The Use of Mass Spectrometry Technology (MALDI-TOF) in Clinical Microbiology

By Fernando Cobo

The Use of Mass Spectrometry Technology (MALDI-TOF) in Clinical Microbiology presents the state-of the-art for MALDI-TOF mass spectrometry. It is a key reference defining how MALDI-TOF mass spectrometry is used in clinical settings as a diagnostic tool of microbial identification and characterization that is based on the detection of a mass of molecules. The book provides updated applications of MALDI-TOF techniques in clinical microbiology, presenting the latest information available on a technology that is now used for rapid microbial identification at relatively low cost, thus offering an alternative to conventional laboratory diagnosis and proteomic identification systems.

Although the main use of the technology has, until now, been identification or typing of bacteria from a positive culture, applications in the field of virology, mycology, microbacteriology and resistances are opening up new opportunities.

• Presents updated applications of MALDI-TOF techniques in clinical microbiology
• Describes the use of mass spectrometry in the lab, the principles of the technology, preparation of samples, device calibration and maintenance, treatment of microorganisms, and quality control
• Presents key information for researchers, including possible uses of the technology, differences between devices, how to interpret results, and future applications
• Covers the topic in a systematic and comprehensive manner that is useful to both clinicians and researchers

• Language: English
• Publisher: Academic Press
• Release date: Aug 3, 2018
• ISBN: 9780128144527

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Preface

Fernando Cobo

Matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) mass spectrometry (MS) is a diagnostic tool for microbial identification and characterization based on the detection of the mass of molecules. The first description of the use of MS technology for bacterial identification was in 1975, but it took a long time for the introduction of this technique in routine microbiology. The introduction of MALDI-TOF MS in microbiological practice has supposed a great revolution due to the enormous benefits gained with the new technology. The main impact of MALDI-TOF MS in clinical diagnosis has been the rapidity in microbial identification at a relatively low cost. On the other hand, direct impact on health care and economic advantages are other characteristics that make this technique relevant in the field.

Currently, the main use of this technology in clinical microbiology is the identification or typing of bacteria from positive cultures. However, the spectrum of microorganisms which were investigated using MALDI-TOF MS permanently increased. Applications in the field of clinical virology, mycology, and microbacteriology, and the study of bacterial resistances have also been developed and are opening up new options of microbiological diagnosis.

Regarding bacteriology, MALDI-TOF MS is widely accepted as the main laboratory method for microorganism identification in the majority of laboratories, and it is possible that in the next years this technique will become the standard diagnostic tool in other microbiological areas.

In this book, experts in the field have contributed with their chapters based on their own experience from different aspects of this exciting new technology. The book is divided into 17 chapters covering all aspects of this new method. Chapters 1 and 2, Proteomics: Technology and Applications, and Basis on Mass Spectrometry: Technical Variants, respectively, involve some generalities about proteomic and the basis on MS. Chapter 3, MALDI-TOF Commercial Platforms for Microbial Identification, discusses the main commercialized MALDI-TOF MS platforms for microbial identification, and Chapters 4–6, Work Procedures in MALDI-TOF Technology; Indications, Interpretation of Results, Advantages, Disadvantages, and Limitations of MALDI-TOF; Quality Control in MALDI-TOF Techniques, respectively, cover the main issues about work procedures in MALDI-TOF MS technology, indications and interpretation of results and quality control. Chapters 7–16, Application of MALDI-TOF for Bacterial Identification; Detection of Bacterial Resistance, Biomarkers, and Virulence Factors Through MALDI-TOF Technology; Direct Identification of Pathogens From Positive Blood Cultures by MALDI-TOF Technology; Use of MALDI-TOF Techniques in the Diagnosis of Urinary Tract Pathogens; Direct Application of MALDI-TOF Mass Spectrometry to Cerebrospinal Fluid for Pathogen Identification; Application of MALDI-TOF in Clinical Virology; Use of MALDI-TOF Mass Spectrometry in Microbacterial Diagnosis; Use of MALDI-TOF Mass Spectrometry in Fungal Diagnosis; Application of MALDI-TOF in Bacterial Strain Typing and Taxonomy; Application of MALDI-TOF in Parasitology, respectively, focus on the main applications of MS technology in clinical microbiology, such as bacteria, fungi, microbacterial identification, as well as the identification of pathogens with this technique from some body fluids. Finally, Chapter 17, Future Applications of MALDI-TOF Mass Spectrometry in Clinical Microbiology, is a short review about the future applications of MALDI-TOF MS in microbiological diagnosis.

I expect that this modest contribution may help the readers to approach this exciting technology which has revolutionized the microbiological diagnosis.

I would like to acknowledge and express my sincere thanks to all contributors and colleagues from Elsevier; without their work and support the publication of this book would have been impossible.

