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Application and Perspectives of MALDI–TOF Mass Spectrometry in Clinical Microbiology Laboratories

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Application and Perspectives of MALDI–TOF Mass Spectrometry in Clinical Microbiology Laboratories microorganisms Review Application and Perspectives of MALDI–TOF Mass Spectrometry in Clinical Microbiology Laboratories Eva Torres-Sangiao 1,2,* , Cristina Leal Rodriguez 3 and Carlos García-Riestra 1,4 1 Escherichia coli Group, Foundation Institute of Health Research—University Hospital Complex of Santiago de Compostela (FIDIS—CHUS), ES-15706 Santiago de Compostela, Spain; [email protected] 2 Clinical Microbiology and Parasitology Lab, University Hospital Marqués de Valdecilla (HUMV), ES-39008 Santander, Spain 3 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark; [email protected] 4 Clinical Microbiology and Parasitology Lab, University Hospital Complex of Santiago de Compostela (CHUS), ES-15706 Santiago de Compostela, Spain * Correspondence: [email protected] Abstract: Early diagnosis of severe infections requires of a rapid and reliable diagnosis to initiate appropriate treatment, while avoiding unnecessary antimicrobial use and reducing associated mor- bidities and healthcare costs. It is a fact that conventional methods usually require more than 24–48 h to culture and profile bacterial species. Mass spectrometry (MS) is an analytical technique that has emerged as a powerful tool in clinical microbiology for identifying peptides and proteins, which makes it a promising tool for microbial identification. Matrix assisted laser desorption ionization– time of flight MS (MALDI–TOF MS) offers a cost- and time-effective alternative to conventional Citation: Torres-Sangiao, E.; Leal methods, such as bacterial culture and even 16S rRNA gene sequencing, for identifying viruses, bac- Rodriguez, C.; García-Riestra, C. teria and fungi and detecting virulence factors and mechanisms of resistance. This review provides Application and Perspectives of an overview of the potential applications and perspectives of MS in clinical microbiology laboratories MALDI–TOF Mass Spectrometry in and proposes its use as a first-line method for microbial identification and diagnosis. Clinical Microbiology Laboratories. Microorganisms 2021, 9, 1539. Keywords: MALDI–TOF MS; microbial identification; proteomics; resistome; disease biomarkers https://doi.org/10.3390/ microorganisms9071539 Academic Editor: Juan Carlos Rodríguez 1. Background Mass spectrometry (MS) was originally developed at the end of the 19th century to Received: 2 June 2021 measure the masses of atoms, and one of its first contributions to science was demonstrat- Accepted: 18 July 2021 ing the existence of isotopes, at the beginning of the 20th century [1]. MS is an analytical Published: 20 July 2021 approach that measures the mass-to-charge ratio (m/z) of chemical compounds and calcu- lates its exact molecular weight. Laser desorption/ionization (LDI), matrix-assisted laser Publisher’s Note: MDPI stays neutral desorption ionization (MALDI) or surface-enhanced laser desorption/ionization (SELDI) with regard to jurisdictional claims in as well as electrospray ionization (ESI) are currently the most widely used ionization published maps and institutional affil- techniques for analyzing chemical structures in biological systems [2]. In the late 1980s, iations. with the introduction of soft ionization, protein analysis developed rapidly, revolutionizing MS. In the late 1990s, the pioneering application of MS in microbiology [1] demonstrated that intact bacterial cells could be distinguished using MALDI coupled to a time of flight (TOF) analyser [3]. These achievements stimulated the fast development of MALDI–TOF Copyright: © 2021 by the authors. MS system approaches as promising tools for the microbial characterization of bacteria [4], Licensee MDPI, Basel, Switzerland. fungi [5], viruses [6], and even nematodes [7]. This article is an open access article The MALDI–TOF MS system performs different proteomic strategies using intact or distributed under the terms and digested proteins. The “top down strategy” is used for direct analysis of intact proteins, conditions of the Creative Commons proteoforms and post-translational protein modifications [8], whereas the “bottom up” is Attribution (CC BY) license (https:// used for mixtures of peptides derived from protein digestion (i.e., peptide sequencing). creativecommons.org/licenses/by/ MALDI–TOF MS systems represent a basic configuration workflow in a linear mode. 4.0/). Microorganisms 2021, 9, 1539. https://doi.org/10.3390/microorganisms9071539 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, x FOR PEER REVIEW 2 of 20 Microorganisms 2021, 9, 1539 2 of 19 MALDI–TOF MS systems represent a basic configuration workflow in a linear mode. Figure 1 