Introduction to MALDI-TOF MS

Sandler Mass Spectrometry Users Group

University of California San Francisco
May 20, 2003
Features of MALDI-TOF MS
Soft ionization - analyze intact biomolecules and synthetic polymers
Broad mass range - analyze a wide variety of biomolecules
Simple mixtures are okay
Relatively tolerant of buffers and salts
Fast data acquisition
Easy to use and maintain, no water or gas hook ups required
High sensitivity, superior mass resolution and accuracy

MALDI: Matrix Assisted Laser Desorption Ionization
hn
Laser
1. Sample (A) is mixed with
excess matrix (M) and dried
on a MALDI plate.
2. Laser flash ionizes matrix
molecules.
3. Sample molecules are ionized
by proton transfer from matrix:
MH
+
+ A M + AH
+
.

AH
+

+20 kV
Variable Ground
Grid Grid
Sample plate
Time-of-flight mass analyzer
+
+
+
+
Source
Drift region (flight tube)
d
e
t
e
c
t
o
r

V
Ions are formed in pulses.
Small ions reach the detector before large ones.
Measures the time for ions to reach the detector.
Calibration of the mass scale
The mass-to-charge ratio of an ion is proportional to the square
of its drift time.
t = Drift time
L = Drift length
m = Mass
K = Kinetic energy of ion
z = Number of charges on ion
2
2
2
L
K t
z
m

Voyager-DE STR MALDI TOF

Camera
Laser
Sample
plate
Pumping
Pumping
Timed ion
selector Reflector
Linear
detector
Extraction
grids
Reflector
detector
Attenuator
Prism
Collision
cell
MALDI TOF Hardware
Laser, Attenuator and Prism
Nitrogen laser at 337 nm, 3 ns wide pulses, 20 Hz.
Laser attenuator varies the intensity of the laser hitting the sample.
Prism deflects the laser beam into the ion source.
Sample Plate and Sample Stage
An accelerating voltage is applied to the sample plate in the range
15-25 kV.
Variable Voltage Grid
A grid 1-2 mm above the sample plate with an additional voltage to
fine- tune ion acceleration
Ground Grid
Grounded surface defines end of acceleration region
Grounded Aperture
Entrance to flight tube
MALDI TOF Hardware
Vacuum System
High vacuum is required to avoid ion collisions
Flight tube
A field free region where ions drift at a velocity
inversely proportional to the square root of their
mass/charge.
Linear Detector
Measures the ion abundance in linear mode (no
reflector used) and sends a signal to the digitizer.
+
e
-
primary ion
e
-
e
-
e
-
L
D
- 1000V
- 100V
L >> D
Ions are detected with a microchannel plate
Microchannel Plate (MCP)
High current detector schematic
Microchannel plate
Fast scintillator
Condenser
Photomultiplier tube
signal
1kV
15kV
Used in linear mode to enhance signal from high mass molecules
MALDI TOF Hardware
Reflector
A single stage gridded ion mirror that subjects the ions to a uniform
repulsive electric field to reflect them. It is tilted by 1 in the DE-
STR to focus the ions on to the detector
Collision Cell
Gas cell for collision induced dissociation (CID) to enhance
fragmentation in PSD analysis
Reflector Detector
Measures ions reflected by the mirror. In the DE-STR this is a 6-10
mm pore size micro-channel plate.
Timed Ion Selector
A velocity selector that allows a single precursor ion of a selected
mass and their fragment ions to pass to the detector. A Bradbury-
Neilson gate is used.
Voyager-DE STR MALDI TOF
Camera
Laser
Sample
plate
Pumping
Pumping
Timed ion
selector Reflector
Linear
detector
Extraction
grids
Reflector
detector
Attenuator
Prism
Collision
cell
The problem: Peaks are inherently broad in MALDI-TOF
spectra (poor mass resolution).
+
+
+
Sample + matrix on target
Ions of same mass, different velocities
The cause: Ions of the same mass coming from the target
have different speeds. This is due to uneven energy
distribution when the ions are formed by the laser pulse.
Can we compensate for the initial
energy spread of ions of the same
mass to produce narrower peaks?
Delayed Extraction
Reflector TOF Mass Analyzer
Step 1: No applied electric field. Ions spread out.
+
+
+
Ions of same mass, different
velocities
Step 2: Field applied. Slow ions accelerated more than fast ones.
0 V.
0 V.
+
+
+
Step 3: Slow ions catch up with faster ones.
20 kV.
20 kV.
0 V.
0 V.
+
+
+
Delayed Extraction (DE) improves
performance
Detector
Ion Source
What is a reflector TOF analyzer?
Reflector (Ion Mirror)
The reflector or ion mirror compensates for the initial energy spread of
ions of the same mass coming from the ion source, and improves
resolution.
A single stage gridded ion mirror that subjects the ions to a uniform repulsive electric
field to reflect them.
0 V. +20 kV
A reflector focuses ions to give better mass
resolution
+
+
Reflector
Resolution & mass accuracy on mellitin
0
2000
4000
6000
8000
C
o
u
n
t
s

2840 2845 2850 2855
Mass (m/z)
Resolution = 14200
Resolution = 4500

Resolution = 18100
15 ppm error
24 ppm error
55 ppm error
1. PSD refers to a method of detecting and measuring
the masses of fragment ions that are formed from a
selected precursor ion.

