MALDI-TOF(/TOF)

MALDI-TOF/TOF
MALDI-TOF/TOF
Mass
massSpectrometry
spectrometry
An Introduction
An Introduction
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF(/TOF) Mass Spectrometry
ion source
2
Time-of-Flight (TOF)
mass analyzer
Introduction MALDI-TOF/TOF; version12/04/2012
detector
MALDI
MALDI
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI stands for ...
Matrix Assisted Laser
Desorption / Ionization
invented by
Tanaka (Nobel prize awarded 2002),
Hillenkamp and Karas
in the mid ´80s
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Introduction MALDI-TOF/TOF; version12/04/2012
How does MALDI work?
Required items:
- a sample to be analyzed
- a matrix substance
- a laser
MALDI preparation / MS analysis:
1.)
2.)
3.)
4.)
Mix sample and matrix solutions in suitable ratio.
Put a sub µl aliquot of this mixture on the target plate.
Let the mixture co-crystallize.
Insert the target plate in the MALDI mass spec
(running under high vacuum).
5.) Shoot with the laser on the MALDI preparation
to generate ions (singly charged predominantly).
6.) Collect, analyze and detect the resulting ions.
& De-protonizing
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Introduction MALDI-TOF/TOF; version12/04/2012
What role does the MALDI matrix play?
The matrix transfers the energy needed for ionization
from the laser light to the sample molecules.
Positive ionization mode:
Sample embedded in
light-absorbing matrix
Sample molecule
Excitation of matrix
molecules by laser light
Laser
Matrix molecule
M
6
Laser energy
Matrix
Desorption/protonation
of sample molecules
MH+
[M+H]+
Introduction MALDI-TOF/TOF; version12/04/2012
Formation of alternative adducts depends on the presence
of respective cations (either being ubiquitary present or
actively added – depending on type of sample):
[M+Na]+; [M+K]+; [M+Cu]+; [M+Li]+; [M+Ag]+
What role does the MALDI matrix play?
The matrix transfers the energy needed for ionization
from the laser light to the sample molecules.
Negative ionization mode:
Sample embedded in
light-absorbing matrix
Sample molecule
Excitation of matrix
molecules by laser light
Laser
Matrix molecule
M
7
Laser energy
Matrix
Desorption/deprotonation
of sample molecules
M-H-
[M-H]-
Introduction MALDI-TOF/TOF; version12/04/2012
Commonly used MALDI matrix substances:
Peptides:
4-Hydroxy-α-cyanocinnamic acid (HCCA)
Proteins:
2,5-Dihydroxyacetophenone (DHAP)
Sinapinic acid (SA)
2,5-Dihydroxybenzoic acid (DHB)
Glycans:
2,5-Dihydroxybenzoic acid (DHB)
Nucleic acids:
3-Hydroxypicolinic acid (HPA)
2,4,6-Trihydroxyacetophenone (THAP)
Why different matrices for different types of sample?
It´s all about
- the amount of energy needed to ionize a particular sample compound
(individual matrices show specific „energy threshold“)
- the stability of a particular sample compound
(too „hot“ matrix may lead to non-desired fragmentation of sample compounds)
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Introduction MALDI-TOF/TOF; version12/04/2012
How does MALDI work?
Fundamental papers on the principle of MALDI:
M. Karas, D. Bachmann, F. Hillenkamp
Analytical Chemistry, 57, 2935-2939 (1985)
K. Tanaka, H. Waiki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida
Rapid Communications in Mass Spectrometry, 2, 151-153 (1988)
R. C. Beavis, B. Chait, K.G. Standing
Rapid Communications in Mass Spectrometry, 3, 233-237 (1989)
M. Karas, M. Glückmann, J. Schäfer
Journal of Mass Spectrometry, 35, 1-12, (2000)
R. Zenobi and R. Knochenmuss
Mass Spectrometry Reviews, 17, 337-366 (1998)
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF
(Time of Flight)
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF
Basic principle of MALDI-Time-of-Flight based mass analysis:
High vacuum 10-7mbar
High vacuum 10-7mbar
Intensity
m/z
+
++
+
+
+
+
+
Acceleration
MALDI
Ion Source
Field free TOF analyzer region
(drift tube)
Time Of Flight (depending on m/z)
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Introduction MALDI-TOF/TOF; version12/04/2012
Detector
+
+
+
+
+
+
+
+
MALDI-TOF
Epot = zeU
2
1/2mv
Ekin =
2
zeU = 1/2mv
This defines the potential energy at which
all ions start from the MALDI target.
This equation defines the kinetic
energy of ions after acceleration into
the flight tube.
In the process of ion acceleration,
energy is preserved but turns from
potential into kinetic energy.
v = 2zeU/m
Transforming this equation shows
the dependency of velocity of moving
ions on their m/z value.
t = L m/2zeU
With the linear velocity v being defined
as Ldrift tube/tflight, the dependency of the ions´
flight time tflight from m/z becomes
obvious.