presents a schematic overview divided into three compartments: (1) the Figure1 presents a schematic overview divided into three compartments: (1) the ionization ionization source system (laser), (2) the mass analyser (TOF), and (3) the ion detector. First, source system (laser), (2) the mass analyser (TOF), and (3) the ion detector. First, the the sample for analysis is prepared by mixing with a matrix, an energy-absorbent, organic sample for analysis is prepared by mixing with a matrix, an energy-absorbent, organic compoundcompound solution. solution. Then, after Then, the after mixture the mixture (matrix (matrix and sample) and sample) crystallize crystallize upon drying, upon drying, the samplethe sample is ionized is ionized using a laser using beam. a laser In beam.the process In the of processdesorption of desorptionand ionization and using ionization the ionizationusing the source ionization system, source the system, molecule thes molecules are converted are converted to gas-phase to gas-phase ions and ions and individuallyindividually charged charged [M+H]+, [M+H]+, so that so they that ca theyn be can manipulated be manipulated by external by external electric electric and and magneticmagnetic fields. fields.MALDI MALDI is based is basedon a soft on ionization a soft ionization method method that preserves that preserves the integrity the integrity of the ofsample the sample without without massive massive fragmentation fragmentation [9]. Soft [9]. ionization Soft ionization allows allows the analysis the analysis of of proteinsproteins and peptides and peptides and large and organic large organic molecules molecules (i.e., polymers, (i.e., polymers, dendrimers) dendrimers) [10], which [10], which tend totend become to become brittle brittleand fragmented and fragmented in other in otherionization ionization methods. methods. Once Oncethe sample the sample moleculesmolecules are ionized, are ionized, the ions the are ions arranged are arranged and separated and separated based basedon their on m their/z ratiom/z usingratio using a massa analyser. mass analyser. For microbiological For microbiological applications applications primarily, primarily, the TOF the mass TOF analyser mass analyser is is used. Last,used. an Last, ion andetection ion detection system system (detector) (detector) measures measures the different the different separated separated ions and ions and createscreates a mass a spectrum, mass spectrum, characterized characterized by m/z by ratiosm/z alongratios with along their with relative their relative abundance. abundance. As a result,As a result, the mass the spectrum mass spectrum represents represents individual individual protein protein profiles profiles (likewise (likewise a peptide a peptide mass fingerprintsmass fingerprints (PMF)) (PMF)) where where different different peaks peakscorrespond correspond to different to different m/z ratiosm/z ratiosthat can that can then bethen searched be searched for protein for protein mass values mass values against against a database a database contai containingning known known microbial microbial isolatesisolates [11]. Therefore, [11]. Therefore, the MALDI–TOF the MALDI–TOF system system identifies identifies microorganisms microorganisms using usingMS to MS to determinedetermine a spectrum a spectrum unique unique for each for protein. each protein. Figure Figure1. Schema 1. Schema showing showing the linear the mode linear work modeflow workflow in a MALDI–TOF in a MALDI–TOF MS system. MS system. Microbial identification through MALDI–TOF MS presents multiple advantages. First, Microbial identification through MALDI–TOF MS presents multiple advantages. it is based on intact proteins, which avoids time-consuming digestions, desalting and other First, it is based on intact proteins, which avoids time-consuming digestions, desalting pre-treatments such as solid phase extraction while keeping good sensibility. Second, it has and other pre-treatments such as solid phase extraction while keeping good sensibility. high transmission efficiency, fast scan rates and very low m/z ratio detection limits [9,12]. Second, it has high transmission efficiency, fast scan rates and very low m/z ratio detection Third, its analysis is low cost, despite the initial investment in expensive equipment [13]. limits [9,12]. Third, its analysis is low cost, despite the initial investment in expensive Because of these characteristics, MALDI–TOF MS has revolutionized clinical diagnosis, equipmentrepresenting [13]. Because a fast andof these an effective characteri methodstics, MALDI–TOF for the identification MS has of revolutionized bacterial profiles at clinicalboth, diagnosis, the genus representing and species a fast taxonomic and an levels.effective This method new approachfor the identification is being establishing of bacterialas
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