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MALDI-TOF Mass Spectrometry in Clinical Analysis and Research

Dandan Li, Jia Yi, Guobin Han, and Liang Qiao*

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ABSTRACT: In the decade after being awarded the Nobel Prize in

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Chemistry in 2002, matrix-assisted laser desorption/ionization time-of-

flight mass spectrometry (MALDI-TOF MS) has been widely used as
an analytical chemistry tool for the detection of large and small
molecules (e.g., polymers, proteins, peptides, nucleic acids, amino acids,
Downloaded via 156.213.193.241 on May 24, 2023 at 18:48:02 (UTC).

lipids, etc.) and for clinical analysis and research (e.g., pathogen
identification, genetic disorders screening, cancer diagnosis, etc.). In
view of the fast development of MALDI-TOF MS in clinical usage, this
review systematically summarizes the most important applications of
MALDI-TOF MS in clinical analysis and research by analyzing MALDI
TOF MS-related reviews collected in the Web of Science database. On
the basis of the analysis of keyword co-occurrence of over 2000 review
articles, four themes consisting of “pathogen identification”, “disease
diagnosis”, “nucleic acids analysis”, and “small molecules analysis” were found. For each theme, the review further outlined their
application implications, analytical methods, and systems as well as limitations that need to be addressed. Overall, the review
summarizes and elaborates on the clinical applications of MALDI-TOF MS, providing a comprehensive picture for researchers
embarking on MALDI TOF MS-related clinical analysis and research.
KEYWORDS: matrix-assisted laser desorption/ionization (MALDI), time-of-flight (TOF), mass spectrometry (MS), clinical application,
pathogen identification, disease diagnosis

1. BACKGROUND diseases. As technology matures, MALDI-TOF MS-based

In 1985, the concept of matrix-assisted laser desorption/ systems, e.g., the MALDI Biotyper by Bruker, the VitekMS
ionization (MALDI) mass spectrometry (MS) was originally by bioMérieux Clinical Diagnostics, the MassARRAY System
by Agena Bioscience, and the Clin-TOF from Bioyong
proposed by Karas et al.1 They adopted organic molecules as
Technology, have been registered as medical devices for
matrices to assist the desorption/ionization of molecules under
clinical uses.
UV laser irradiation. In 1988, mass analyzer time-of-flight
In recent years, a considerable number of review articles
(TOF) was coupled to MALDI by Koichi Tanaka and
have been published to summarize MALDI-TOF MS clinical
colleagues for the analyses of macromolecules, especially
applications. On the basis of a search of the Web of Science
proteins.2 After that, MALDI-TOF MS was developed for
database, reviews with MALDI-TOF as a theme in the last five
generating and analyzing ions from a variety of molecules,
years (2018−2022) mostly focused on the applications of
particularly large, nonvolatile, and thermally labile compounds
microbiology such as microbial species identification,3,4
such as proteins, polymers, and oligonucleotides, which
antimicrobial susceptibility testing,5,6 and bloodstream in-
provides the basis for the use of MS in biomedical research.
fection diagnosis.7 Emphasis is often placed on specific
In the decade since being awarded the Nobel Prize in
clinically relevant application scenarios that are accompanied
Chemistry in 2002, MALDI-TOF MS has been largely
by in-depth discussions with very few reviews covering
developed for proteomics/metabolomics studies through the different clinical application directions. This review incorpo-
qualitative and quantitative analysis of proteins/metabolites. rates several clinically relevant applications of MALDI-TOF
Modern life sciences have been greatly facilitated by the
MALDI-TOF MS technology. Due to the advantages including
easy operation, high throughput, and high tolerance to Received: March 31, 2022
contamination (i.e., salts, buffers, detergents), MALDI-TOF Revised: July 15, 2022
MS-based instruments have been widely used in many clinical Accepted: July 15, 2022
scenarios to help clinical specialists make medical decisions. Published: July 27, 2022
For instance, MALDI-TOF has been developed for the
diagnosis of bloodstream infections and neurodegenerative
© 2022 The Authors. Published by
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385 ACS Meas. Sci. Au 2022, 2, 385−404
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Figure 1. MALDI-TOF mass spectrometer. Target plate is placed in the vacuum chamber of a mass spectrometer. Spots to be analyzed are shot by
a laser to desorb and ionize the sample and matrix molecules from the target plate. Cloud of ionized molecules is accelerated into the TOF mass
analyzer, toward the detector. Lighter molecules travel faster, followed by progressively heavier analytes. Mass spectrum is generated, representing
the number of ions hitting the detector over time. Separation is by the mass-to-charge ratio, but because the charge is typically single, separation is
effectively by molecular weight.
with high significance. Four themes of MALDI-TOF MS are
summarized through a systematic analysis of keywords from
published MALDI MS-related reviews collected in the Web of
Science database. For a deeper understanding, the application
implications, analytical methods, and systems as well as the
limitations that need to be addressed are outlined and
discussed. This review aims to provide a general guide for
MALDI-TOF MS users to develop potential MALDI-TOF
MS-based analysis methods in the field of clinical analysis and
research.
