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MALDI-TOF mass spectrometry: Theory and principles
Peter Roepstorff
Protein Research GroupDepartment of
Biochemistry and Molecular BiologyUniversity of Southern Denmark
roe@bmb.sdu.dkwww.protein.sdu.dk
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
Matrix-assisted Laser Desorption/Ionization (MALDI)Time-Of-Flight (TOF)mass spectrometer
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 =
Linear time-of-flight mass spectrometer.
mass: > >
20 kVd
mv2
2 Ek= q U = ze U =
dv
t =
d2
2Ue t2 = m/z
Linear time-of-flight mass spectrometer.
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
Delayed ion extraction (DE)
U (kV) : 20
020
m/z : = ;V0 ( ) > V0 ( )
t tif
Tif = time of ion formation
Delayed ion extraction (DE)
U (kV) : 20
01819.7
19.2U = 0.5 kV
t = td
Td = delayed extraction
Delayed ion extraction (DE)
U (kV) : 20
018
V ( ) > V ( )
+ 0.5 kV
Delayed ion extraction (DE)
U (kV) : 20
018
V ( ) = V ( )
+ 0.2 kV
Delayed ion extraction (DE)
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
Matrix-assisted Laser Desorption/Ionization (MALDI)Time-Of-Flight (TOF)mass spectrometer
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?