Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a soft ionization technique that enables the analysis of large, non-volatile biomolecules such as proteins, peptides, lipids, and oligonucleotides. It is standard in proteomics, clinical microbiology, and polymer analysis.

In MALDI, the analyte is co-crystallized with a matrix compound, typically a small organic acid that absorbs strongly at the laser wavelength. Common matrices include α-cyano-4-hydroxycinnamic acid for peptides and sinapinic acid for proteins. A pulsed UV laser (typically 337 nm nitrogen or 355 nm Nd:YAG) irradiates the sample, causing rapid heating and desorption of matrix and analyte ions. The matrix facilitates ionization through proton transfer, producing predominantly singly charged ions with minimal fragmentation.

The time-of-flight analyzer separates ions based on their mass-to-charge ratio by measuring the time required to travel through a field-free flight tube. Smaller ions reach the detector faster. The flight time is proportional to the square root of m/z. Delayed extraction and ion reflectron improve resolution by compensating for initial energy spread.

MALDI-TOF MS is used for peptide mass fingerprinting in protein identification, where the masses of tryptic peptides are matched against theoretical digests from protein databases. In clinical microbiology, intact bacterial cells are analyzed directly, and the resulting protein mass spectrum is compared against reference libraries for species identification. This approach has revolutionized clinical microbiology by enabling rapid identification within minutes.

Limitations include poor reproducibility for quantitative analysis, matrix interference in the low mass range, and reduced sensitivity for analytes below 500 Da. MALDI imaging extends the technique to map the spatial distribution of biomolecules directly on tissue sections.