To put the basic MALDI-TOF separation principle into simple words:
The larger its m/z, the slower an ion will fly, the longer the measured flight time will be.
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Linear mode
Intensity
m/z
+
++
+
+
+
+
+
Acceleration
IS1
MALDI
0kV
Ion Source
Field free TOF analyzer region
(drift tube)
Time Of Flight (depending on m/z)
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Introduction MALDI-TOF/TOF; version12/04/2012
Linear detector
+
+
+
+
+
+
+
+
MALDI-TOF: Linear mode
Observation:
Ions of identical mass arrive at the detector at slightly different time points,
causing peak broadening (i.e. limiting the resolution)
+++
++
++
+
Uaccel
0kV
MALDI
Ion Source
14
Δt
1040
1045
1050
m/z
Field free TOF analyzer region
(drift tube)
Introduction MALDI-TOF/TOF; version12/04/2012
++
Linear detector
Peak broadening effect,
limiting the resolution
in resulting mass spectrum.
MALDI-TOF: Linear mode
Limited in resolution due to spatial spread and energy spread
Spatial spread:
Initial energy (=speed) spread:
- initial movement of ions towards different
directions
- heterogeneous secondary reactions (ion-ion; ionneutral)
- ions are desorbed from different zcoordinates due to heterogeneity in size of
matrix crystals
Int.
For all
m/z!
500
1000
Reference:
W. Ens, Y. Mao, F. Mayer, K.G. Standing,
Rapid Communications In Mass Spectrometry, 5, 117-123 (1991)
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Introduction MALDI-TOF/TOF; version12/04/2012
v[m/s]
MALDI-TOF: Resolution
Example spectrum containing 3 singly charged [M+H]+ signals at m/z
922Da, 923Da and 924Da:
100%
922.009
FWHM=0.073
50%
923.013
924.016
921.0
922.0
Resolution
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Introduction MALDI-TOF/TOF; version12/04/2012
923.0
924.0
= m/Δm
= 922.009/0.073
= 12630
m/z
MALDI-TOF
How to improve resolution???
 Optimized matrix preparation homogeneity
(minimizing spatial spread)
 Pulsed ion extraction for efficient ion focusing
(minimizing initial energy spread)
 Further ion focusing by means of a reflector TOF
setup (minimizing remaining energy spread)
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF
How to improve resolution???
 Optimized matrix preparation homogeneity
Spatial
spread



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Introduction MALDI-TOF/TOF; version12/04/2012
Resolution
MALDI-TOF:
How to improve resolution???
 Pulsed ion extraction for efficient ion focusing in the
MALDI ion source
Ion source
+
Epot
+
+
+
IS1
Field-free drift region
IS2
Detector
Pulsed Ion Extraction
(PIE)
t>tdelay
+
+
t<tdelay
1040
1045
1050
m/z
x[cm]
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Introduction MALDI-TOF/TOF; version12/04/2012
1041
1046
1051
m/z
MALDI-TOF:
How to improve resolution???
 Pulsed ion extraction for efficient ion focusing in the
MALDI ion source
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF
How to improve resolution???
 Further ion focusing by means of a reflector TOF setup
Ion source
+
+
+
+
IS1
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Field-free drift region
IS2
+
Reflector
detector
Introduction MALDI-TOF/TOF; version12/04/2012
+
+
+
+
+
Reflector
(ion mirror)
Linear
detector
MALDI-TOF
How to improve resolution???
IS1
1040
1045
1050
m/z
+
+
Reflectron
detector
IS2
1041
+
+
1046
Introduction MALDI-TOF/TOF; version12/04/2012
+
Linear
detector
+
Reflectron
(ion mirror)
1051
m/z
PIE compensates
- initial energy spread
- initial spatial spread
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MALDI-TOF,
reflector + PIE
Field-free
drift region
MALDI-TOF,
linear + PIE
+
+
Ion source
MALDI-TOF,
linear
M1
+
+
1046
1050
m/z
Reflector compensates
- remaining energy spread
MALDI-TOF
Linear vs. reflector mode
FAQ:
If MALDI-TOF performed in reflector mode gives so much better
resolution - why then use linear mode at all???
Answer:
Linear mode is used whenever analytes are not stable enough to
survive the energetic stress inside the reflector (ions are decelerated/
re-accelerated in the reflector by a high kV electric field within
nanoseconds!).
This effects especially larger molecules, e.g. intact proteins. They may
undergo serious fragmentation, which results in either badly resolved
spectra (peak fronting due to non-resolved fragments) and/or drastic
loss in sensitivity (low mass fragments will miss the reflector
detector).