2. BASIS OF MALDI-TOF MS
2.1. Instrumental Basis of MALDI-TOF MS
During MALDI MS detection of biomolecules, the sample to
be analyzed is placed on a conductive plate together with an
organic matrix (Figure 1). Under laser irradiation by an nS-
class pulsed laser (typically a nitrogen laser at a wavelength of
337 nm or a Nd:YAG laser at a wavelength of 355 nm), the
matrix molecules absorb the laser energy and convert it to
electronically excited energy, instantly transforming the solid
mixture of the matrix and analyte into a gaseous state. After
sample desorption, charge transfer occurs, causing ionization of
the analyte in collisions between the uncharged neutral
molecules, the matrix ions, the protons, the electrons, and
the metal cations. Then, ions produced by photoablation and
photoionization are accelerated by an electric field into the
mass analyzer for mass-to-charge ratio (m/z) analysis.
Although the ionization mechanism of the MALDI process
remains a subject of ongoing debate, it is generally recognized
that the matrix plays a crucial role. The matrix used in MALDI
is usually an organic molecule capable of absorbing laser
energy and dispersing sample molecules. Common matrices
include 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxy-
trans-cinnamic acid (HCCA), sinapinic acid (SA), etc. These
matrixes are suitable for nitrogen and Nd:YAG lasers.8 The
analyte is usually dissolved in a solvent containing the matrix
and deposited on a conductive plate for drying and
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crystallization. It I believed that the analyte ions are carried
in the matrix crystal and then desorbed into the gas phase as
matrix−analyte ion pairs under laser irradiation.9−12 Active
species like electrons and radicals are produced during the
process as well.10 As a result, secondary reactions between the
analyte, the matrix, and the active species take place in the
plume, ultimately producing analyte ions. MALDI is
characterized to generate mainly singly charged ions.
Generally, TOF is selected as a suitable mass analyzer to
couple with the MALDI ion source (Figure 1). The high-
throughput characteristic of the TOF mass analyzer makes it a
perfect match for the MALDI ion source, a pulsed ion source
at high frequency. For mass spectrometers equipped with a
TOF mass analyzer, sample ions are extracted by a delayed
potential pulse (delayed ion extraction) into the TOF tube,
which is a field-free region, and the counterions are driven back
to the target plate. The extracted sample ions will pass through
the TOF tube at speeds inversely proportional to the square
root of their m/z values. By recording the time of the flight
pass, the m/z of the ions can be calculated. With the principle,
the TOF mass analyzer is not limited in mass range, which also
makes it an ideal mass analyzer to couple with MALDI, which
generates singly charged ions with large m/z. The resolution of
a TOF mass analyzer depends on the length of the flight path
and can be optimized with techniques of delayed ion extraction
and reflectron. In reflectron TOF, a contrary electric field is
placed at the end of the TOF tube to push the ions back but
with a single angle from the original axial direction. In such a
way, ions with the same m/z but different kinetic energies due
to the initial energy distribution generated during the MALDI
process can be corrected to reach a much higher analysis
resolution than the linear TOF. When one TOF mass analyzer
is not sufficient to analyze the target analytes in a complex
mixture, coupling a second TOF mass analyzer helps to
perform tandem MS analysis. The first TOF mass analyzer is
then used to select the precursor ions, which are fragmented in
a collision cell and then analyzed by the second TOF mass
analyzer to obtain a tandem mass spectrum.
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2.2. Common Analytical Strategies by MALDI-TOF MS
The experimental method of MALDI-TOF MS can be
summarized as follows. First, the sample is mixed with a
suitable matrix material and dropped onto a clean MALDI
sample plate. For surface-assisted laser desorption/ionization
(SALDI), a variant of the typical MALDI, solid nanomaterial is
used as the matrix with a more homogeneous sample
distribution (Figure 2).13 After drying, samples were analyzed
Figure 2. Typical workflow of sample preparations for analyses by
MALDI-TOF MS and SALDI-TOF MS. In conventional MALDI-
TOF MS, the analytes and chemical matrices are mixed and dried to
form cocrystals, whereas analytes are coated homogeneously and
distributed evenly on a layer of solid matrix in SALDI-TOF MS.
