

TIS

to collision cell

from ion source

m1m2m3

m1

m3

m2

m1

m2

+

-

Switch down time calculatedby low mass gate geometry

Switch up time calculated byhigh mass gate geometry

TTL Pulse5 V0 V

0 V

-950 V

+950 V

TIS

Single Gate

TTL Pulse5 V0 V

0 V

-950 V

+950 V

TIS

Single Gate

m1

m3

m2

TOF 1

m1m2m3 m1m2m3 m2m1 m2

m3

0

0

m1m2m3 m1m2m3m1

m2

m3

m1m2m3

m1

m3

m2 m2 m2

Deceleration

D. Suckau et al, Anal Bioanal Chem. 2003 Aug;376(7):952-65. Epub 2003 Jun 27.

TIS R=1000 is achievable at the laser desorption threshold.

In practice R= 200-400 because the laser is operated at elevated intensity to induce LID

Precursor selection.

1163.0 1171.2 1179.4 1187.6 1195.8 1204.0

Mass (m/z)

1.5E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

4700 Reflector Spec #1 MC[BP = 1156.6, 76544]11

78

.58

58

11

82

.64

36

12

00

.57

19

MS

1165.0 1170.6 1176.2 1181.8 1187.4 1193.0

Mass (m/z)

1.3E+4

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y

4700 MS/MS Precursor 1178.59 Spec #1 MC=>BC=>SM5[BP = 1022.4, 21983]

1179.0

479

1183.0

741

MS/MS

1176.0 1178.6 1181.2 1183.8 1186.4 1189.0

Mass (m/z)

2660.7

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y

4700 MS/MS Precursor 1182.66 Spec #1 MC=>BC=>SM5[BP = 1184.1, 2661]

1183.1

360

1179.0

516

MS/MS

4 Da Resolution at TIS focus plane

High-Resolution Data Control of laser intensity

Control of collision energy (1KeV –3KeV)

Control of collision gas pressure

Four different collision gases

Two independent mechanisms are involved in the dissociation of the precursors

-Metastable decay (PSD)

-Collision-induced dissociation (CID)

Controlled Ion Fragmentationin the 4700

69.0 543.4 1017.8 1492.2 1966.6 2441.0

Mass (m/z)

4.2E+4

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y

<<2313 msms CID_air>> 4700 MS/MS Precursor 2313.26 Spec #1[BP = 1452.7, 41831]1452.6946

263.1505

129.1077

1254.6135

358.171772.087686.1018 1580.7642620.2817242.1882 399.1692

136.0854 641.2974392.1894 1693.8330958.4935 1285.5808 1925.8577614.3875284.1345102.0669 2185.02491355.6604867.4082 1193.5931425.2157 1570.6647 1816.62601032.5645602.2458 2013.7970

69.0 543.4 1017.8 1492.2 1966.6 2441.0

Mass (m/z)

4.5E+4

0

10

20

30

40

50

60

70

80

90

100

% Int

ensit

y4700 MS/MS Precursor 2313.26 Spec #1 MC[BP = 1452.7, 45239]

1452.6924

1254.6080

1580.7705

1693.8365620.2562 1028.532778.3201 1285.5742 1925.8468856.5000263.1375 2167.0061491.2277 1065.4902 1306.6072 1570.6768634.2697 867.3965 1816.659372.0821 1997.7178199.1069 342.1868

Tryp lactogl. 2313 MSMS

PSD

CID (air)

Sensitivity of the 4700

702.0 1194.8 1687.6 2180.4 2673.2 3166.0

Mass (m/z)

862.6

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1=>NF0.7=>MC[BP = 731.2, 863]

731.

2305

741.

2336

796.

2419

754.

2336

840.

2522

1627

.862

5

1245

.583

3

1581

.900

1

1449

.840

9

1713

.988

3

1537

.882

7

1934

.120

8

1802

.062

1

1669

.959

4

2047

.083

9

2313

.259

3

1846

.050

018

90.0

583

2630

.178

2

2952

.550

5

2722

.194

3

3113

.247

6

71 312 553 794 1035 1276

Mass (m/z)

482.1

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity4700 MS/MS Precursor 1245.58 Spec #1=>SM5=>MC[BP = 1234.1, 482]

1234.1117

154.1026

73.1265 197.1366

294.101386.0922 199.1283 857.3294460.2661 704.3442 945.3309589.3196147.4250 227.1264 328.1614431.1519

Lactogl. 1245 MSMS (low amol): 30.000 spectra.

Lactogl. MS (low amol)

Resolution in MS - mode

Resolution in MS/MS mode

Bruker UltraFlex TOF-TOF

MS/MS Voltages

Timed ion selector operation

TIS

from ion source

+

-

Switch down time calculatedby low mass gate geometry

Switch up time calculated byhigh mass gate geometry

TOF CID

0

0

LIFT

TOF 2

Few ns

0.0

0.5

1.0

1.5

2.0

2.5

4x10

Inte

ns. [a

.u.]

250 500 750 1000 1250 1500 1750 2000 2250

m/z

y8

y7y3

y2

y5

y4

y6

y10

y12 y13

MH+

(MH+) - 98

b3

b4

b8

b9

b10b12 b13

y8*

y9

y10*

y11

y11*

b5

b6b7

b15

b16

b17

(MH+) - 80

b14b11

[H]

G S H Q I S L D N P D pY Q Q D F F P K

-80y2y3y4y5y6y7y8y10y11y12y13

b3 b4 b8 b9 b10 b11 b12 b13b5 b6 b7 b14 b15 b16 b17

y9

MS/MS of the synthetic phosphopeptide

1363.945

1398.000

1407.926

1379.978

0.0

0.5

1.0

1.5

2.0

4x10

Inte

ns. [a

.u.]

1360 1370 1380 1390 1400 1410 1420

m/z

V2V1CID CellTIS

MS/MS VoltagesB

ruke

rA

BI

1245.59

TrypLac 1 pmol, MS

TrypLac 1 pmol, 1245.5 MSMS

MS and MS/MS of tryptic lactogl.

Beta-Lactoglobulin, score 95

TPEVDDEALEK, m/z 1245.5

Result of the 1245.58 MS/MS spectrum

Advantages of MALDI MS/MS analysis?

Fast and simple sample preparation.

Only a single sample preparation is needed for PMF and subsequent MS/MS analysis.

It is possible to reanalyze interesting samples.

Sensitive

Relative tolerant towards contaminants as salt

Offline LC MALDI experiments.

Advantages of LC-MS/MS on a MALDI platform

• LC decoupled from MS– Mass analysis can be faster or slower than separation – Wider selection of flowrates, solvents and modifiers

• MS decoupled from MS/MS– Can look at the entire LC run before having to decide

which precursors to select for MS/MS– Enables selection of precursors at the optimal elution

time

• LC trace is ”frozen” on the MALDI plate– Alows further analysis asking new questions– Alows for result-dependent analysis using RDA

software– More confident IDs and better sequence coverage

Thank you

To Nicolai Bache and Sabrina Laugesen

for preparing slides

Questions?

Documents

MALDI-TOF mass spectrometry: Theory and principles