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF
Linear vs. reflector mode: Cytochrome C (MWavg=12360Da)
12361.0
1250
12355.9
1000
12368.1
1000
750
12366.0
12356.9
1500
12364.0
2000
12358.0
1500
Reflector mode: High resolution
R=30,000
Intens. [a.u.]
12360.9
Intens. [a.u.]
Linear mode: Low resolution
R=1,500
500
500
250
0
12340
12350
12360
12370
12380
m/z
Spectrum shows one broad peak
representing the envelope of the
non-resolved isotope peaks.
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Introduction MALDI-TOF/TOF; version12/04/2012
12340
12350
12360
12370
12380
m/z
Spectrum shows all the isotope
peaks well separated from each
other.
MALDI-TOF
Where do these isotope peaks originate from?
Most elements are found in nature in form of different so called isotopes. Isotopes
of an element have nuclei with the same number of protons (the same atomic
number) but different numbers of neutrons.
However, both, protons and neutrons contribute to the weight of an atom, which
explains the difference in weight of isotopes.
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF
Where do these isotope peaks originate from?
Isotope
1-H
2-H (Deuterium)
12-C
13-C
14-N
15-N
16-O
18-O
19-F
23-Na
31-P
32-S
34-S
35-Cl
37-Cl
39-K
79-Br
81-Br
Mass
1.007825
2.014000
12.00000
13.00336
14.00307
15.00011
15.99491
17.99916
18.99840
22.98977
30.97376
31.97207
33.96787
34.96885
36.96590
38.96371
78.91834
80.91629
[%] Abundance
99.985
0.015
98.90
1.10
99.63
0.37
99.76
0.20
100
100
100
95.03
4.22
76.77
31.98
93.26
50.69
49.31
Elements that are found in nature in form of only one single isotope, are
called monoisotopic elements.
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF
Where do these isotope peaks originate from?
For a given molecule, the individual isotopes of all the elements contained in it finally yield a
characteristic intensity distribution of isotopic masses. This is shown below by means of the
isotopically resolved mass spectra of 3 compounds being different in size:
Element composition: C112H164N29O34S2
Monoisotopic mass [M+H]+: 2524.1510
Average mass [M+H]+: 2525.8196
2525.2
2526.2
5405.6
2524.2
5409.6
1000.5
Element composition: C253H363N55O75S
Monoisotopic mass [M+H]+: 5404.6075
Average mass [M+H]+: 5407.9984
5406.6
5407.6
5408.6
Element composition: C41H69N13O14S
Monoisotopic mass [M+H]+: 1000.4880
Average mass [M+H]+: 1001.1409
5410.6
5411.6
5412.6
1001.5
1002.5
2528.2
2529.2
1003.5
998
1000
1002
5404.6
2527.2
1004
1006 m/z
2522
2524
2526
2528
2530
2532
m/z
5400
5404
5408
5412
5416
m/z
The monoisotopic mass is the sum of the masses of all the atoms present in a molecule
using the mass of the most abundant isotope for each element.
The average mass of a molecule is the sum of elemental masses using the average weighted
over all stable isotopes of each element contained in the molecule.
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Calibration strategies
x104
Step 2) Internal re-calibration
(optionally)
Sample spectrum
870.530
Intens. [a.u.]
Step 1) External calibration
Step 1.2
3
x104
Sample spectrum
870.530
4
Intens. [a.u.]
5
2211.066
4
- apply calibration fit to
sample spectra obtained
from near neighbour spots
3
2
1
Step 1.1
*
3208.436
m/z
x10 4
Calibrant spectrum
1672.921
757.397
0
1500
2000
2500
3000
3500
m /z
- calibrants of known mass cover mass range of
interest
- m/z vs. flight time is fitted using a polynom of
varying order (depending on size of mass range
to be calibrated and number of available calibrant
signals, resp.)
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1000
1250
1500
1750
2000
2250
2500
2750
3000
3250
842.509 Da (trypsin artefact)
2211.104 Da (trypsin artefact)
3657.937
2932.578
2093.079
2
1000
750
* denotes compounds of known identity/mass
1758.941
4
0
m/z
1296.685
1046.542
6
2465.196
Intens. [a.u.]
*
3250
2363.150
3000
2097.090
2750
1959.958
2500
1797.888
2250
1565.869
2000
1517.871
1750
1645.805
1500
1291.708
1373.788
1250
1419.807
1000
944.562
750
2211.066
0
1181.557
3208.436
2363.150
2097.090
1959.958
1565.869
1797.888
1517.871
1419.807
1181.557
944.562
1
1645.805
1291.708
1373.788
5
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Introduction MALDI-TOF/TOF; version12/04/2012
Internal re-calibration allows for
- optimum mass accuracy due to compensation
of spot-to-spot heterogeneities that typically
cause mass errors after external calibration
MALDI-TOF: Calibration strategies
When to use which calibration polynom?