Reprinted with permission from ref 16. Copyright 2013 Springer
Nature.
by MALDI-TOF MS. Under laser irradiation, the analyte
molecules are ionized by protonation or deprotonation in a
thermal plume of ablation gas and accelerated into the mass
analyzers or hybrid mass analyzers for separation and
detection.14,15
MALDI-TOF MS has been widely used in many
biochemical analyses, such as bacterial typing, proteomics,
and MS imaging, and in polymer science as well as inorganic
chemistry, e.g., nanomaterials characterization. Due to its high
speed and sensitivity, it is often used to confirm the existence
of a target molecule,16 to discriminate or type species based on
their mass fingerprint,17 to image the distribution of molecules
in biological samples (e.g., cells and tissues),18 and to identify
compounds in a nontargeted manner by coupling with
separation methods, such as gel electrophoresis, capillary
electrophoresis, and liquid chromatography via an automatic
sample depositing system, and tandem MS strategies.19 MS
imaging is based on the mapping of the corresponding ion
intensities while determining the spatial distribution of many
molecules in a sample. Because organic matrices deposited on
the sample can cause diffusion of the analytes, altering their
original distribution and reducing the spatial resolution, several
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matrix-free ionization platforms have been developed to
address this problem, including inorganic matrix and nano-
photonic platforms.20
Surface-enhanced laser desorption/ionization-time-of-flight
mass spectrometry (SELDI-TOF MS) is an extension of
MALDI-TOF MS. Compared with MALDI, the main
difference of SELDI is that the proteins of interest can be
sequestered and purified before laser ionization by interacting
with premodified substances on the surface of ProteinChip
based on biological or chemical affinities.13 For SELDI,
samples such as serum and urine can be spotted without
extra treatment. Followed by surface extraction by Protein-
Chip, nonspecific substances and contaminants can be
removed by subsequent on-spot washing and better crystal-
lization of the matrix and target molecules can be achieved,
which delivers higher specificity and sensitivity in subsequent
analysis.
3. KEYWORD CO-OCCURRENCE OF MALDI
MS-ASSOCIATED REVIEW ARTICLES
Developed applications of MALDI MS with the highest level of
interests were summarized by analyzing keywords in published
reviews related to MALDI. The Web of Science database was
selected for the research work, and VOSviewer (The Centre
for Science and Technology Studies, CWTS), software for the
construction of relationships between the structure, the
evolution, and the collaboration of knowledge domains, was
used for keyword co-occurrence analysis.
3.1. Methods of Keywords Co-Occurrence Analysis
The steps of keyword co-occurrence analysis mainly include
reference searching, keyword cleaning, data importing, and
analysis of included keywords. First, MALDI-associated
reviews were screened from the Web of Science database.
The searching method was “Topic = MALDI, Document Type
= Review Articles, Database = Web of Science Core
Collection”. A total of 2401 records were represented on the
Web site and added to a marked list. The information was
exported in the form of plain text followed by keyword co-
occurrence analysis using VOSviewer on 18 October 2021. It
should be noted that some critical words need to be “cleaned”
before co-occurrence analysis because the same object may be
expressed differently in different articles with the usage of
abbreviations, symbols, and so on. On the basis of preliminary
co-occurrence analysis, the following substitutions were
adopted. “Matrix-assisted laser desorption/ionization (maldi),
matrix-assisted laser desorption, assisted-laser-desorption/
ionization, laser-desorption-ionization, laser-desorption ioniza-
tion, desorption-ionization, desorption ionization, maldi-tof-
ms, maldi-tof ms, maldi-tof, maldi-ms, maldi, laser-desorption/
ionization-time, laser-desorption/ionization-, laser-laser-de-
sorption/ionization, laser-desorption/ionization, and assisted
laser-desorption” were replaced with “l-d/i”. ‘High perform-
ance liquid chromatography, high-performance liquid chroma-
tography, performance liquid chromatography, liquid-chroma-
tography” were replaced with “liquid chromatography”.
