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