Compass for Flex series 1.1 and 1.2:
Min. number of calibrant peaks
Calibration polynom to be used
external calibration:
2
4
linear
quadratic
internal re-calibration:
(external pre-calibration: quadratic)
1
quadratic
Compass for Flex series 1.3 or higher:
Min. number of calibrant peaks
Calibration polynom to be used
external calibration:
3
4
6
linear
quadratic
cubic enhanced
internal re-calibration:
(external pre-calibration: quadratic)
1
4
6
linear correction
quadratic
cubic enhanced
internal re-calibration:
(external pre-calibration: cubic enhanced)
1
4
linear correction
cubic enhanced
Note: For optimum mass accuracy, calibrants in general have to cover the
entire mass range that is to be calibrated. Extrapolation of calibration functions
will always have a negative effect on the resulting mass accuracy.
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Typical applications
Gel-based proteomics:
Protein ID by peptide mass fingerprinting (PMF)
- reflectron MALDI-TOF
- mass range: 700 ... 4000Da
1) 2D gel separation of proteins
2) Comparative image analysis
3)
4)
5)
6)
Biological state A
Excision of regulated spots
Enzymatic digestion
MALDI-TOF-PMF
Protein ID by database search
(MASCOT, Phenyx etc.)
Database
1
2
4
x104
870.530
Intens. [a.u.]
3
5
4
Biological state B
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Introduction MALDI-TOF/TOF; version12/04/2012
2211.066
3208.436
2363.150
2097.090
1959.958
1565.869
1797.888
1645.805
1419.807
1517.871
1
1181.557
944.562
2
1291.708
1373.788
3
0
750
1000
1250
1500
1750
2000
2250
2500
2750
3000
3250
m/z
MALDI-TOF: Typical applications
Gel-based proteomics:
Protein ID by peptide mass fingerprinting (PMF)
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Typical applications
TLC-MALDI Coupling
•
Product:
•
TLC-MALDI adapter target
for autoflex/ultraflex
(# 255595)
•
dedicated wizard driven
software for Compass 1.2 or
better
•
ImagePrep (option) for
matrix application
TLC-MALDI is a BRUKER patent
•
Simple to use, even MALDI
novices can just do it!
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Typical applications
TLC-MALDI Coupling
TLC-MALDI setup dialog:
•Wizard driven software
interface for simple
analysis setup
•Multiple chromatographic
lanes
•Defined step raster along
with or orthogonal to the
chromatographic axis
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Typical applications
MALDI Tissue Imaging
80µm
Tubuli
160 µm
Scale bar: 1mm
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Introduction MALDI-TOF/TOF; version12/04/2012
240 µm
Rat Testis
MALDI-TOF: Typical applications
MALDI Tissue Imaging: MALDI Imaging and H&E Staining from
the Same Section
Rat Testis
Blood vessel
Tubuli
3457 Da
5455 Da
10261 Da
200 µm
6263 Da
2396 Da
Image Resolution: 20 µm due to smartbeam-II laser
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Typical applications
Profiling of microorganisms:
Workflow
Fast and easy operation from single colony to
result
MALDI Biotyper
software identifies
species
Smear colony
on MALDI target
Unknown
microorganism
microflex LT to
generate MALDI-TOF
mass spectrum:
The proteomics
fingerprint
For Research Use Only.
Not for Use in Any Diagnostic Procedures.
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Typical applications
Intens. [a.u.]
Profiling of microorganisms:
Aspergillus fumigatus
3000
2000
Intens. [a.u.]
1000
0
Bacillus subtilis
8000
6000
4000
2000
0
Candida albicans ATCC 10231
1.0
0.8
0.6
0.4
0.2
0.0
Escherichia coli DH5alpha
2500
2000
1500
1000
500
0
3000
4000
5000
6000
7000
8000
9000
10000
m/z
Fungi, yeast, gram+ and gram- bacteria
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Typical applications
2638.547
2550.494
2462.441
2374.390
2198.285
2110.233
2022.178
1934.126
2286.337
1758.022
1581.918
2000
1493.864
4000
1669.970
6000
1846.074
8000
1405.811
Intens. [a.u.]
Polymer characterization: Polyethylene glycol distribuion
0
1200
1400
1600
1800
Introduction MALDI-TOF/TOF; version12/04/2012
2000
2200
2400
2600
2800
m/z
MALDI-TOF: Typical applications
Polymer characterization: Polyethylene glycol distribuion
Data evaluation for Reflector Mode
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF: Typical applications
Polymer characterization: Separated PEO/PPO Copolymer
Fraction 10
Fraction 8
50
50
(8)
(10)
30
20
30
20
10
10
0
10
20
30
40
50
30
20
10
0
0
(12)
40
No of EO units
40
No of EO units
No EO units
40
Fraction 12
50
0
0
10
No of PO units
20
30
No of PO units
40
50
0
10
20
30
No of PO units
Weidner S.M., Falkenhagen J., Maltsev S., Sauerland V., Rinken M. Rapid Commun. Mass Spectrom.