“Tandem mass-spectrometry” and “mass-spectrometry” were
replaced with “mass spectrometry”. “Imaging mass spectrom-
etry” was replaced with “mass spectrometry imaging”. “Breast-
cancer” was replaced with “breast cancer”. “Alzheimers-disease,
Alzheimer’s disease, Alzheimer disease” were replaced with
“Alzheimer”, and “lung-cancer, lung cancer” were replaced with
“lung disease”. For keyword co-occurrence analysis, we created
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Figure 3. Network visualization of keyword co-occurrence analysis using the keywords with the highest co-occurrence among all MALDI MS-
related reviews exported from the Web of Science Core Collection. Size of the circles is proportional to the number of occurrences. Distance
between different circles is inversely proportional to their relationship. Closely related keywords are marked with the same color according to the
cluster analysis. Web of Science search covers review articles published from January 1, 1994 to October 18, 2021.

Figure 4. Overlay visualization of keyword co-occurrence network with publication year. Most frequently occurring keywords in all MALDI MS-
related reviews were exported from the Web of Science Core Collection. Size of the circles is proportional to the number of occurrences. Distance
between different circles is inversely proportional to their relationship. All circles are colored according to the timeline bar. Web of Science search
timeline runs from January 1, 1994 to October 18, 2021.

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a map based on the bibliographic area and read data from
bibliographic database files. All cleansed texts were imported,
and the minimum occurrence threshold of keywords was set as
20. A total of 177 keywords that met the criteria among 12 480
keywords were obtained. According to the results of keyword
co-occurrence analysis, the most important clinical applications
of laser desorption/ionization were summarized.
3.2. Network Visualization of Keywords in MALDI
MS-Associated Review Articles
The results of the network visualization are shown in Figure 3.
In the network visualization, a circle represents a keyword
label. The size of the circle is proportional to the number of
occurrences. The lines between the keywords represent the
relevance of the keyword labels. In general, the closer two
keyword labels are located to each other, the stronger their
correlation is. Depending on the results of the cluster analysis,
neighboring keywords share a common color.
As shown in Figure 3, relatively significant keywords among
all 177 keywords appear in the network. Through cluster
analysis, the most important MALDI-related keywords were
divided into six main sections with different color labels. The
red section occupies the largest area. The keyword
“identification” dominates the red section, and most of the
remaining keywords including “microbial or microbiology”,
“infections”, “antibiotics”, and “resistance” are related to
clinical microbiology. Keywords such as “diagnosis” and
“discrimination” are mostly used to represent research
purposes ranging from pathogen species identification to
bloodstream infection diagnosis. Most of the themes in the
blue section focus on disease diagnosis such as “biomarker
discovery”, “lung disease”, “ovarian-cancer”, “breast cancer”,
and “Alzheimer”. In the blue-green section, the clinically
relevant keyword “nucleic acid” can be observed with few
surrounding branches. In the purple section, the keyword
“small molecules” can be found. The keywords in the green
section are mostly related to techniques, such as electro-
phoresis, chromatography, mass analyzer, and fragmentation.
The keywords in the yellow section are mostly related to
biological research, like proteomics and metabolomics. Above
all, the four most important themes of MALDI in clinical
applications can be summarized. They are “pathogen
identification”, “disease diagnosis”, “nucleic acids analysis”,
and “small molecule analysis”.
3.3. Dynamic Changes of Research Focus on MALDI MS
The results of dynamic changes of research focus on MALDI
MS are represented in the form of overlay visualization. The
keyword distribution is the same as the network visualization
except that the items are colored differently. A color bar is
represented in the bottom right of the visualization. The color
indicates the publication year of the review. For instance,
reviews in yellow were published around or after 2016, while
reviews in dark blue were published around or before 2010. A
lighter color means a more recent publication, and a dark color
represents an older publication. By overlaying the visual-
izations, Figure 4 shows a timeline of MALDI MS-based
developments and the main periods of research on each of the
important keywords associated with MALDI MS.
In Figure 4, the color changing from purple to green to
yellow is proportional to the proximity of the review in which
the review was published. The yellow-filled “pathogen
identification” section has attracted intensive attention from
researchers in the last five years. The green-filled “small
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molecule” has been a popular theme since 2015. “Cancer” is a
green keyword accompanied by some nearby keywords
describing specific cancer types such as breast cancer and
ovarian cancer in a darker color, demonstrating a rise of the
MALDI-based biomarker discovery and disease diagnosis over
the past decade. “Small molecule” in light green is also a
relatively young theme surrounded by many light green topics
over the last 10 years. The purple-decorated “nucleic acid”
indicates the relatively early use of MALDI in the analysis of
nucleic acids at least 10 years ago. From the timeline of
MALDI MS-based developments, we can observe a clear
change of the application development focus on the technique
from biological analysis/research to clinical analysis/research.