2. Fragment ions are mainly formed by unimolecular
decomposition after the precursor ions are fully
accelerated (after they exit the sourcehence post-
source decay)

3. Fragment ions are separated and detected in the
reflector.
Fundamentals of Post Source Decay
(PSD)
Laser
Reflector
Source
Linear
detector
Reflector
detector
Decay can
occur at any
point along here
Decomposition occurs in the flight tube
No of
ions
Internal energy
Only a small fraction of the precursor ions have enough
energy to fragment during their lifetimes.
Internal energy of precursor ions
For peptides the efficiency of PSD fragmentation is amino acid composition and
sequence dependent.
There are two ways to increase the amount of
fragmentation: both act to increase the
precursor ions internal energy.

Use higher laser intensity

Use a collision cell
Increasing PSD Fragmentation
PSD fragment ion velocities are the
same as their precursors
+
+
All three of these species
travel at the same velocity in
the flight tube until they reach
the reflector.
Why? Velocity is determined by initial acceleration. Initial
energy = 20 keV. Bond energies = ~ 10 eV, so breaking a
bond has a very minor effect on velocities.
+
Timed Ion Selector (TIS)
The TIS is a Bradbury-Neilson gate, which is a type of
velocity selector. It allows only selected precursor ions and
their fragments to pass through to the reflector.
+
+
-
-
Gate closed:
alternating
potentials on
wires
Gate open:
wires at ground
potential
Ions
Timed Ion Selector operation
TIS off
TIS on
Gate open
Gate closed
Effect of the timed ion selector
The intact molecular ion has translational kinetic energy
equal to:
KE = 1/2 Mv
2
where:
KE = kinetic energy (= zeV)
M = mass
v = velocity

Before fragmentation
The translational kinetic energy of a fragment ion is
Post source fragmentation

M
m
KE KE
M m
where
KE
M
= precursor kinetic energy
KE
m
= fragment kinetic energy
M = precursor mass
m = fragment mass
Precusor and PSD fragment ions take
different paths in the normal reflector
Reflector
detector
Reflector
+20 kV
0 V.
Fragment ion
formed by PSD
Intact
precursor ion
+
+
How are PSD fragment ions that are
traveling at the same speed as the
precursor ion but contain reduced kinetic
energy made to arrive at the detector so
that they are focused?
By varying the steepness of the
voltage gradient in the reflector across
the fragment ion mass range.
Consider an ion (MH
+
) that can decompose into two
fragments, A and B.
Either of the following reactions can occur:
Assume MH
+
= 1,000 Da, AH
+
= 700 Da, and BH
+
= 300 Da
PSD mirror ratio setting
MH
+
AH
+
+ B
MH
+
A + BH
+

MH
+

BH
+

AH
+

MH+ ( 1,000) correctly focused
AH+ (700) Poorly focused
BH+ (300) Poorly focused
At mirror ratio = 1.00
MH
+

BH
+

At mirror ratio = 0.7
MH+ ( 1,000) not focused
AH+ (700) correctly focused
BH+ (300) Poorly focused
AH
+
BH
+

AH
+
& MH
+
MH+ ( 1,000) not focused
AH+ (700) not focused
BH+ (300) correctly focused
At mirror ratio = 0.3
MR=0.61
MR=0.71
MR=0.80
(MR= mirror ratio)
Resolution decreases as the fragment ions
penetrate less into the mirror
A PSD spectrum is taken in stitches
591 753 915 1077 1239 1401
Mass (m/z)
0
4.7E+4
0
10
20
30
40
50
60
70
80
90
100
%

I n
t
e
n
s
i t
y
Stitched PSD[BP = 354.1, 59562]
784.3
1181.7
756.3
619.3
1182.8
1296.7
767.4
650.3
646.3
739.3
742.2 1137.7
1164.8 881.5 1000.7
1046.6
1229.2
59.0 164.2 269.4 374.6 479.8 585.0
0
6.0E+4
0
10
20
30
40
50
60
70
80
90
100
%

I n
t
e
n
s
i t
y
Stitched PSD[BP = 354.1, 59562]
354.1
269.2
(-His)
(-Tyr) (-Pro)
255.1
382.2
513.2
112.1
(-Val)
364.1
506.3
416.1
115.1
235.1
534.0
156.2
138.1 326.1343.1
272.2
400.1 426.1 489.3
133.0 217.2
313.0 59.0
95.0 251.1 337.1 81.0 195.1 370.2
140.1
PSD Spectrum of Angiotensin I, MH
+
= 1296.7 Da
Composite of the focused mass regions from several spectra acquired with different
mirror ratios
Characteristics of CID
(collison induced dissociation)
Immonium ion signals are enhanced with collision
gas; use routinely below fragment mass 200.

Collisions can induce fragmentation of ions that do
not decompose under normal PSD conditions.

Side chain fragmentation may allow one to
distinguish between Leu and Ile.

Effect of CID on immonium ions
(GRF Lys-C peptide KLLQDILSR; MH+ = 1085.667)
CID to distinguish between Ile and Leu