2007; 21: 2750-2758
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Introduction MALDI-TOF/TOF; version12/04/2012
40
50
MALDI-TOF/TOF
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF:
Location of the various MS/MS Processes
Timed
Sample Target
Ion Selector
(TIS)
CID
LID
ISD
Gridless DE
MALDI Source
Collision
Cell
Ion Source
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Introduction MALDI-TOF/TOF; version12/04/2012
Source 2
(Lift)
MALDI-TOF/TOF:
Principal scheme:
Reflector
LIFT
Ion source 1
PCIS
Ion source 2
CID cell
Reflector
detector
Ion path in TOF1 region (linear TOF)
Ion path in TOF2 region (reflector TOF)
Ion source 1 = MALDI ion source
Ion source 2 = LIFT re-acceleration cell
PCIS = PreCursor Ion Selector
PLMS = Post LIFT Metastable Suppressor
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Introduction MALDI-TOF/TOF; version12/04/2012
PLMS
MALDI-TOF/TOF:
Analysis of a mixture containing 3 compounds (green,red, blue)
being different in mass:
TOF1
region
Source 1
CID
cell
PCIS
v1 < v2 < v3
Molecular ions are separated in TOF1 according to their mass.
Part of the ions undergo fragmentation in TOF1 region induced by:
- metastable laser induced decay (LID)
- collision induced decay (CID)
Most important:
Fragments and their precursor ions travel at the same velocity.
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF tandem MS:
LID vs. CID
LID: Laser-Induced Dissociation
Most straight forward way to peptide backbone fragmentation (b,y-type
ions).
Used for protein identification by means of peptide sequencing.
CID: Collision-Induced Dissociation (high energy)
Additional side chain cleavages. Higher relative intensity of internal
fragments. Overall shift of average fragment size towards lower mass.
Used as an option in special applications, e.g.:
- denovo sequencing (enhanced immonium ions)
- differentiation of isobaric aminoacids L of I by respective side chain
cleavages
- detailed glycan analysis (monomer linkage positions determined by products of
specific crossring cleavages)
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF tandem MS:
Analysis of a mixture containing 3 compounds (green,red, blue)
being different in mass:
Selecting the red precursor ion for fragment analysis by MS/MS:
Source 1
PCIS
Rejected
PCIS = PreCursor Ion Selector
(calibrated for time/mass correlation)
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF tandem MS:
PCIS = PreCursor Ion Selector
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF tandem MS:
Analysis of a mixture containing 3 compounds (green,red, blue)
being different in mass:
Red precursor ion selected for fragment analysis by MS/MS:
LIFT
Source 1
Ion Source 2
Ion gate
Rejected
Ion Source 2 (LIFT):
Re-acceleration of fragments and
remaining molecular ions.
Re-focusing (resolution!!!) of the ions.
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Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF tandem MS:
FAQ: Why re-acceleration of ions in Ion Source 2 (LIFT)?
Answer: To minimize the energy spread of the metastable fragments and, thus,
to capture on the detector the complete fragment ion spectrum at one
fixed reflector voltage.
+19 keV
19-27 keV
0 - 8 keV LIFT
Source 1
Source 2
A simple calculation example considering a precursor ion of [M+H]+ = 1000Da:
Initial acceleration in source 1
Precursor [M+H]+
Fragment f1 [M+H]+
Fragment f2 [M+H]+
=
=
=
=
8kV
1000Da
500Da
100Da
Ekin precursor
Ekin f1
Ekin f2
= 8keV (100%)
= 4keV (50%)
= 0.8kV (10%)
Reacceleration by 19kV narrows down this energy spread:
After reacceleration, even the smallest
fragment ions have more than 70% of
the energy of the precursor and, thus,
will be captured on the detector.
49
Introduction MALDI-TOF/TOF; version12/04/2012
Ekin precursor
Ekin f1
Ekin f2
= (8+19)keV = 27keV (100%)
= (4+19)keV = 23keV (85%)
= (0.8+19)kV = 19.8keV(73%)
MALDI-TOF/TOF tandem MS:
FAQ: Why do we need any re-acceleration of ions in Ion Source 2 (LIFT)?
Post Source Decay (PSD)
high reflector voltage
N2 laser
trigger
diode
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
++
+
+
++
+ +
+ +
+
+
+
+
+
+
Only parent and high m/z fragments
are focused onto detector
50
Introduction MALDI-TOF/TOF; version12/04/2012
neutral fragments pass
through reflector while
fragment ions of differing
energies penetrate to differing
depths of the reflectron
hit spacebar to reduce reflector voltage
MALDI-TOF/TOF tandem MS:
Post Source Decay (PSD)
medium reflector voltage
N2 laser
trigger
diode
+
+
+
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+ ++
+
+ ++
+
+
+
+
+
+
+
+
+
+
+
+
Only medium m/z fragments
are focused onto detector.
