The Ongoing Revolution of MALDI-TOF Mass Spectrometry for Molecular Diagnostics

Abstract

MALDI-TOF MS is bringing an ongoing revolution to clinical microbiology, and to anticipate possible future possibilities this chapter provides a primer on the basics of the MALDI-TOF MS. While the first commercial MALDI-TOF MS instruments appeared around 1990, the first papers on the identification of bacteria, moulds and yeasts appeared in the period between 1994 and 2001. The first commercial software tools for automated identification appeared around 2004 (e.g. Biotyper 1.0, Bruker). The core of the MALDI-TOF MS technology consists of three functional units, being an ion source, a mass analyser and an ion detector. The starting point of the MALDI analysis is the ionization of the sample in the ion source. As the acronym MALDI indicates, a laser pulse supplies the energy required for ionization. The basic principle underlying separation of ions according to their mass over charge ratio is exploiting the law of conservation of energy. By ionizing the sample in a region with an electric field present, the ions obtain an elevated potential energy, which is transferred into kinetic energy, i.e. an impulse towards the time-of-flight (TOF) tube. Since direct detection of a few or single ions arriving on the back flange of the TOF tube is impossible, somehow its impact needs to be amplified. In MALDI-TOF MS, devices are used to amplify and detect the single-ion signals. A number of processing steps need to be performed to obtain a signature fit for interpretation. Owing to the large sensitivity of the ionization process to the local spot morphology, spectra resulting from single-ionization events feature an extreme variation in signature content. Results stemming from different locations at the sample spot are combined to combat this variation. A relation must be established between the spectra produced by MALDI-TOF MS and the identification of the organisms to produce relevant diagnostic information. This is a classical pattern recognition task. Future directions of MALDI-TOF for clinical microbiology laboratories are aimed at increased efficiency, better performance and additional functionality. One of the biggest drawbacks of MALDI-TOF MS is the need for biomass (a subcultured isolate) as starting material. Even though MALDI-TOF MS enables a significant speed-up of the identification process itself, the total turnaround time for an analysis will not reduce below the time needed for culturing. Culturing and isolation need to be made redundant to reduce the turnaround time. Single-cell MALDI was developed to achieve this short turnaround time. Single-cell MALDI is able to generate interpretable spectra from as little as a single microbial cell. Since the samples are analysed cell by cell, analysis of mixtures is feasible. Furthermore, since a reliable identification requires less than 10² cells, direct analysis (i.e. without an enrichment step) of clinical samples may be possible as well for a number of sample types.