Matrix-Assisted Laser Desorption/ionization (MALDI-TOF) : MD Exam (2011) Notes

MD Exam (2011) Notes
Matrix-assisted laser desorption/ionization (MALDI-TOF)

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in
mass spectrometry, allowing the analysis of biomolecules (biopolymers such as proteins,
peptides and sugars) and large organic molecules (such as polymers, dendrimers and other
macromolecules), which tend to be fragile and fragment when ionized by more conventional
ionization methods.
It is most similar in character to electrospray ionization both in relative softness and the ions
produced (although it causes many fewer multiply charged ions).
The ionization is triggered by a laser beam (normally a nitrogen laser). A matrix is used to
protect the biomolecule from being destroyed by direct laser beam and to facilitate
vaporization and ionization.
Developed in 1988 by Professor Hillenkamp
Designed to enhance mass-spec. by solving two main problems
 Thermal instability and low volatility

 Large and heavy biomolecules
Principle of MALDI-TOF mass spectrometry
Laser bursts volatize the matrix carrying along with it sample DNA, which is then propelled
by an electric field down a flight tube to the detector. The time of flight is proportional to the
mass/charge ratio; identically charged molecules with small masses have short times of flight,
larger masses have longer times of flight.
 Matrix-assisted laser desorption/ionization time of flight mass spectrometry

 Ionizes molecules via laser pulses
 Separates molecules according to mass to charge ratio
 Mainly used for detection of large biomolecules
First, the polymer needs to be dissolved in a solvent. Proteins are usually dissolved in water,
sometimes acetonitrile is added. For other biomolecules, other solvents may need to be
found. Also, some MALDI specs do not need to have the sample in solution; it can be in a
solid state. Next, after the compound is in solution, a matrix needs to be added. Similar to
AA, different matrices work better with different compounds. However, all matrices need
absorb UV radiation. Some common matrices are trans-cinnamic acid or 2,5-
dihydroxybenzoic acid. Matrix is generally added in an amount that is greater than 10^4
times the sample, ensuring that the matrix absorbs a majority of the radiation rather than the
sample. This will prevent unwanted sample fragmentation. Another important aspect of the
matrix is that it serves to isolate polymers from one another. Finally, the matrix serves as a
source of protons for the sample to ionize.
After the sample has been prepared, the solution is loaded into the sample chamber, which is
then vacuum pumped to evacuate all of the air in the chamber. At this time, the solvent
evaporates, leaving the sample in a dispersed compound containing both the matrix and the
sample.
At this point, the laser shoots short pulses of light, in the 330-360 nm range, at the sample,
causing it to essentially explode. The matrix is vaporized, and the sample is ionized into the
+1 or –1 state. No one is really sure of the mechanism of this ionization, but for some reason
it occurs, and the +/- 1 state is found. It is also at this point that the polymers do something
unusual. They evaporate. Usually, the polymers are too big and heavy to evaporate, but at
these high temperatures and low pressures, evaporation or desorption occurs. Thus, we are
left with ionized polymers in the gaseous phase.
Ions are accelerated by electrodes at opposite end of tunnel of a known length. If it is a

negative ion, it accelerates to the cathode; positive ions accelerate towards the anode. The
charge depends on the type of molecule being analyzed as well as the matrix used. This
electrical force is used to accelerate the particles down tunnel towards the detector at the far
end. Since all of the ions have the same charge, their acceleration, and thus the time it takes
to reach the detector are completely dependent upon the mass of the fragment.
Once the molecules reach the detector, a peak is registered. These peaks are recorded via a
high speed recording mechanism. The size of the peak is proportional to the number of
molecules that reach the detector at a given time interval.
Uses of MALDI-TOF
 Used to characterize and identify large molecules
 Used in pharmaceutical for QC, monitoring of enzyme reactions
 Used in DNA sequencing for forensics
 Used to identify different strains of viruses and bacteria to help develop vaccines
Mass Range 1-400,000 Da
Detection Limit 10-15 – 10-18 moles
Accuracy 0.1 – 0.01%
Sensitivity 1 uL containing 100 fmol – 10 pmol
Sample Less than 1 pmol

Consumption
Advantages
1. A very small sample size is needed. As little as 1 pmol can give very resolved spectra.
2. It is often difficult to get large biomolecules/polymers into the gas phase to use in mass
spec. Using the low temperatures and high pressure, it is possible to get large molecules into
the gas phase with fragmenting them.
3. Unlike SEC (Size Exclusion Chromatography), samples aren’t compared to any other
standards; they are absolute measurements of mass.
Disadvantages
1. It is often difficult to find suitable matrices to isolate specific polymers. The matrix
not only has to isolate the molecule, but also must absorb large amounts of UV
radiation so that the sample is not fragmented, but instead vaporized.
2. Being that this is a “soft” ionization technique, a majority of the energy is used
volatizing the matrix rather than exciting the ions. Thus, it is possible for ions to relax
from the excited state after some collisions.
Due to the extreme sensitivity, contaminants can easily interfere with the produced spectra.
For example, trace amounts of buffer salts will produce such intense peaks that they will
effectively drain out the peaks of the sample. Thus, when these contaminants can be
removed, the sample should be prepared at a very high concentration

Matrix-Assisted Laser Desorption/ionization (MALDI-TOF) : MD Exam (2011) Notes

Uploaded by

Copyright:

Available Formats

Matrix-Assisted Laser Desorption/ionization (MALDI-TOF) : MD Exam (2011) Notes

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Matrix-Assisted Laser Desorption/ionization (MALDI-TOF) : MD Exam (2011) Notes

Uploaded by

Copyright:

Available Formats

MD Exam (2011) Notes

Matrix-assisted laser desorption/ionization (MALDI-TOF)

Developed in 1988 by Professor Hillenkamp

Designed to enhance mass-spec. by solving two main problems

 Thermal instability and low volatility

Principle of MALDI-TOF mass spectrometry

 Matrix-assisted laser desorption/ionization time of flight mass spectrometry

Ions are accelerated by electrodes at opposite end of tunnel of a known length. If it is a

 Used to characterize and identify large molecules

 Used in pharmaceutical for QC, monitoring of enzyme reactions

 Used in DNA sequencing for forensics

Mass Range 1-400,000 Da

Detection Limit 10-15 – 10-18 moles

Accuracy 0.1 – 0.01%

Sensitivity 1 uL containing 100 fmol – 10 pmol

Sample Less than 1 pmol

You might also like