Lower m/z fragments are still
focused away from the detector
51
Introduction MALDI-TOF/TOF; version12/04/2012
neutral fragments and higher
m/z fragments pass through
reflector while lower m/z
fragment ions penetrate more
deeply into the reflectron
hit spacebar to reduce reflector voltage
MALDI-TOF/TOF tandem MS:
Post Source Decay (PSD)
low reflector voltage
N2 laser
trigger
diode
+
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
++ +
+
+
+
+
+
+
+
+
+
+
Lowest m/z fragments
are focused onto detector.
Higher m/z fragments all
pass through the reflector
52
Introduction MALDI-TOF/TOF; version12/04/2012
neutral fragments and higher
m/z fragments pass through
reflector while only the lowest
m/z fragment ions will be
reflected
hit spacebar to continue
MALDI-TOF/TOF tandem MS:
Reacceleration and focusing potentials applied in the
Ion Source 2 (LIFT):
Lift 1
19 kV
17 kV
Ground
53
Introduction MALDI-TOF/TOF; version12/04/2012
Lift 2 Ground
MALDI-TOF/TOF tandem MS:
Reacceleration and focusing potentials applied in the
Ion Source 2 (LIFT):
Lift 1
19 kV
17 kV
Ground
54
Introduction MALDI-TOF/TOF; version12/04/2012
Lift 2 Ground
MALDI-TOF/TOF tandem MS:
Reacceleration and focusing potentials applied in the
Ion Source 2 (LIFT):
Lift 1
19 kV
17 kV
Ground
55
Introduction MALDI-TOF/TOF; version12/04/2012
Lift 2 Ground
MALDI-TOF/TOF tandem MS:
Reacceleration and focusing potentials applied in the
Ion Source 2 (LIFT):
Lift 1
19 kV
17 kV
Ground
56
Introduction MALDI-TOF/TOF; version12/04/2012
Lift 2 Ground
MALDI-TOF/TOF tandem MS:
Reacceleration and focusing potentials applied in the
Ion Source 2 (LIFT):
Lift 1
19 kV
17 kV
Ground
57
Introduction MALDI-TOF/TOF; version12/04/2012
Lift 2 Ground
MALDI-TOF/TOF tandem MS:
58
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF tandem MS:
Analysis of a mixture containing 3 compounds (green,red, blue)
of different mass:
Source 1
PCIS
LIFT
Source 2
Rejected
PLMS:
Post LIFT Metastable Suppressor
deflects remaining non-fragmented
precursor ions to avoid undesired
metastable decay in TOF2 region,
which would yield broad fragment
peaks detected at wrong mass.
59
Reflector
detector
Introduction MALDI-TOF/TOF; version12/04/2012
PLMS
Reflector
MALDI-TOF/TOF tandem MS:
Analysis of a mixture containing 3 compounds (green,red, blue)
being different in mass:
Source 1
PCIS
LIFT
Source 2
Rejected
Reflector
detector
60
Introduction MALDI-TOF/TOF; version12/04/2012
Rejected
PLMS
Reflector
MALDI-TOF/TOF tandem MS:
[Abs . Int. *10^ 3]
2.4
PLMS OFF
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
740
760
780
800
820
840
860
880
900
920
940
m/z
[Abs . Int. *10^ 3]
20
y 7
18
b 6
16
PLMS ON
14
12
10
8
a 6
6
b-17 6
4
2
a-17 6
b 7
y 6
a 7
0
740
760
780
800
820
840
m/z
61
Introduction MALDI-TOF/TOF; version12/04/2012
860
880
900
920
940
MALDI-TOF/TOF tandem MS:
Example MS/MS spectrum obtained from a peptide:
y12
y11
y10
y8
y9
y7
y6
y5
y4
y3
y2
y1
Asp-Ala-Phe-Leu-Gly-Ser-Phe-Leu-Tyr-Glu-Tyr-Ser-Arg
H+
62
b1
b2
b3
b4
b5
b6
Introduction MALDI-TOF/TOF; version12/04/2012
b7
b8
b9
b10
b11
b12
H+
MALDI-TOF/TOF: Typical applications
Analysis of posttranslational modifications: Phosphorylation
-98Da
?
?
FQSEEQQQTEDELQDK
63
Introduction MALDI-TOF/TOF; version12/04/2012
Neutral loss of
H3PO4
MALDI-TOF/TOF: Typical applications
Analysis of posttranslational modifications: Phosphorylation

FQSEEQQQTEDELQDK
64
Introduction MALDI-TOF/TOF; version12/04/2012
-98Da
Neutral loss of
H3PO4
Modified threonin is obviously a wrong
guess, as all the aminoacid residues
following the threonin do not match the
detected fragment signal pattern!!!
