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Advancements in MALDI-TOF MS for Quick
Identification of Microbial Pathogens: A
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Comprehensive Synopsis
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Copyright © 2024 PHD Assistance. No part of this document may be published without permission of the author
Abstract
Every year, plant diseases caused by plant pathogens significantly impair food yield, resulting in
large economic losses throughout the world. Accurate detection and Identification of plant pathogens
is critical for plant pathogen diagnostics and, therefore, plant disease management. Diagnostics and
disease management methods necessitate the simultaneous Problem Identification and
quantification of a diverse variety of pathogenic and nonpathogenic microorganisms. Over the last
decade, the fast development of matrix-assisted laser-desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS) methods for microbe characterization has resulted in much-
enhanced detection and Identification of microorganisms. MALDI-TOF MS is used in the biological
sciences to evaluate particular peptides or proteins immediately desorbed from intact bacteria, fungal
spores, nematodes, and other microorganisms. Taxonomic Identification of microorganisms by
MALDI-TOF MS is based on the capacity to record biomarker ions in a broad m/z range that are

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unique to and typical of particular bacteria. Recent advancements in mass spectrometry have
sparked increased interest in the study of increasingly complex microbial communities. Such

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research is in its early stages, but it has the potential to provide light not just on interactions between
microbes and their host plants but also on interactions between diverse microbial species living in
conjunction with plants. The mass spectrometry community has recently attempted to make data
from large-scale mass spectrometry studies publicly available in the form of a consolidated
repository. With such a resource, MALDITOF MS might be used as a universal approach for
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detecting plant pathogens and non-pathogens. The effects of experimental synopsis settings are well
established; repeatable spectra may be acquired using computerized database searches, and genus,
species, or strain can quickly identify microorganisms.

MALDI-TOF MS as an alternative to molecular approaches for the quick Identification of plant

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microbes
Pathogenic bacteria, fungi, nematodes, and viruses constitute a persistent hazard to plant
propagation material production. Reliable and appropriate technologies for screening numerous
pathogens are critical for implementing successful disease control programs. Thus, early detection
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and Identification of plant-associated microbes is an essential component of successful disease

control and is especially crucial in the context of foreign plant material importation. Rapid
Identification of plant-associated bacteria allows for the application of suitable management
measures prior to disease development or spread. Because most plant-based foods have a short
shelf life, it is critical that any potentially contaminated portion be identified as soon as feasible and
as accurately as possible to minimize delays and costly losses. Traditionally, the most common
methods for identifying plant pathogens depended on which are complex and needed a high level of
taxonomic expertise, professional training, and experience [1]. The overall identification time,
including traditional culture time (morphology of cell colonies and slide culture), is about 2-14 days,
depending on the varying growth rates of various species; this frequently delays illness management.
However, in recent years, several techniques for plant pathogen diagnosis have been introduced,
including the use of monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), which
have significantly increased the speed of in vivo detection of pathogenic antigens and DNA-based
techniques, such as polymerase chain reaction (PCR), which allow regions of the pathogen's
genome to be amplified several millionfold.

Copyright © 2024 PHD Assistance. No part of this document may be published without permission of the author
PCR, which was introduced in the mid-1980s, is currently utilized to study plant-microbe interactions
[3]. Its application in horticulture and agriculture is quickly growing, and the availability of such
diagnostic assays benefits a variety of fields. With many nations' borders opening and growing free-
trade agreements, fast testing for probable contamination with quarantine organisms is critical.
Furthermore, the need for quick and economical pathogenic detection tests is growing in order to
enable prompt application of control measures. However, certain requirements must be followed
before novel detection methods may be used in academic and scientific settings. These rules can be
separated into two categories: technical and economic requirements. While technical requirements
are necessary for the creation of any effective diagnostic procedure, economic factors are also
significant.

This review paper highlights the challenges and accomplishments in the development and
deployment of specialized MALDI-TOF MS in plant pathology. Figure 1 displays the MALDI-TOF MS

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methodologies used to characterize plant-associated bacteria. Fungi, bacteria, nematodes, viruses,
and virus-like organisms are the four basic types of plant-associated microorganisms. The

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Identification of plant-associated microorganisms (pathogens/non-pathogens) in the first three groups
using MALDI-TOF MS will be explored in this study.
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Fig. 1 Pictorial illustration of strategies for rapid characterization of plant-associated microorganisms

by MALDI-TOF MS and computational data processing (Sechul Chun et al. 2022)

MALDI-TOF mass spectrometry and plant microorganisms

1.Bacterial Identification using mass spectrometry

It has long been understood that studying plant diseases would be impossible without a
comprehensive definition of the microorganisms.As a result, tremendous effort has been expended
since its start to improve current techniques for microbial characterization continuously. MALDI-
TOF MS was developed two decades ago to detect biomarkers in target bacterial samples. MALDI-
TOF MS has been successfully applied to a wide range of microbial applications, including bacterial
RNA and DNA analysis, detection of recombinant proteins, characterization of unknown proteins,
bacterial proteomics, detection of virulence markers, and very fast bacterial characterization at the
genus, species, and strain levels.

