**4. Mass spectrometry in microbiological diagnosis**

Since its discovery, over a hundred years, mass spectrometry has been a useful tool to un‐ derstanding the chemistry of proteins and biological processes involved. However, until the discovery of soft ionization techniques such as MALDI (Matrix-assisted laser desorption/ Ionization) and ESI (electrospray ionization) this methodology could be used as a routine tool in laboratories. [59].

Recently, mass spectrometry (MS) has entered to microbiology laboratories, offering a fast and reliable identification of microorganisms based on proteomic analysis.

#### **4.1. Mass spectrometer**

The mass spectrometer, in summary, is supported on the fragmentation of proteins to small peptides or other biomolecules to smaller molecules and then be ionized, these molecules are separated by the acceleration of ions in an electric field and then detected in based on their charge/mass ratio, in a gas phase state to produce a corresponding electrical signal to detect ions [60, 61].

A mass spectrometer is mainly composed of three elements in a vacuum atmosphere: an ionization source, a mass analyzer and detector.

#### *4.1.1. Ionization source*

The result of applying a source of ionization in a sample was the production of electrically charged ionized particles that gain or loss electrons in a gas phase.

There are several ionization processes that can be employed for the same purpose, to pro‐ duce ionized peptides [62] among these processes are MALDI (Matrix -assisted laser desorp‐ tion/Ionization) and ESI (electrospray ionization) that are most known.

#### *4.1.2. MALDI (desorption / ionization matrix-assisted laser)*

*3.4.1. SOMAmers applications*

10 Common Eye Infections

tool in laboratories. [59].

**4.1. Mass spectrometer**

detect ions [60, 61].

*4.1.1. Ionization source*

plied for microbiological diagnosis in the future.

ionization source, a mass analyzer and detector.

charged ionized particles that gain or loss electrons in a gas phase.

**4. Mass spectrometry in microbiological diagnosis**

Comparison between proteome of healthy and diseased tissues from human using Somas‐ can, can provide major knowledge of the biology of the disease and may lead to the discov‐ ery of new highly specific biomarkers for diagnosis, prognosis and therapeutic targets for

Somascan premium has been used for the discovery of biomarkers for the detection of meso‐ thelioma in the population exposed to asbestos. SOMAmers reagent showed better perform‐ ance with respect to the ELISA test. Also the system is used to discover biomarkers for the detection of non-small cell lung cancer [57]. Moreover the system can be applied in tumor tissue lysate to obtain biomarkers associated with the disease as well SOMAmers same re‐ agents can be used for histochemical evidence [58]. The SOMAmers represents an effective tool for biomarker discovery in different areas such as oncology, neurology, cardiovascular and metabolic diseases. To microbiological purposes as those related to the detection of agents in microbial infections SOMAmers represent a good alternative tool that may be ap‐

Since its discovery, over a hundred years, mass spectrometry has been a useful tool to un‐ derstanding the chemistry of proteins and biological processes involved. However, until the discovery of soft ionization techniques such as MALDI (Matrix-assisted laser desorption/ Ionization) and ESI (electrospray ionization) this methodology could be used as a routine

Recently, mass spectrometry (MS) has entered to microbiology laboratories, offering a fast

The mass spectrometer, in summary, is supported on the fragmentation of proteins to small peptides or other biomolecules to smaller molecules and then be ionized, these molecules are separated by the acceleration of ions in an electric field and then detected in based on their charge/mass ratio, in a gas phase state to produce a corresponding electrical signal to

A mass spectrometer is mainly composed of three elements in a vacuum atmosphere: an

The result of applying a source of ionization in a sample was the production of electrically

and reliable identification of microorganisms based on proteomic analysis.

the development of new drug treatment and it will improve personalized medicine.

In this method, the sample is soaked in an organic matrix which is crystallized with air and is irradiated by a laser, matrix most used are the acid α-cyano-4-hydroxy-trans cinnamic acid, 2,5-dihidrobenzoic acid or sinapinic acid [59].