MALDI-TOF/TOF: Typical applications
Analysis of posttranslational modifications: Phosphorylation
-98Da

?
?
FQSEEQQQTEDELQDK
65
Introduction MALDI-TOF/TOF; version12/04/2012
Neutral loss of
H3PO4
MALDI-TOF/TOF: Typical applications
#2
3079.242
2905.286
2570.243
2492.274
2649.235
2344.307
2113.902
2247.949
1927.827
1805.168
1639.950
1249.633
1138.503
803.902
1006.994
927.501
0.50
0.25
#4
1740.855
0.75
1439.821
#1
1.00
1399.701
1) Acquire PMF.
2) Run PMF database search.
3) Data dependent MS/MS acquisition
#3
1880.946
1.25
2045.061
1.50
1479.811
x105
1567.762
Intens. [a.u.]
Gel-based proteomics:
Protein ID based on PMF + datadependent MS/MS
0.00
750
1000
1250
1500
1750
2000
2250
2500
2750
3000
m/z
Successful
protein ID by PMF
Protein ID by PMF
failed
- acquire MS/MS of matching
peptides for confirmation of
protein ID
- acquire MS/MS of most
abundant peptides
- acquire MS/MS of non-matching
peptides (identify further proteins,
unexpected modifications etc.)
PFF database search
(combined MS/MS data)
66
#1
#2
#3
#4
DeNovo sequencing
with subsequent
MS BLAST homology search
(in case of non- or partially
sequenced taxa)
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF: Typical applications
Gel-based proteomics:
Protein ID based on PMF + datadependent MS/MS
Advantages of MALDI-TOF/TOF in gel-based
proteomics:
+ speed (PMF + PFF analysis typically < 2min
measuring time per gel spot)
+ amol sensitivity
+ automated, data dependent MS/MS workflow
+ improved significance of combined (PMF+PFF)
data sets compared to PMF only data
67
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF: Typical applications
1880.946
1.25
1439.821
1.00
3079.242
2570.243
2905.286
2492.274
2649.235
2247.949
2344.307
1927.827
2113.902
1639.950
1399.701
1805.168
803.902
927.501
0.50
1249.633
1740.855
0.75
0.25
2045.061
1479.811
1.50
1567.762
x105
1138.503
Optional pre-fractionation on protein level (gel; LC; IP etc.)
Enzymatic digestion
nanoLC of resulting peptide mixture
LC fraction collection on MALDI plate
off-line MALDI-TOF/TOF analysis
1006.994
1)
2)
3)
4)
5)
Intens. [a.u.]
LC-based proteomics: LC-MALDI analysis of complex samples
0.00
750
1000
1250
1500
1750
2000
2250
2500
2750
3000
m/z
68
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TOF/TOF: Typical applications
LC-based proteomics: LC-MALDI analysis of complex samples
+ reduced ion suppression due to separation upfront to the mass
spectrometer
+ no time constraints during data acquisition (no time pressure
caused by LC speed)
+ increased depth of analysis (more protein IDs from complex
mixtures; increased sequence coverage for individual
proteins)
+ data dependent, non-redundant MS/MS precursor selection (esp.
useful for non-isobaric SILE experiments)
+ LC separation is „frozen“ on the MALDI plate and, thus, allows
archiving of LC separated samples for later re-analysis
- a single LC-MS/MS experiment, when compared to online-ESI, is
rather time consuming
69
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TDS:
Top-down sequencing of intact proteins
by MALDI-TOF/TOF
70
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TDS:
Top-Down Sequencing of intact proteins
N T
E R M S E Q U E N C E C T
E R M
+ MALDI matrix (1,5-DAN, sDHB, SA)
3004.160
2874.129
2764.341
2807.335
2651.262
2589.216
2504.188
2376.140
2532.212
2134.974
2263.060
2291.041
500
2162.955
2034.870
1878.804
750
1933.827
1000
1804.798
1250
2433.158
2006.893
1500
1777.766
Intens. [a.u.]
In Source Decay (ISD)
250
0
1800
Bruker MALDI-TOF
71
Introduction MALDI-TOF/TOF; version12/04/2012
2000
2200
2400
2600
2800
3000
m/z
MALDI-TDS:
Top-down sequencing of intact proteins
Intact
protein
N T
E R M S E Q U E N C E C T
E R M
N
N T
72
N
N
N
N
N
T
T
T
T
T
E
E
E
E
E
MALDI-ISD
R
R M
R M S
R M S E
Introduction MALDI-TOF/TOF; version12/04/2012
..►
..