Chapter One

Proteomics

Technology and Applications

Raquel Nancy Ballesté,    Clinical Laboratory Department, Hospital de Clinicas, University of the Republic, Montevideo, Uruguay

Abstract

Proteomics is a recent discipline that has exponentially gained interest in last years. It constitutes the large-scale study of proteins and is aimed to analyze, identify, and characterize the cellular proteome.

The most used techniques for the study of the proteome include spectrophotometric and chromatographic systems, mass spectrometry, protein microarrays and bioinformatics, together with them, the development of new technological platforms allows the identification and sequencing of proteins with unprecedented levels of speed, sensitivity, and specificity.

Proteomic studies have acquired a predominant role in different applications in human pathology; among them, the area of clinical microbiology with bacterial identification has revolutionized the microbiological diagnosis in the clinical laboratory, and alongside this, the development of biomarkers in oncological diseases.

Proteomics has an enormous potential in the permanent development of new tools applicable in human pathology.

Keywords

Proteomics; mass spectrometry; clinical applications of proteomics; bioinformatics in proteomics

1.1 Introduction

Proteins are complex organic molecules, formed by amino acids arranged in long rows or polypeptide chains maintained by peptide bonds. They are vital parts of all living organisms, constituting the main components of the metabolic pathways of cells, being essential for cellular functioning and, therefore, for life [1].

Proteins have an enormous structural heterogeneity, many of which are enzymes that catalyze different chemical reactions vital for cell metabolism; others have a structural role, as cellular cytoskeleton proteins that maintain cell structure and form are fundamental in cell communication, signal transduction pathway, in the immune response, in the maintenance of cellular homeostasis, and in the cell cycle, among others.

The term proteomics was referred to in 1997 as an analogy with genomics (the study of genes). The word proteome is the fusion of protein and genome, and was taken by Marc Wilkins in 1994. The proteome is the complete complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or system [2,3].

The proteome of an organism is a highly dynamic element, because its components vary depending on the tissue, cell, or cell compartment studied, and these, in turn, can change due to alterations in their environment, such as stress situations, action of drugs, energy requirements, or their physiological state (normal or pathological). Therefore, the description of the proteome allows to have a dynamic image of all the expressed proteins, at a given moment and under certain concrete conditions of time and environment [4].

The systematic study and comparison of the proteome in different metabolic and/or pathological situations allows to identify those proteins whose presence, absence, or alteration correlates with certain physiological states.

The fact that the same gene can give rise to different protein forms and these, in turn, can interact with other proteins forming protein complexes, or that the proteins present different posttranslational modifications giving rise to diverse molecular forms that can be present simultaneously, makes the proteome represent a level of complexity superior to that of the genome and that the analysis of the proteome is an even more challenging task. Unlike the genome, the proteome of an organism is dynamic, with spatiotemporal changes throughout its life cycle [5].

The Human Proteome Project has generated a map of the molecular architecture based on proteins and peptides of the human body, which is crucial to help determine the biological and molecular functions, as well as advance in the development of new applications for diagnosis, treatment, and monitoring of different pathologies [6].

1.2 Definition and Importance of the Proteomics

Proteomics is the large-scale study of proteins, in particular, their expression, structure, and function. Proteomics, goes beyond the mere cataloging of proteins, trying to establish, ultimately, its structure, biological activity, mode of action, cellular localization, posttranslational modifications, and interaction with other proteins or molecules [4,5].

It can also be defined as the set of techniques or technologies aimed at obtaining functional information of all proteins and is aimed at the analysis, identification, and characterization of the cellular proteome. Proteomic studies have acquired a predominant role in different applications in human pathology in recent years.

Proteomics offers highly complementary information to genomics; if we take into account that most of the biological functions are carried out by proteins, proteomics offers a new and different view of the disease. The basic difference between genomics and proteomics is that while the first one focuses in the study of the entire genetic heritage of an organism (both nuclear and extranuclear, coding or noncoding), the second focuses on the parts of the genome that are translated into proteins. These two disciplines also differ in their nature: while the genome remains relatively static, the proteome is dynamic [3,4]. The set of proteins that are expressed not only varies from one cell to another but also depends on the interactions between the genome and the environment at a specific time, so that any genome can potentially give rise to an infinite number of proteomes [4].

Currently you can find several areas of development in proteomics [4,7]:

1. Functional proteomics: study and characterization of a specific group of proteins.

2. Proteomics of expression: quantitative study of protein expression patterns between samples that differ in some variable.

3. Structural proteomics or cellular map: refers to the study of the subcellular localization of proteins and protein–protein interactions.

Despite its complexity, the rapid and remarkable development in recent years has led to its application in different pathologies or clinical proteomics, which deals with the systematic and exhaustive identification of protein patterns of disease and the application of those data to patients (study of the disease, susceptibility, prevention, selection of therapies, monitoring of treatments, etc.).

