MALDI-TOF Mass

Spectrometry
LAB 02
MALDI-TOF Mass Spectrometry
• Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-
TOF) mass spectrometry has advanced the microbial identification in
microbiology and other fields.
• This technology offers rapid, accurate, and cost-effective identification
of microorganisms, including bacteria, fungi, and viruses.
• MALDI-TOF works on the principle of Mass Spectrometry where
molecules are ionized and analyzed by detector to give electrical
peaks on the base of mass to charge ratio (m/z).
• MALDI-TOF MS is based on the ionization of microbial proteins,
typically ribosomal proteins, and their analysis by time-of-flight mass
spectrometry.
Working Principle of MALDI-TOF Mass Spectrometry
• Sample Preparation: A small amount of microbial colony is mixed with
a matrix solution (usually a low molecular weight organic acid like α-
cyano-4-hydroxycinnamic acid, CHCA).
• The matrix-sample mixture on a target slide is dried. The matrix
absorbs UV light and assists in the ionization of the microbial proteins.
• Desorption/Ionization: A laser pulse (UV) irradiates the sample,
causing the matrix to absorb the laser energy, resulting in the
desorption and ionization of the sample molecules into the gas phase.
Working Principle of MALDI-TOF Mass Spectrometry
• Ion Acceleration: The ionized molecules are accelerated in an electric
field towards the mass analyzer. The ions are separated based on their
mass-to-charge (m/z) ratio.
• Time-of-Flight Analysis: The time it takes for the ions to reach the
detector is measured. Smaller ions reach the detector faster than
larger ions, allowing for the generation of a mass spectrum (in form of
electrical peaks).
• Data Analysis: The mass spectrum, which represents the protein
fingerprint of the microorganism, is compared to a database of known
spectra for identification.
Workflow of MALDI-TOF MS in Microbial Identification
• Isolation of Microorganism: Pure colonies of the microorganism are
isolated on an agar plate.
• Sample Preparation: A single colony is transferred to the target plate
and mixed with the matrix solution.
• Desorption/Ionization: The sample-matrix mixture is irradiated with a
laser to produce ionized molecules.
• Mass Spectrometry Analysis: The ionized molecules are analyzed based
on their time-of-flight.
• Identification: The resulting mass spectrum is compared with a
reference database to identify the microorganism.
Advantages of MALDI-TOF MS
• Speed: Identification can be completed in minutes once the sample is
prepared, significantly faster than traditional biochemical methods.
• Accuracy: High accuracy and reproducibility in identifying a wide
range of microorganisms.
• Cost-Effective: Reduces the need for costly reagents and consumables
used in conventional methods.
• Broad Spectrum: Capable of identifying bacteria, fungi, and some
viruses, as well as differentiating between closely related species.
• Minimal Sample Requirement: Requires only a small amount of
microbial culture (single colony).
Applications of MALDI-TOF MS
• Clinical Microbiology: Rapid identification of pathogens directly from
clinical samples, aiding in timely and appropriate treatment.
• Environmental Microbiology: Identification of microorganisms in soil,
water, and other environmental samples.
• Food Microbiology: Detection and identification of spoilage organisms
and foodborne pathogens.
• Pharmaceutical Microbiology: Ensuring sterility and identifying
contaminants in pharmaceutical products.
• Research: Studying microbial diversity, phylogeny, and microbial
community dynamics.
Basic Key Terms
• In MALDI-TOF mass spectrometry, CHCA is commonly used as the
matrix for both bacterial and fungal samples. Formic acid (FA) is often
added to fungal samples to enhance protein extraction and improve
ionization efficiency.
• The matrix is a chemical compound that absorbs laser energy and
assists in the desorption and ionization of analyte molecules, typically
enhancing the ionization process while protecting the analytes from
fragmentation.
• In MALDI-TOF mass spectrometry, desorption refers to the process
where the matrix absorbs laser energy and transfers it to the sample,
causing the analyte molecules to be released from the solid phase
into the gas phase without decomposing (fragmentation).