►
ISD
fragments
M
R M
E
T E
C T E
E C T E
C E C T E
R
R
R
R
R
M
M
M
M
M
MALDI-TDS:
Top-down sequencing of intact proteins
N T
E R M S E Q U E N C E C T
N
M
E
E
R
T
M
C
S E
E C
Q
N
U
E
E
U
N N-terminal fragments (c-type ions)
fragments (z- and/or y-type ions)
N C-terminal
T
M
R M
N
N
N
N
N
R
R
R
R
R
T
T
T
T
T
E
E
E
E
E
R
R M
R M S
R M S E
MALDI-ISD
..►
Introduction MALDI-TOF/TOF; version12/04/2012
..
►
73
T
R
E R M
E
T E
C T E
E C T E
C E C T E
M
M
M
M
M
N
Q
MALDI-TDS:
MALDI-ISD-TOF spectrum of Cytochrome C (horse)
Heme
Acetyl
49 N-term AA (N-terminus modified)
44 C-term AA
Spectrum
acquired
on Bruker Ultraflex III TOF/TOF
74
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TDS applications:
Monoclonal antibodies
IgG heavy chain: Aim of analysis: - N-terminal pyroGlu???
- C-terminal lysine deleted???
PyroGlu
K???
Matching N-terminal fragments confirm
N-terminal pyroGlu modification.
Matching C-terminal fragments confirm
absence of C-terminal K.
Spectrum
acquired
onMALDI-TOF/TOF;
Bruker Ultraflex
III TOF/TOF
75
Introduction
version12/04/2012
MALDI-TDS applications:
PEGylated therapeutic peptides
Aim of analysis: Determination of PEGylation site
Peptide sequence: Acetyl-WVTHR*LAGLLSR*SGGVVK*KNFVPTDVGPXAF
PEG polymer:
~500mer (approx. 22kDa)
Linear MALDI-TOF spectrum showing MW distribution of the PEGylated
peptide:
C. Yoo, D. Suckau, V. Sauerland, M. Ronk and M. Ma,, J. Am. Soc. Mass Spectrom., 20 (2), 2009, 326-333.
76
Introduction MALDI-TOF/TOF; version12/04/2012
MALDI-TDS applications:
PEGylated therapeutic peptides
Aim of analysis: Determination of PEGylation site
Peptide sequence: Acetyl-WVTHR*LAGLLSR*SGGVVK*KNFVPTDVGPXAF
PEG polymer:
~500mer (approx. 22kDa)
MALDI-ISD-TOF spectrum of the PEGylated peptide:
Ac-WVTHR*LAGLLSR*SG
GVVK*KNFVPTDVGPXAF
PEG
~500mer
Breakdown of N-terminal
fragment series clearly
identifies
PEGylation site
Yoo et al., 2008 JASMS
77
Introduction MALDI-TOF/TOF; version12/04/2012
Top-Down Protein Analysis
PMF „Bottom-Up“
ISD „TOP-Down“
ABCDEFGHIKLMNOPQ
Digest
FGHIK
OPQ
DE
LMN
ABC
Peptides can not be localized
unreliable PTM detection
78
Introduction MALDI-TOF/TOF; version12/04/2012
MS/MS(/MS)
ABCDEFGHIKLMNOPQ
Sequence and relationship to
intact protein mass, termini,
PTMs maintained
Advanced MALDI-TDS: T3 sequencing:
Further fragmentation (MS3) of ISD fragments by LID
ISD
Source 1
LID
LIFT
PCIS
Source 2
ISD fragments
undergo further
metastable decay
in TOF1 region
Rejected
79
Introduction MALDI-TOF/TOF; version12/04/2012
Reflector
detector
Rejected
PLMS
Reflector
MALDI-T3-TOF/TOF spectrum of ISD fragment c13
(m/z=1516.9Da) from Cytochrome C (horse)
Spectrum acquired on Bruker ultrafleXtreme
Abs. Int. * 1000
a
G
b
y
K
4.5
E
K
146.1239
y1
G
K
K
K
I
F
628.2450
b6
946.5142
y8
72.0549
a1
443.1678
b4
3.0
314.1500
b3
2.5
2.0
761.4441
y6
633.3813
y5
286.1074
a3
571.2834
b5
1.5
G
Q
1116.5572
a 10
4.0
3.5
V
K
K
997.4887
b9
884.3361
b8
1243.7509
b 11
1144.6240
b 10
1074.6167
y9
756.3251
b7
1371.8814
b 12
1343.7556
a 12
543.2209
a5
1.0
V
0.5
0.0
200
400
600
800
1000
1200
1400
m/z
T3 sequencing provides access to the very terminal aminoacid sequence, which is
commonly not accessible by ISD (due to occurence of matrix clusters and other
components occupying the low mass region in MS mode).
80
Introduction MALDI-TOF/TOF; version12/04/2012
www.bruker.com
81
Introduction MALDI-TOF/TOF; version12/04/2012

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