Through clinical proteomics, protein patterns can be defined to generate information of clinical value about the susceptibility, diagnosis, prognosis, and therapy of a certain disease. For this to have a real impact on health, protein patterns should be identified and selected, validated in population studies, and extrapolated to clinical practice. It is also possible to identify specific proteins associated with specific pathologies, which allow to diagnose the pathology in question or predict its evolution; these proteins are known by the generic name of biomarkers [8].

Proteomics is currently a priority line of research in the field of biology, with the growth in the number of research projects aimed at the study of proteomes in a systematic way, which has led to the emergence of new technologies on a large scale.

1.3 Technical Methods in Proteomics

The same characteristics that give proteins their fundamental role as effector molecules of cellular function (structural and functional heterogeneity, chemical diversity, among others) also hinder their experimental analysis.

Presently there is no unique flowchart for the proteomic analysis of a sample. This is because variables, such as complexity, protein separation method, protein concentration, and stability added to the technological platform available for analysis and especially to the type of biological question intended to answer, are the basic parameters that determine the choice of a study strategy or another. There is therefore no single methodology or gold standard for the study of proteomes; consequently, proteomics research is the result of the application of a set of diverse techniques that allow the study of proteins.

The methodology for the proteomic study of a sample consists essentially of the following stages [4]:

1. Isolation and separation of proteins,

2. Analysis of the structure of the separated proteins,

3. Use of computer databases to identify the characterized proteins.

1.3.1 Isolation and separation of proteins

Biological samples are a complex mixture of proteins; usually these samples from tissues or biological fluids (blood, urine, cephalo-spinal fluid, saliva, etc.) are separated mainly by chromatographic and/or electrophoretic techniques, which are robust, versatile technologies with high capacity resolution [9,10].

The most used are spectrophotometric and chromatographic systems. Electrophoresis on one-dimensional (SDS-PAGE) and two-dimensional (2-D PAGE) polyacrylamide gels is based on a separation of the proteins in function of the charge followed by a separation based on their molecular weight, capillary electrophoresis, high-performance liquid chromatography (HPLC), affinity chromatography, and ion chromatography exchange [10,11].

The types of HPLC normally used are those of normal phase, reverse phase, molecular exclusion, ion exchange, and based on bioaffinity. Normal phase, reverse phase and ion exchange HPLC separate by polarity; the one of molecular exclusion by size; and those of bioaffinity due to the different capacity of biologically active substances to form stable, specific and reversible complexes.

These isolation techniques can be used separately, altogether or in continuous flow systems, such as the multidimensional protein isolation systems technology which contains reversed phase and ion exchange in a conjugated form. Besides, another recent development in HPLC is the operation of systems of micro-HPLC and HPLC for nano-HPLC (with nano flow), presenting very high sensitivity. The use of nano-HPLC allows the separation of peptides before placing them towards the source of ionization as well as the removal of small amounts of pollutants that interfere with the analysis [10,11].

The liquid chromatography (LC) is a physical method of separation based on the distribution of the components of a mixture between two phases: a fixed or stationary and another mobile. The technology of liquid chromatography coupled mass spectrometry (LC-MS) is used for the separation of peptides of synthetic peptides, native peptides, or enzymatic proteolysis. This technology allows the separation of complex mixtures of peptides and their simultaneous analysis by mass spectrometry (MS). At present, the LC-MS system is one of the most powerful tools of the proteomic analysis based on MS.

1.3.2 Analysis of the structure of the separated proteins

Producing ions in gas phase is relatively easy for volatile compounds of low molecular weight; however, the application of MS in the field of proteins is relatively recent. This is due to the difficulty of obtaining ions of macromolecules in gas phase, because the components and proteins are compounds of low volatility and high molecular weight; therefore, the use of special ionization techniques is necessary.

The identification of proteins is performed in its vast majority by means of mass spectrometers (MSs). These instruments allow obtaining ions from organic molecules, separating and detecting them according to their mass/charge. MSs consist of three components: ionization source, mass/charge analyzer, and detector [11,12].

There are different types, the most frequently used are:

MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight): This is commonly used for the identification of proteins by peptide fingerprint and the study of the protein profiles of a sample and protein interactions. It is worth highlighting the development of the imaging technique, which allows a 2D visualization of the distribution of trace metals, metabolites, surface lipids, peptides, and proteins, directly in the tissues without a prior fixation or extraction [13,14].

SELDI-TOF (Surface Enhanced Laser Desorption Ionization–Time of Flight: desorption): This instrument uses the same technology as the MALDI-TOF, incorporating the function ability of the plate (ion exchange, reverse phase, antibodies, DNA, other proteins, etc.) in which the sample is loaded. The comparison of the proteins retained in the plate among different samples can be used to search for biomarkers. This technique is an adaptation for the clinical proteomics of fluids [15].

