MALDI-TOF identification (overview)

Principle

• Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
• Microbial proteins (mainly abundant ribosomal proteins, roughly 2,000–20,000 Da) are co-crystallized with a matrix and ionized by a laser.
• Ions fly through a vacuum tube; time of flight depends on mass-to-charge ratio, with lighter ions arriving first.
• The resulting spectral fingerprint is compared with a reference database to identify the organism.

Specimen

• Fresh colonies from solid media; validated protocols also exist for positive blood culture broth and some direct specimens.

Reagents and equipment

• Target plate, matrix (commonly α-cyano-4-hydroxycinnamic acid, CHCA), 70% formic acid, ethanol and acetonitrile for extractions.
• Instrument platforms (e.g. Bruker MALDI Biotyper, bioMérieux VITEK MS) with manufacturer databases.

Procedure

• Direct smear: spread a thin film of colony on a target spot and overlay matrix; let dry.
• On-plate formic acid overlay improves results for yeasts and some Gram-positives.
• Full tube extraction (ethanol/formic acid/acetonitrile) for molds, mycobacteria and difficult organisms; also inactivates hazardous organisms.
• Run calibration standard, then samples; results in minutes.

Results

• The software gives a score or confidence value; on the Bruker system a score ≥2.0 is typically high-confidence species level and 1.7–1.99 genus level (manufacturer and lab criteria apply).

Quality control and pitfalls

• Calibrate with a bacterial test standard and run QC organisms per manufacturer and local policy.
• Cannot reliably separate some close relatives: E. coli vs Shigella, S. pneumoniae vs S. mitis/oralis group (some systems), Bacillus cereus group members.
• Mixed colonies, too thick or too thin smears and organisms missing from the database cause failures or errors.
• Does not routinely provide susceptibility results.

Clinical use

• Rapid, low-cost identification of bacteria, yeasts, molds and mycobacteria, reducing turnaround time by a day or more.