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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
AH+
+20 kV
+
V
Ions are formed in pulses. Small ions reach the detector before large ones. Measures the time for ions to reach the detector.
detector
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
Attenuator
Laser
Linear detector
Collision cell
+
L
eee- e
-100V
L >> D
1kV 15kV
Attenuator
Laser
Linear detector
Collision cell
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
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.
Ion Source
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.
+ +
0 V.
+20 kV
Reflector
15 ppm error
Resolution = 18100
6000
Resolution = 14200
24 ppm error
Counts
4000
Resolution = 4500
2000
55 ppm error
0
2840
2845
2850
2855
Mass (m/z)
Laser
Source
Reflector
No of ions
Internal energy
For peptides the efficiency of PSD fragmentation is amino acid composition and sequence dependent.
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.
TIS on
Gate closed
Before fragmentation
The intact molecular ion has translational kinetic energy equal to:
KE = 1/2 Mv2
where:
m KE m = 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.
Assume MH+ = 1,000 Da, AH+ = 700 Da, and BH+ = 300 Da
MH+ ( 1,000) correctly focused AH+ (700) Poorly focused BH+ (300) Poorly focused
MH+ ( 1,000) not focused AH+ (700) correctly focused BH+ (300) Poorly focused
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
1 64.2
269 .4
3 74.6
479.8