Question

What is MALDI-TOF? How does it work? What can it tell us?

Answer 1

MALDI represents matrix assisted laser desorption/ ionisation and TOF represents time-of-flight. It is a mass spectrometry technique where charged fragments are produced and their m/z (mass to charge) ratio is determined. Here the ion is produced by MALDI and the TOF is the mass analyser.

Working of MALDI-TOF:

The basic principle of MALDI is to separate the individual analyte molecules from each other which is made possible by making their solid solution in a matrix material. This matrix is composed of a particular material in large excess. The choice of the matrix is variable. Polar analytes go well with polar matrix while the non polar analytes with the non polar matrix. Now the matrix-analyte solution is deposited onto a solid surface referred to as the target. Now this target is hit upon by a laser, usually UV is used but others can also be preferred depending on the experiment. The laser striking on a particular spot causes thermal ablation where the matrix molecules absorb the energy further enabling the analyte molecules to transit into gaseous phase along with them. It results in a protonation or deprotonation of the analyte molecules; however mostly they emerge as single positively charged ions. Hence different fragments with varied m/z ratio is created. These are then separated using a time of flight. TOF is a process which employs the principle of difference in time taken by molecules with different m/z ratio to travel the same distance. Practically, the ion species generated are made to travel in a field-free acceleration path starting at the same time. The lighter ions with less m/z ratio will disperse faster and reach the detector quicker than the heavier ones with higher m/z ratio. This lag is time is correlated to identify the fragments and the data is finally computed in the respective MS profile using a software.

A reflectron is also used in this experiment. This is because all the ions with same mass which are supposed to reach the detector at the same point of time, don't receive the same initial pulse, and may have slight difference in their kinetic energy. This can lead to different time taken by those with same mass. Hence a reflectron which comprises of a ring of electrode maintained at a high voltage, is placed in the path which reflects the ions at a specific angle in their flight tube. This serves the purpose since the ions moving faster i.e. those with higher energy, penetrate a greater depth inside the reflectron, hence spend more time there instead of getting deflected, while those with large mass or moving slower cannot penetrate much inside. This difference in surface penetrations finally allows the ions of same mass to reach the detector almost at the same time. So a better resolution is obtained. However, the usage of reflectron is limited due to stability issues.

What can MALDI-TOF tell us/ its significance:

This is a mass spectrometry technique that how determine the m/z ratio of the ionic samples generated out of the analyte. The analytes determined include biomolecules like peptides, oligonucleotides etc.

The degree of completion of synthesis of a molecular species can be checked during their commercial synthesis by comparing their mass with the established database.

Being a soft ionisation technique, it is used to determine the intact mass of the proteins, then peptide mass fingerprinting can be done to determine the mass of peptides of the new protein synthesized and compared with the theoretical database. Oligonucleotides synthesized during the assays to be used in a variety of molecular biology experiments like primers, drugs, probes etc. can also be checked if their complete synthesis has been done.