Transcript Introduction to MALDI-TOF MS - University of California

Introduction to MALDI-TOF MS
Sandler Mass Spectrometry User’s 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
Laser
Sample plate
1. Sample (A) is mixed with
excess matrix (M) and dried
on a MALDI plate.
hn
2. Laser flash ionizes matrix
molecules.
AH+
3. Sample molecules are ionized
by proton transfer from matrix:
MH+ + A  M + AH+.
Variable Ground
+20 kV
Grid
Grid
Time-of-flight mass analyzer
Source
Drift region (flight tube)
+
+
+
V
•Ions are formed in pulses.
•Small ions reach the detector before large ones.
•Measures the time for ions to reach the detector.
detector
+
Calibration of the mass scale
The mass-to-charge ratio of an ion is proportional to the square
of its drift time.
2
m 2t K
 2
z
L
t
L
m
K
z
=
=
=
=
=
Drift time
Drift length
Mass
Kinetic energy of ion
Number of charges on ion
Voyager-DE STR MALDI TOF
Sample
plate Extraction
grids
Attenuator
Prism
Reflector
Timed ion detector
selector
Reflector
Laser
Collision
cell
Camera
Pumping
Pumping
Linear
detector
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.
Ions are detected with a microchannel plate
primary ion
-1000V
+
ee-
L
e- e
-100V
D
L >> D
Microchannel Plate (MCP)
High current detector schematic
Used in linear mode to enhance signal from high mass molecules
Fast scintillator
Microchannel plate
Condenser
Photomultiplier tube
signal
1kV 15kV
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 DESTR 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 BradburyNeilson gate is used.
Voyager-DE STR MALDI TOF
Sample
plate Extraction
grids
Attenuator
Prism
Reflector
Timed ion detector
selector
Reflector
Laser
Collision
cell
Camera
Pumping
Pumping
Linear
detector
The problem: Peaks are inherently broad in MALDI-TOF
spectra (poor mass resolution).
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.
Sample + matrix on target
Ions of same mass, different velocities
+
+
+
Can we compensate for the initial
energy spread of ions of the same
mass to produce narrower peaks?
Delayed Extraction
Reflector TOF Mass Analyzer
Delayed Extraction (DE) improves
performance
0 V.
Ions of same mass, different
velocities
+
+
+
0 V.
Step 1: No applied electric field. Ions spread out.
20 kV.
+
+
+
0 V.
Step 2: Field applied. Slow ions accelerated more than fast ones.
20 kV.
Step 3: Slow ions catch up with faster ones.
+
+
+
0 V.
What is a reflector TOF analyzer?
A single stage gridded ion mirror that subjects the ions to a uniform repulsive electric
field to reflect them.
Detector
Ion Source
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 reflector focuses ions to give better mass
resolution
+
+
0 V.
+20 kV
Reflector
Resolution & mass accuracy on mellitin
Resolution = 18100
15 ppm error
8000
6000
Resolution = 14200
Counts
24 ppm error
4000
Resolution = 4500
2000
55 ppm error
0
2840
2845
2850
Mass (m/z)
2855
Fundamentals of Post Source Decay
(PSD)
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 source—hence postsource decay)
3. Fragment ions are separated and detected in the
reflector.
Decomposition occurs in the flight tube
Laser
Source
Decay can
occur at any
point along here
Reflector
detector
Reflector
Linear
detector
Internal energy of precursor ions
Only a small fraction of the precursor ions have enough
energy to fragment during their lifetimes.
No of
ions
Internal energy
For peptides the efficiency of PSD fragmentation is amino acid composition and
sequence dependent.
Increasing PSD Fragmentation
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
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
“Gate open”
TIS on
“Gate closed”
Effect of the timed ion selector
Before fragmentation
The intact molecular ion has translational kinetic energy
equal to:
KE = 1/2 Mv2
where:
KE = kinetic energy (= zeV)
M = mass
v
= velocity
Post source fragmentation
The translational kinetic energy of a fragment ion is
m
KEm  KE M  
M
where
KEM = precursor kinetic energy
KEm = fragment kinetic energy
M = precursor mass
m = fragment mass
Precusor and PSD fragment ions take
different paths in the “normal” reflector
Reflector
detector
Intact
precursor ion
+
+
Fragment ion
formed by PSD
0 V.
Reflector
+20 kV
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.
PSD mirror ratio setting
Consider an ion (MH+) that can decompose into two
fragments, A and B.
Either of the following reactions can occur:
MH+
AH+ + B
MH+
A + BH+
Assume MH+ = 1,000 Da, AH+ = 700 Da, and BH+ = 300 Da
At mirror ratio = 1.00
AH+
BH+
MH+
MH+ ( 1,000) correctly focused
AH+ (700) Poorly focused
BH+ (300) Poorly focused
At mirror ratio = 0.7
BH+
AH+
MH+
MH+ ( 1,000) not focused
AH+ (700) correctly focused
BH+ (300) Poorly focused
At mirror ratio = 0.3
BH+
AH+ & MH+
MH+ ( 1,000) not focused
AH+ (700) not focused
BH+ (300) correctly focused
Resolution decreases as the fragment ions
penetrate less into the mirror
MR=0.80
MR=0.71
MR=0.61
(MR= mirror ratio)
A PSD spectrum is taken in “stitches”
PSD Spectrum of Angiotensin I, MH+ = 1296.7 Da
Composite of the focused mass regions from several spectra acquired with different
mirror ratios
Stitched PSD[BP = 354.1, 59562]
100
90
(-Tyr)
(-P ro)
354.1
269.2
(-H is)
6.0E +4
255.1
80
382.2
%Intensity
70
60
364.1
50
40
81.0 95.0
506.3
416.1
115.1
30
20 59.0
513.2
112.1
(-V al)
235.1
138.1 156.2
133.0
140.1
272.2
195.1
217.2
251.1
534.0
326.1343.1
313.0
400.1
337.1
426.1
489.3
370.2
10
0
59.0
164.2
269.4
374.6
0
585.0
479.8
Stitched PSD[BP = 354.1, 59562]
100
4.7E +4
784.3
90
80
1181.7
756.3
619.3
1182.8
%Intensity
70
60
50
1296.7
767.4
650.3
40
30
20
646.3
739.3
742.2
881.5
10
0
591
753
1000.7
915
1046.6
1077
Mass (m /z)
1137.7
1164.8
1229.2
1239
0
1401
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
CID to distinguish between Ile and Leu
(GRF Lys-C peptide KLLQDILSR; MH+ = 1085.667)