# MALDI-Based Mass Spectrometry in Clinical Testing: Focus on Bacterial Identification
## 3 Measurement Principles and Characteristics
(2) The mass spectrometer irradiates the MALDI sample plate with a laser to ionize the proteins (mainly ribosomal proteins) in the bacteria, and the mass spectral pattern is obtained from the mass/charge ratio (\(m/z\)) of the constituent proteins and the intensity from the time of flight to the detector. (3) The obtained MALDI mass spectrum pattern is compared to a database for identification. The MALDI matrix used for bacterial identification is typically \(\alpha\)-cyano-4-hydroxycinnamic acid (CHCA), which works by (1) absorbing laser energy efficiently, (2) supplying protons to the sample and promoting ionization, and (3) preventing sample decomposition. differs depending on the type of molecule, the ratio of the peak intensities of different ions does not necessarily reflect the concentration ratio of the molecules in the sample. Still, it is possible to compare samples of a given substance. Since bacterial identification uses a mass spectrometer, an analysis method based on pattern matching is used. The major advantage of the MALDI-TOF MS identification method is that it can be used to identify not only general bacteria, but also anaerobic bacteria, acid-fast bacteria, yeast-like fungi, and filamentous fungi. Rapid identification of bacterial species using MALDI-TOF MS and support for appropriate use of antimicrobial agents based on the results can lead to shorter time to effective antimicrobial therapy, shorter hospital stays, and lower mortality rates. These effects also lead to a reduction in mortality. The sample preparation procedure is as follows: bacteria are scraped from the colonies on the culture medium and applied to the MALDI target plate of the mass spectrometer. A drop of \(\alpha\)-cyano-4-hydroxycinnamic acid (CHCA) matrix solution is added to the bacteria on the MALDI target plate; after drying of the solution, the sample is assessed by MALDI-TOF MS. The analytical performance of MALDI-TOF-MS can be improved by using the matrix solution that is most suitable to the compound to be analyzed and the purpose of analysis. DHBA (2,5-dihydroxybenzoic acid) can be applied to compounds such as proteins, peptides, lipids, and carbohydrates. However, there are cases where needle-like mixed crystals are produced, and the sample is severely localized. \(\alpha\)-Cyano-4-hydroxycinnamic acid (CHCA) is suitable for sensitive analysis of peptides and is widely used in proteomic studies by MALDI-MS or MALDI-TOF-MS. Sinapinic acid (SA) is also suitable for protein analysis and is useful in microbial analysis. Preparation of MALDI matrix solution was performed. The following is an example of such a process. The matrix solution consisted of pre-dissolved Bruker Matrix HCCA, portioned (Bruker Daltonics). The HCCA matrix was dissolved to a final concentration of 5 mg/mL in a mixture of 50% acetonitrile, 47.5% water, and 2.5% trifluoroacetic acid. A spot of matrix solution is added to the MALDI sample spot. The samples are desorbed from the MALDI target plate surface by pulsed laser irradiation, ionized, and accelerated in the mass spectrometer, under the influence of the electric field. The ionized protein (bacterial component) flies toward the detector, and the protein mass is determined as a function of its arrival time. As a result, peaks with different masses are detected. The MALDI mass spectrum pattern for each bacterium is different. The amount of bacterial sample required is several micrograms, and identification is possible with \(10^{4}\) to \(10^{5}\) bacteria. Measurement time is only a few minutes, which is a great advantage for rapid bacterial identification. In the sample, the peaks observed in the MALDI mass spectrum are derived primarily from bacterial proteins. Proteins derived from bacterial ribosomes are said to account for 50-70% [32]. This bias reflects that fact that ribosomal proteins are highly expressed, easily ionized, and easily detected by TOF MS in the molecular weight range of approximately 4000-15,000 Da.