Mass Spectrometry
Separation principle:A particle with a certain mass (m) and a certain charge (z) behaves in a predictable manner when exposed to a electromagnetic (EM) field. By measuring this response, the ratio of the two variables (m/z) can be determined.
First approach:Ionize the particleAccelerate the particle by an electric fieldModify the flight-path of the particle with a magnetic fieldMeasure the effect of the magnetic field
The First Mass Spectrometer:The Magnetic Sector
chamber of ionized particles
Acceleration
magnet
Atmospheric pressure Vacuum
Photographic plate
(m/z)1
(m/z)2
(m/z)3
Other Types of Mass Spectrometers Quadropole
only ions of known m/z can pass through at a given EM field generated by four poles (quadropole).
Ion Traponly ions of known m/z can reach the detector, others are kept spinning in circles by an EM field generated by four poles.
Time of Flightthe accelerated ions travel a known length and the time it takes is directly related to the m/z ratio.
Ion Cyclotron (Fourier Transform)similar to Ion Trap, six poles instead of four, the decay of the induced electric current of all the spinning ions is measured and transformed to frequency distribution, which is directly related to the m/z ratio.
Time of Flight Mass Spectrometer
ionized particles
acceleration Field free region detector
Time m/z
Inte
nsity
Can only be performed in high vacuum to minimize collisions between ions of different mass or other gaseous moyeties
Vacuum
Atmospheric pressure
Matrix Assisted Laser Desorption Ionization
• produces mainly singly charged ions
• pulsed ionization, perfect match to TOF MS
• a “soft” ionization, good for sensitive biomolecules
• fairly tolerant of impurities such as salts and detergents
laser
sample
gaseous plume
+
++
+
+
+ - -
-
---
--
-
--
-
-
++
+ +
+
+
chargeformation
laser ablation
Sample Clean-Up
Ethanol Precipitation
GenoPure
ZipTipnon-autoclaved water
Single Base Extension
Primer
ddC
ddT
ddA
ddG
273.3
288.2
297.2
313.2
15
16
9
m/z
m/z
m/z
m/z
m/z
Single Base ExtensionPrimer W1156 (22mer)
TGCTATACCAAGTTTCCATAAA
AA
C
TATA
CC
A
C/A
W1156Sample 709C
ΔM=312.7 Da
W1156Sample 712C
ΔM1=313.0 Da
ΔM2=297.1 Da
W1156Sample 713C
ΔM=296.5 Da
W1156Control (no DNA)
ΔM=301.3 Da
Results of primer W1156
From FP experiments: 709C – Homozygote, type B (ddG)712C – Heterozygote713C – Homozygote, type A (ddA)
Mass difference error compared to theoretical masses:
(ΔMmeas- ΔMddNTP)/ ΔMddNTP709C 713C Control
ddG 0.2% 5% 0.2% 5% 4%ddA 5% 0.3% 5% 0.1% 1.4%ddT 9% 3% 8% 3% 5%ddC 14% 8% 14% 9% 10%
712C
Primer W1488 (34mer)
W1488 Sample 713C
ΔM=296 Da
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