# MALDI-Based Mass Spectrometry in Clinical Testing: Focus on Bacterial Identification
## 3 Measurement Principles and Characteristics  
A nitrogen laser (337 nm) is often used to excite the mixed crystal. In such a case, a material with an absorption band at 337 nm is used for the matrix. When the homogeneously mixed crystal of the sample matrix is irradiated with high-density laser light, the matrix first absorbs the laser photons and reaches an excited state. At the same time, the temperature of the crystal rises, and when the mixed crystal reaches the evaporation point, the sample and the matrix evaporate (sublimate). This fact means that, even if the sample does not sublimate well on its own, the sample will sublimate with the help of the matrix. Next, proteins in the evaporated sample undergo chemical ionization by the matrix in a highly excited state. TOF refers to time of flight, one of the ion separation techniques, along with magnetic field and ion trap. The ionized sample is accelerated by an electric field and different masses will gain different velocities. Since ions travel a fixed distance, the molecular weight is determined by measuring the time of flight of each ion. The mixture of sample and MALDI matrix on the MALDI sample plate is desorbed and ionized by a pulsed laser irradiation. The ions formed are accelerated by the electric field applied on the sample plate, and measured by a detector. The arrival time of ions is finally converted to a mass spectrum. In MALDI-TOF MS, the linear mode is used to identify microorganisms, while the reflectron mode is used to detect the ionized components. The linear mode is used for microbial identification in MALDI-TOF MS, because it is more sensitive and can measure proteins with a high molecular weight. Microbial identification is performed in positive ion mode (measures proteins). The optimal and minimum number of laser shots necessary to acquire high-quality spectra in a short time (96 samples/30 min) was automatically determined by the instrument. The main peaks observed in the mass spectrum were derived from the fungal proteins, and their molecular weights ranged from 4000 to 100000000000000000000000000000000000000000000000  
15,000 Da, which are easily detectable using TOF MS. Therefore, the measurement range for microbial identification was approximately 2000-20,000 Da, where the protein-derived peaks are observed. MALDI-TOF MS has become an effective measurement technique in clinical microbiology today [27, 28, 29]. As mentioned earlier, MALDI Biotyper(r) from Bruker Daltonics, AXIMA(r) Microbial Identification System from Shimadzu, and VITEK(r) MS from Sysmex BioMerieux are the currently available bacterial identification systems. Here, the principles of measurement and operation are introduced using MALDI Biotyper(r) as an example (Figure 2). MS consists of three basic steps: ionization, mass separation, and detection [9, 30, 31]. The sample molecules are charged (ionized) to form molecular ions in a gaseous state. The generated ions are sent to the mass separation section, where the ions are separated according to their mass and charge (\(m/z\)) ratio using an electric or magnetic field in a vacuum. The separated molecular ions are converted to electrical signals by the mass detector. The MALDI mass spectral data are displayed in a MALDI mass spectrum graph, where \(m/z\) is plotted on the horizontal axis and the signal intensity of the ions is plotted on the vertical axis. The signal indicating each ion is called the peak. Since ionization efficiency  
Figure 2: Flow of bacterial identification by MALDI-TOF MS (example using MALDI Biotyper®). For identification by mass spectrometry (MS), (1) the colony is applied to a MALDI sample plate to be irradiated with a laser. The MALDI matrix is added and dried, and the MALDI sample plate is inserted into the mass spectrometer.