32.17: MALDI-TOF Mass Spectrometry

Mass spectrometry is a powerful characterization technique that can identify and separate a wide variety of compounds ranging from chemical to biological entities, based on their mass-to-charge ratio (m/z). The instruments that allow this detection, known as mass spectrometers, have three components: an ion source, a mass analyzer, and a detector. These spectrometers differ based on the nature of their ion source and analyzers.

Matrix-assisted laser desorption ionization (MALDI) is a commonly used ionization source in mass spectrometers. It enables the soft ionization of biological molecules like peptides, lipids, and saccharides without fragmenting them. This means they can be analyzed intact as the sample integrity is maintained.

In MALDI, the sample is mixed with a compatible matrix that is organic molecules. When the laser strikes the sample, the matrix functions as a mediator for energy absorption, yielding the intact sample analyte ions. The matrix molecules energetically ablate from the sample surface, absorb the laser energy and carry the sample molecules into the gas phase. The sample molecules are usually ionized during the ablation process to carry a single positive charge.

The desorbed sample ions carrying the charge are then directed towards the mass analyzer, usually a time-of-flight (TOF). The ions pass through a field-free drift region, which allows separation based on their mass/charge ratio, allowing the smaller ions to reach the detector first.

MALDI-TOF instruments are often connected with a reflectron that reflects ions increasing the ion flight path. This increased flight time between ions of different m/z allows better sample resolution where ions of the same mass reach the detector simultaneously. MALDI sources can be coupled with other analyzers, including triple quadrupoles and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. The MALDI-FT-ICR is known for its high mass resolution, wherein the sample ions carrying the charge move to an ICR cell and cyclotron in a magnetic field while separating on m/z.

With a couple of limitations, like, the possibility of photodegradation by the laser for some sample types, absorption of the laser by a few fluorescent analytes that could affect the results, an acidic matrix that could alter sample nature, etc., overall, this technique has vast applications. It can analyze protein samples isolated by electrophoresis or chromatography, peptides, carbohydrates, DNA, identify microorganisms from clinical samples and ascertain biomolecules in tissues, among many others.

MALDI-TOF mass spectrometry is an analytical technique for detecting and characterizing biological samples such as peptides, carbohydrates, and DNA.

MALDI-TOF contains an ionization source to generate gaseous ions and a mass analyzer for ion detection based on the ion’s time of flight.

First, the sample peptides are mixed with a matrix and loaded onto the target plate.

When struck by a laser, the sample desorbs, producing positively charged ions. That is why this technique is called matrix-assisted laser desorption ionization.

Ions then move into the mass analyzer, where they are sorted, depending on the time required to travel through the length of the flight tube.

This time of flight is proportional to the square root of the mass-to-charge ratio. Among the ions with the same charge, smaller ions move faster.

The detector collects these ions and passes the information to a computerized system. It generates a peptide mass fingerprint to identify the unknown protein by comparing the peptide mass with a database library.