In MALDI, the protein or peptide of interest is coprecipitated with the organic compound which is capable of absorbing laser light of an appropriate wavelength. The laser allows to prepare the compound fragmentation and disruption of the crystalline matrix generating a cloud of particles, these particles capture electrons and therefore remain as negatively charg‐ ed ions in most cases (Figure 3A) [59, 62].

**Figure 3.** Schematic representation of A) MALDI (Matrix-assisted laser desorption/ionization) and B) ESI (Electrospray ionization) method. Protein samples must be ionized before they pass through mass spectrometer.

#### *4.1.3. ESI (electrospray ionization)*

In this process, the sample is dissolved in an organic solvent, this mixture passes through a fine capillary tube that is maintained in an electric field produced by an electrode near to the capillary and other on the detector, this mixture is sprayed to form high load of tiny drop‐ lets of the solvent that evaporates quickly, thus produce a series of gaseous ions that result from protonation of side chains such as Arginine and Lysine, these ions fragmented by elec‐ tric field are then detected (Figure 3B) [63].

#### *4.1.4. Mass analyzer*

Is the main component of the mass spectrometer, the charged fragments (ions and radical ions), are accelerated and deflected by a strong magnetic field that affects their travel result‐ ing in a curvilinear path. Ions and radical ions are collected, detected and quantified with high accuracy and sensitivity, depending on the mass/charge ratio (m/z). [64].

There are several analyzers; however the most common type is the TOF (Time of flight). This analyzer defines a flight zone through which the ions are accelerated by acquiring a high kinetic energy, and during this trip will be separated according to their ratio mass/ charge (m/z). Most of ions generated have a single charge (z = 1), so that the ratio m/z is equal to m. The length of time for each ion in reaching the detector is called flight of time and depends on this ratio (Figure 4) [59].

#### *4.1.5. Ion detector*

At the end of the fragmentation and separation of ions from a sample by MALDI or ESI, ions impact on the detector. The fragments after flowing through the pipe in electric field (TOF) are deflected and detected, not-charged fragments are not deflected by the field and lost in the pipe walls, but the charged fragments are recorded by the detector, and mass are calcu‐ lated from the flight of time. In many cases, before the detector is the reflector, which in‐ crease the resolution of the technique (Figure 4) [65, 69].

**Figure 4.** Scheme of Mass Spectrometer. Mass analyzer characterized and separated ions according to their mass/ charge ratio (m/z). Ions detector generate mass spectrum for every ion detected. MALDI and ESI mass spectrum ob‐ tained are shown.

The mass spectrum of a compound is typically represented as a bar graph with the masses in the X axis, and the intensity or relative abundance of the ions of the m/z reaching the de‐ tector in the Y axis. The highest peak is assigned as 100% of intensity known as the base peak, and the main peak or molecular ion is the peak corresponding to the unfragmented radical cation (Figure 4) [65].

#### **4.2. Fingerprinter obtaining and analysis**

from protonation of side chains such as Arginine and Lysine, these ions fragmented by elec‐

Is the main component of the mass spectrometer, the charged fragments (ions and radical ions), are accelerated and deflected by a strong magnetic field that affects their travel result‐ ing in a curvilinear path. Ions and radical ions are collected, detected and quantified with

There are several analyzers; however the most common type is the TOF (Time of flight). This analyzer defines a flight zone through which the ions are accelerated by acquiring a high kinetic energy, and during this trip will be separated according to their ratio mass/ charge (m/z). Most of ions generated have a single charge (z = 1), so that the ratio m/z is equal to m. The length of time for each ion in reaching the detector is called flight of time

At the end of the fragmentation and separation of ions from a sample by MALDI or ESI, ions impact on the detector. The fragments after flowing through the pipe in electric field (TOF) are deflected and detected, not-charged fragments are not deflected by the field and lost in the pipe walls, but the charged fragments are recorded by the detector, and mass are calcu‐ lated from the flight of time. In many cases, before the detector is the reflector, which in‐

