9/6/2022
Matrix Assisted Laser Desorption
Ionization - Time of Flight
◦ The application of mass spectrometry to the identification of microorganisms
◦ Mass spectrometry is an analytical technique in which chemical compounds
MALDI-TOF are ionized into charged molecules and the ratio of their mass to charge (m/z)
is measured. Each compound has a unique m/z ratio profile.
◦ MALDI is the ionization technique
Analytic Techniques
◦ TOF is the mass spectrometry analyzer
This Photo by Unknown Author is licensed
under CC BY
MALDI
Ionization step
◦ MALDI was developed in the 1980s and made it possible to apply mass spectrometry to
proteins.
◦ It converts peptides into ions by adding or removing one or more protons
◦ It is a “soft ionization” meaning the ionization process does not cause a significant loss of
sample integrity and it is the best choice for proteins
◦ MALDI creates single charged ions (+), making interpretation easier
◦ It does not require an initial chromatography separation step (seen in traditional GC-MS)
◦ Intact microorganisms can be directly processed to MALDI-TOF without pretreatment
because most vegetative bacteria are lysed following exposure to water, organic solvent
and/or strong acid in the MALDI matrix.
◦ When resistant microorganisms such as some viruses, bacterial spores, and yeast cells have
to be analyzed by MALDI, strong organic acids and/or alcohols are usually added in
pretreatment steps.
This Photo by Unknown Author is licensed under CC BY
MALDI: Steps
◦ The sample is embedded in or mixed with the matrix.
◦ The sample-matrix mixture is fixed to a metal plate, with multiple spots for samples
◦ The sample and matrix crystalize as they dry and then they are hit with a very brief
laser pulse.
◦ Rapid heating of the irradiated spot causes it to become vibrationally excited.
◦ The matrix molecules absorb the laser energy and carry the analyte molecules into
the gas phase.
◦ During this process, the analyte molecules are ionized by being protonated with
the nearby matrix molecules.
◦ The most common MALDI ionization format is for analyte molecules to carry a
single positive charge.
This Photo by Unknown Author is licensed under CC BY-SA
This Photo by Unknown Author is licensed under CC BY-SA
1
9/6/2022
TOF
◦ Ionized molecules are accelerated through a charged field, which starts the
separation based on their mass to charge ratio (m/z)
◦ Then they enter the flight tube (no electric field)
◦ The m/z ratio of an ion is measured by determining the time required for it to
travel the length of the flight tube
◦ The lighter ions will arrive earlier at the detector than the heavier ones, each
will have a different TOF.
◦ The intensity of each ion is also recorded
◦ Based on the TOF information, a characteristic spectrum called peptide mass
fingerprint (PMF) is generated for the sample
This Photo by Unknown Author is licensed under CC BY-SA This Photo by Unknown Author is licensed under CC BY
Interpretation Applications
◦ Using PMF matching, the mass spectrum of unknown microbial isolates is ◦ Shortens the diagnostic time, often by days
compared with the mass spectra of known microbial isolates contained in the ◦ Average time from sample prep to results is 30 minutes; run time is about 2
database, which is constantly updated by manufacturers. minutes
◦ For species level identification of microbes, a typical mass range m/z of 2–20 ◦ Cultures are typically first grown out on media, then colonies picked and
kDa is used, which represents mainly ribosomal proteins (60–70% of the dry identified
weight of a microbial cell) along with a few housekeeping proteins.
◦ Can be used to identify bacteria, fungi and yeast
◦ MALDI-TOF MS identification is independent of culture conditions (media
types) ◦ MALDI-TOF MS is suitable for high-throughput and rapid microbial identification
at low costs in medical diagnostics, biodefense, environmental monitoring,
and food quality control.
This Photo by Unknown Author is licensed under CC BY-SA
This Photo by Unknown Author is licensed under CC BY-SA