3.4. Conclusions of Keywords Co-Occurrence Analysis on
MALDI MS-Associated Review Articles
After the keyword clustering analysis of over 2401 review
articles, four of the most important clinical applications of
MALDI MS were summarized consisting of “pathogen
identification”, “disease diagnosis”, “nucleic acids analysis”,
and “small molecules analysis”. In combination with the results
of the overlay visualization, their respective years of popular
development were concluded. Before 2012, the development
of MALDI MS in “nucleic acids analysis” wa emphasized, but
there have been relatively fewer studies related to this theme in
the following years. From 2011 to 2021, the themes of “disease
diagnosis” and “small molecules analysis” attracted much
attention, with the former being a more extended theme. Since
2015, “pathogen identification” with the use of MALDI MS is
the most focused theme, with many recent derivative topics.
However, as many individual studies were not included in the
published review articles, the results of overlay visualization
only reflect the relative temporal distribution density of the
focused applications. Nevertheless, because of the high number
of review articles included in the analysis, the directions of
representative clinical applications of MALDI were mostly
covered.
4. CLINICAL APPLICATION AND POTENTIAL OF
MALDI-TOF MS
4.1. Pathogen Identification by MALDI-TOF MS
Through keyword co-occurrence analysis (Figure 3), “patho-
gen identification” can be extracted as a dominant theme in the
microbiology applications of MALDI-TOF MS. The red
section of Figure 3 is led by “identification”, with the
remaining keywords relating to “microorganism or micro-
biology”, “infection”, “antibiotic”, and “resistance”, all of which
are related to clinical microbiology. Verb keywords like
“identify” and “diagnose” were used most frequently from
the identification of pathogen species to the diagnosis of
bloodstream infections. Pathogen detection based on MALDI-
TOF MS nucleic acid analysis is not discussed in this section
and will be presented in section 4.3.
In clinical practice, many diseases are caused by pathogenic
infections, such as bloodstream infections. Some bacteria are
resistant to antibiotics, and the level of resistance increases
over time, which can pose a significant threat to infection
control in patients, particularly in surgery, hemato-oncology,
and intensive care. The clinical microbiology laboratory plays a
key role in patient care. It not only provides definitive
knowledge of the cause of infection but also provides
antimicrobial susceptibility data to the physicians. Hospitals
or routine microbiology laboratories need rapid and reliable
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Figure 5. MALDI-TOF MS-based workflow for microbial identification.
methods to identify pathogens or detect antimicrobial
resistance of bacteria to reduce mortality caused by
inappropriate or delayed treatment.
In the past, bacterial culture and biochemical tests were the
dominant methods for identifying pathogens with different
phenotypic characteristics, including bacteria, yeasts, and fungi.
Methods based on classical morphology and staining were also
used for bacterial identification. Due to long turnaround times
and cumbersome steps, these methods cannot satisfy the
requirement of fast and accurate identification of pathogenic
bacteria by considering the current requirement of clinical
microbiology laboratories. Since the 1960s, molecular diag-
nostic methods, such as 16S or 18S rRNA (rRNA) gene
sequencing, real-time polymerase chain reaction (PCR) assays,
multilocus sequencing typing (MLST), etc.,21 have been used
for bacterial identification. Although molecular technology-
based methods have largely reduced the detection time, there
is a need to reduce the cost of detection with simpler
operations for large-scale clinical applications. With such
motivation and the technique characteristics of MALDI-TOF
MS, bacterial cell molecular mass fingerprinting by MALDI-
TOF MS has been developed during the past years to realize
fast and accurate identification of bacteria and has been widely
used in clinical microbiology laboratories. Pathogen identi-
fication by MALDI-TOF MS is one of the most successful
applications of the technique in the clinical scene.