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Bacteria have a diverse set of proteins that allow them to proliferate in the plant and effectively infect
it. Bacterial taxonomy may be divided into three parts:
a. characterization;
b. classification; and
c. nomenclature.

The usual strategy for microbiological Identification has been phenotypic characterization. However,
during the last 50 years, the emphasis on bacterial characterization has changed from phenotyping to
genotyping.

MALDI-TOF MS is increasingly being used in microbiology taxonomic difficulties, such as detecting

cryptic species in large batches of related isolates. However, it is currently not possible to determine
conclusive similarity values for mass spectral signals acquired from non-specific isolates, i.e., a
species-separating threshold. This is not totally unexpected, given that the same principles for
designating microbial species do not apply to all taxa. Furthermore, past biases have characterized

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live microorganisms using criteria that are not always congruent with contemporary ideas on how
bacterial species should be described.

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Extensive research has also been conducted to identify plant-beneficial bacteria and their application
in agriculture to lessen the impact of pathogens that cause a range of plant illnesses. Plant growth-
promoting rhizobacteria (PGPR) are a diverse collection of bacteria found in the plant rhizosphere that
contribute to enhanced yields of crops, vegetables, and other economically important plants. PGPR
stimulates beneficial activity that is responsible for the manufacture of phytohormones and volatile
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organic compounds that are accessible as plant nutrients, and there have been multiple findings that
imply a reduction of phytopathogenic soil bacteria, fungi, viruses, and nematodes.

For applications that do not need quick or real-time analysis, the use of chromatographic techniques
prior to mass analysis has also been considered. On-line and off-line chromatographic methods have
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been integrated with MS. Typical of this work is that published by Fox et al., who used gas
chromatography-MS to separate two closely related Bacillus spp., Bacillus anthracis and B. cereus,
which are difficult to distinguish phenotypically or genotypically. Cain et al. used chromatography (off-
line) and MALDI-TOF MS to differentiate bacteria based on protein fractions extracted from damaged
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cells. This research was significant for using proteins as biomarkers rather than smaller chemicals.

MALDI mass spectrometry-based bacterial analysis has matured in recent years, and numerous
excellent papers that clarify the many methodologies involved in bacterial characterization have been
authored using reported data. These biorecognition technologies can identify infections quickly and
accurately. Within minutes, nanoparticles are coupled with biomolecules such as antibodies,
antibiotics, adhesion molecules, and complementary DNA sequences for pathogen identification.
Device miniaturization has resulted in scientific laboratories on tiny chips that can detect diseases
using portable, hand-held biosensors. The nanoparticle's versatile chemistry, unusual optical
characteristics, and strong ferromagnetic responses have led to its employment as an efficient tool in
several biodetector systems.

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Fungus detection using mass spectrometry
Until the early 1990s, most biological research was focused on in vitro examinations of specific
components, such as genes and proteins. In the early and mid-1990s, this method switched to in vivo
and molecular large-scale research, beginning with structural genomics and transcriptomics studies,
then moving on to proteomics and, more recently, metabolomics. All of these represent the
methodological foundations of current systems biology [31]. Because no one technique can explain
the complexity of living organisms, each approach that contributes must be validated and recognized
as part of an interdisciplinary, integrative study at several levels, ranging from the gene to the
phenotype via proteins and metabolites. Proteomics science is constantly being renewed, and in the
last ten years, there has been a massive increase in the number of new strategies and procedures, as
well as techniques, with continuous improvements made at all stages of the procedure, beginning in
the laboratory (tissue and cell fractionation, protein extraction, depletion, purification, separation, mass
spectrometric analysis) and ending at the computer (algorithms for protein recognition and

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bioinformatics tools for data analysis).

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Wheat leaf rust, caused by the fungus Puccinia triticina, has also been studied using proteomic
analysis [37]. Rust infections reduce cereal crop yields by a large amount each year [38]. The
proteomes of both the host and the pathogen were examined during illness progression to gain a
better understanding of the problem at the molecular level. Using 2DE (with isoelectric focusing, pH 4-
8) and mass spectrometric analysis, a susceptible line of wheat infected with a virulent race of leaf rust
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was compared to mock-inoculated wheat [37]. Schmidt and Kallow used MALDI-TOF MS to identify
the closely related indoor wood-decay fungus Serpula lacrymans, S. himantioides, Coniophora
puteana, C. marmorata, Antrodia vaillantii, and A. sinuosa [39].