**Figure 4.** Scheme of Mass Spectrometer. Mass analyzer characterized and separated ions according to their mass/ charge ratio (m/z). Ions detector generate mass spectrum for every ion detected. MALDI and ESI mass spectrum ob‐

high accuracy and sensitivity, depending on the mass/charge ratio (m/z). [64].

tric field are then detected (Figure 3B) [63].

and depends on this ratio (Figure 4) [59].

crease the resolution of the technique (Figure 4) [65, 69].

*4.1.4. Mass analyzer*

12 Common Eye Infections

*4.1.5. Ion detector*

tained are shown.

The actual data acquisition with MALDI-TOF-MS is nowadays generally performed in an automated manner. That is, the laser focus scans the sample in a predefined pattern and ac‐ cumulates a mass spectrum from a defined number of laser pulse cycles, generally several hundreds to yield a representative average mass spectrum. The raw spectrum is generally processed to yield a mass fingerprinter that contains the information about peak apex *m/z* values, thus reducing the size of individual files considerably. The essential step for species identification is the comparison of the mass fingerprinter of the sample, to be identified to a database containing reference mass fingerprints, for example MASCOT, SWISS-PROT [66].

#### **4.3. Spectrometry in the diagnosis of microbiological specimens**

The first application of this technique was the study of the chemical structures of organic compounds in the area of Structural Chemistry and also the identification of compounds in the field of organic chemistry, eventually, the use of mass spectrometry spread to biology, geology and recently the clinical and medical area.

In recent years, this technique has been applied as a routine method in the microbiology lab‐ oratory, as a useful tool for the identification of microorganisms using the bacterial colony directly and proteins extracted from the microorganism.

Once obtained mass spectrum and it is compared with a database, software assigns identifi‐ cation and a reliable value of such identification.

MALDI-TOF has been the most used technique in the microbiological diagnosis for bacterial identification; some databases are used for identification profiles. This technique has been useful to obtain the profile of microorganisms for diagnosis using colonies directly from the culture media [67].

One advantage of the technique results from the culture obtained from a sample or sample directly, in this connection, trials have been performed to determine the functionality of the art regarding the identification, has been performed identification of bacteria and yeast us‐ ing MALDI-TOF, allows for quick low-cost diagnosis compared to conventional techniques as Vitek-II, API and biochemical tests, and it is known that these technologies are validated by comparing the technique by identifying the microorganisms from the samples at the spe‐ cies level and it must be matched [68].

Recently, the mass spectrometer has taken a major challenge to modernize and facilitate its use by the coupling of other techniques such as Vitek, an automated method of identifying microorganisms. In recent years several studies have been performed in routine clinical lab‐ oratory using this new technology: Vitek ® MS Biomeriuex.

This mass spectrometric technique is based on MALDI-TOF coupled to a Vitek-II and Myla, a database which receives the fingerprint of the sample from culture or sample directly and then identifies the organism. This technique has been used identifying fungi from clinical samples. Results from this study identifying 18 fungi with a quick and inexpensive strategy, since it does not require a prior extraction of proteins. These tests were perfomerd on clinical isolates from 20 patients, which were also evaluated using Vitek 2. Comparison between re‐ sults from Vitek-MS and Vitek 2 correlated in 93% [69].

Ferreira *et al.,* analyzed 294 facultative anaerobic and aerobic isolates obtained from differ‐ ent clinical samples, using conventional microbiological methods compared to conventional microbiological methods. In the analysis they concluded that bacterial clinical isolates iden‐ tification obtained by MS MALDI-TOF shows excellent correlation with identification ob‐ tained by conventional microbiological methods. Moreover, MS MALDI-TOF allows the identification of bacteria from colonies grown on agar culture plates in just a few minutes with a very simple methodology and hardly any consumable cost [70].