ESI (Electro Spray Ionization): This equipment is especially useful for peptide sequencing, because it allows the ionization of macromolecules fragmented by peptide bonds (weaker bonds), thus obtaining the amino acid sequence [16].

The proteins after separation can be analyzed by MS according to different treatments. Commonly, proteins are cut out from the 2D gel where they have been isolated, digested with a protease to produce a set of peptides, and these peptides are subsequently analyzed by MS MALDI-TOF type. This technique is particularly useful for obtaining the mass spectrum of the set of peptides, known as peptide finger-printing.

The peptides produced after a contaminant removal process are analyzed by ESI MS in combination with a triple Q (Quadrupole) or an ION TRAP, allowing the real-time study of individual peptides present in the mixture, without the need to separate them from the rest, also called as fragmentation spectrum or tandem mass spectrometry (MS/MS) [17,18].

The peptide fingerprints are characteristic of each of the proteins that allow identifying one by one in the database using bioinformatic techniques. In the same way, the MS/MS spectra are also characteristic of each of the peptides; however, none of the two techniques (peptide fingerprint or fragmentation) is of universal applicability.

In the MS, new devices are beginning to be used, which are based mainly on the hybrid combination of already known techniques. For example, devices based on the combination of a double Q and a TOF (qQ-TOF) significantly improve the resolution of the fragmentation spectra and the combination of the MALDI ionization with the qQ-TOF analyzer, or the IONTRAP allows fragmenting peptides directly once obtained from the peptide fingerprint [19,20].

The MSs called MALDI-TOF/TOF combines all the advantages of conventional MALDI-TOF spectrometry with the ability to produce peptide fragmentation spectra quickly and consistently.

There are also MSs based on the coupling of a 2D LC system with an ION TRAP or qQ-TOF analyzer. This procedure, capable of identifying thousands of peptides in a single experiment, allows analyzing automatically the components of very complex peptide mixtures. This technique (also called shotgun-proteomics) has been applied to the direct identification of whole proteomes without the separation of their components by electrophoresis [21].

Another variant of this methodology allows studies of differential expression of proteins in the proteome. This technique, known as isotope coded affinity tag, is based on the use of protein reagents in the form of stable isotopes, which allow to determine the relative proportion of the peptides derived from the proteomes to be compared. This procedure through specific software allows the automatic and simultaneous identification of relative changes in protein concentrations and the nature of proteins that undergo differential expression.

Finally, these methodologies, together with affinity selective purification procedures, allow the analysis of modified peptides, such as phosphopeptides, from whole proteomes, which has given rise to the concept of phosphoproteome.

The Array or Protein Biochips allows the detection, characterization, and quantification of protein, as well as the study of the functional qualities of proteins and their interactions, both between them and with molecules of DNA or lipids. Unlike the nucleic acids, the proteins have neither homogeneous structure nor specific union pattern, but every protein possesses a few particular biochemical characteristics; therefore, the development of the microarrays of proteins is still before technical difficulties [22,23].

1.3.3 Utilization of computer databases to identify the characterized proteins

The interpretation of mass spectrum is the most difficult part of this methodology, as it involves the knowledge of the chemical and biochemical processes applied to protein samples, with the knowledge of bioinformatics for the search and identification of proteins in database banks, mainly because it is based on the correlation of values and arithmetic differences of atomic mass units with the molecular structure of proteins [24,25].

Proteomic analyses generate a large amount of data to analyze, which are assisted by softwares that face the spectra obtained from different databases [24]. The development of bioinformatic tools, such as the introduction of new algorithms, has been key to manipulate these collections of data. This field allows the manipulation of data on a large scale, developing programs and searching tools. These tools have been applied with great success in MS data processing in peptide fingerprint analysis (protein identification), peptide fragmentation fingerprint (identification of the peptide sequence), and de novo sequencing [24,25,26].

1.4 Proteomics’ Role in the Clinical Laboratory

A few years ago the molecular technique and the proteomics technique were only obtained in complex laboratories. Recently we are witnessing a generalization in the use of both techniques begging to generalize in the field of diagnosis and being available in laboratories for daily practice. This, on the one hand, is a great advance for clinical laboratories, on the other hand, an enormous responsibility in the selection and correct application of mentioned methodologies. Clinical laboratories have a fundamental role in the incorporation of technology, the selection of equipment, the technique(s) to be implemented, the application of these techniques in different pathological processes, the verification and validation of the selected method, the control of quality of the preanalytical, analytical, and postanalytical processes, and the cost–benefit evaluation of the incorporation of the new