Identification of plant-parasitic nematodes using mass spectrometry

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Plant parasitic nematodes, which are also hidden enemies of plants, frequently cause symptoms that
are readily misunderstood. These microscopic creatures are cropping system pests rather than single-
crop pests, especially in tropical locations where cropping intensity is high, and circumstances support
the development of a large nematode population. Parasitic nematodes can significantly impair crop
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development and output in a variety of commercially important crops [42]. Endoparasitic root
nematodes may cause massive crop output losses and have thus been widely investigated. Most
economically important plants are vulnerable to attack by one or more plant-parasitic nematode
species, and their interactions with the plant host can range from transitory grazing by root hair
feeders to the highly complex host-pathogen interactions of endoparasites and gall-inducing
nematodes.

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Morphologically, nematodes are quite similar. Caenorhabditis elegans and Caenorhabditis briggsae
are two free-living worms whose Identification would be difficult for most skilled nematode
taxonomists. Despite their physical similarities, a recent analysis of 338 orthologous genes
(homologous genes generated from the same gene in the last common ancestor) revealed that the
two species diverged 80-110 million years ago [49]. This breaking happened 5-45 million years
before the mouse and human lineages separated [50]. While most of us are comfortable making the
man-mouse distinction, this example sends a strong warning to those who must identify nematode
species. Clearly, cryptic species must exist in the phylum Nematoda, and molecular methods may be
the only way to identify them. Protein, lipid, carbohydrate, and DNA analysis, as well as molecular
techniques such as protein electrophoresis, restriction fragment length polymorphism (RFLP),
amplified fragment length polymorphism (AFLP), dot blot assays, and variation in internally
transcribed spacer (ITS) sequences detected by polymerase chain reaction, have all been used to
identify nematodes [51, 52].

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Mass spectrometric imaging

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Mass spectrometry advancements have resulted in new exploratory investigations of molecular
interactions in intact tissue, similar to studies undertaken decades ago using intact tissue for
metabolic research, but now with exact molecular specificity. MALDI mass spectrometric imaging
(MALDI-TOF MSI), in particular, allows the researcher to examine the spatial distribution of sugars,
metabolites, and lipids in plant tissues. The capacity to monitor many substances within a cell or
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tissue at the same time is very appealing. MALDI-TOF MSI has the benefit of not requiring the
creation of an antibody linked with a fluorescent or transmission electron microscopy probe, as well
as the fact that MALDI-MS equipment is widely used. The MALDI-TOF MSI technique has been
utilized effectively in plant biology.

Chemical approach before MALDI-TOF MS

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Analytical approach
Several analytical procedures have been employed to identify bacteria in a variety of biological
materials [16, 67]. TEM, MALDI-TOF MS, combined nanoparticles/luminescence-based assays
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[68], and magnetic bead-based immunological assays [69] are among these approaches.
Regardless of the detection approach, the nanoparticle must successfully and specifically target a
specific bacterium (or, more generally, a biomolecule). This is only possible if its surface is
adequately functionalized with substrates capable of selectively and firmly engaging with surface
groups on the biomolecule of interest. Antibody-modified nanoparticles have been effectively
employed in microbial cell and biomolecule labelling investigations in combination with MALDI-TOF
MS [16].

MALDI matrices approach

The mechanism of ion generation in the process of desorption/ionization under the action of laser
pulses has been systematically summarized [11]. Thus, the success of MALDI measurement is
partly empirical and remains highly dependent on the type of matrix and appropriate target sample
preparation prior to MALDI-TOF MS analysis. In MALDI-MS analysis, matrix selection is critical. The
following are the characteristics of an ideal matrix:

Copyright © 2024 PHD Assistance. No part of this document may be published without permission of the author
 1. absorption at the laser wavelength
2. ability to dissolve or co-crystallize with the sample
3. low volatility
4. ability to suppress analyte decomposition and
5. ability to promote ionization of the analytes.

A matrix can be either solid or liquid. In MALDI analysis, the most widely employed solid matrices are
derivatives of weak organic acids, such as -cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA),
and 2,5-dihydroxybenzoic acid (2,5-DHB). The use of matrix additives is a straightforward technique to
improve the quality of MALDI spectra for certain data analytics, either by suppressing sodium adducts
or boosting analytical sensitivity and resolution. Armstrong et al. [75] were the first to employ ionic
liquids (ILs) as matrices successfully. Because of their unique features (low volatility and excellent
solvents for a wide range of compounds), ILs are well suited for use as MALDI matrices. A second

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generation of IL matrix for MALDI-TOF MS has recently been created [76].

Conclusion

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A topic of great interest that impacts humanity and has major ramifications for agricultural plants,
resulting in plant illnesses, has been evaluated using MALDI-TOF MS. The results and advancements
made using MALDI-TOF MS for detecting bacterial, fungal, and viral pathogens have been described.
The reasons for the poor interest in MALDI technology deployment for precise, quick detection of plant
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diseases have been examined.

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