4.1.1. Bacteria and Fungi Identification by MALDI-
TOF MS. The first attempts to identify bacteria by mass
spectrometry were made in 1975 by Anhalt et al.22 In the
1990s, MALDI-TOF mass spectrometry was introduced for
the identification of bacteria. Since then, studies assessed the
feasibility of MALDI-TOF MS in identifying bacterial
species.23−25 In 1994, Cain et al. found that bacteria could
be differentiated using water-soluble protein MALDI-TOF MS
fingerprints.26 In 1996, Liang et al. reported that bacteria could
be distinguished at the species level by MALDI-TOF MS.26 In
the same year, Holland et al. suggested that intact Gram-
negative bacteria could be detected in their overall state by
MALDI-TOF MS without the need for protein extraction.27
Inspired by this work, Claydon et al. subsequently demon-
strated that MALDI-TOF MS could generate characteristic
spectra of Gram-negative and Gram-positive bacteria in the
intact state within minutes.28 Since then, MALDI-TOF MS-
based bacterial identification has been fully developed in terms
of sample preparation, database optimization, and software
improvements. Until recently, bacterial mass fingerprinting
systems were incorporated into commercial instruments for
routine clinical applications, including VitekMS developed by
BioMérieux, MALDI Biotyper developed by Bruker Daltonics
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Corp., etc. During MALDI-TOF MS-based bacterial identi-
fication, whole bacterial cells or whole cell extracts are
deposited on a sample spot of the MALDI target plate,
overlayered with a matrix, and then subjected to MS analysis.
Cellular molecular mass fingerprints are obtained, mainly from
the highly abundant small proteins or polypeptides, especially
ribosomal proteins. It was found that different species could
have a distinctive mass pattern. By pattern matching, it is
possible to identify bacteria at the species level.
With the initial focus of MALDI-TOF MS-based micro-
organism identification on bacteria, the application was
subsequently extended to the identification of yeasts and
fungi, such as filamentous fungi, in a similar whole cell mass
fingerprinting way. Currently, MALDI-TOF MS has been
applied in yeast identification and developed as a routine tool
for filamentous and dimorphic fungi identification. VitekMS
and MALDI BiotyperCA systems are two systems with licenses
awarded by the Food and Drug Administration (FDA) for the
identification of yeasts and filamentous fungi. For both
systems, extensive application in yeast identification is claimed
based on individual established databases.29 The performance
of the two systems is similar but not identical; thus, many
comparative studies have been conducted focusing on species
with the growths of databases. The correct identification,
misidentification, and cutoff of isolates have frequently been
assessed at the species, genus, or group level since 2010. For
instance, VitekMS Knowledge Base v3.0 was used for the
identification of 319 mold isolates (43 genera), and an
identification accuracy of 67% was obtained. The identification
accuracy increased to 77% with the addition of a modified
SARAMIS database from Shimadzu Scientific Instruments.30
The selection of isolates, sample sources, and sample
preparation methods may correspond to the differences in
the assessment results of the FDA-cleared systems.31−34 For
the following up-gradation of databases, the focus is on
extending the range of species, subspecies, or strains and the
reference quality of all clinically relevant fungi. In addition to
optimizing databases, commercial databases, databases modi-
fied by personals, and user-developed databases can be used
together, alternatively, or additionally to meet individual
requirements.29
Three main approaches for microbial identification in
clinical laboratories were summarized by Hou et al. regarding
the sample preparation methods.35 The sample can be
prepared by bacterial culture, where bacterial single colonies
on solid agar plates were transferred on a MALDI target plate
for analysis. In an alternative strategy, liquid samples such as
positive blood culture and body fluids (blood, urine, sputum)
can be directly identified after multistep sample preparation
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and extraction protocols without plate culture. For fungi, e.g.,
mycobacterium spp., they can be identified after specific
extraction methods. The prepared samples are dropped on the
MALDI target plate and then analyzed by MALDI-TOF MS
for the spectral acquisition of samples to be identified. Then,
commercial databases or self-built databases that contain the
spectra of standard strains are used to match with the sample
spectra for microbial identification. The final identification
results are determined according to the spectral similarity
between standard strains and analytes (Figure 5).
4.1.2. Antimicrobial Resistance Detection by MALDI-
TOF MS. In addition to pathogen identification, it is also
necessary to assess the antimicrobial resistance of a pathogen
to guide the clinical treatment of infectious cases. Antimicro-
bial resistance can be caused by various mechanisms: the
formation of enzymes that can blunt or catabolize antibiotics,36
the generation of novel efflux pumps and the alteration of the
cell membrane,37 the generation of protective proteins on the
action site of antibiotics,38 the alteration of the enzymes that
can decrease the sensitivity of bacteria against antibiotics, and
the alteration of metabolic pathways related to the response
against antibiotics.39 MALDI-TOF MS has the potential to
detect different antimicrobial resistance mechanisms by
analyzing antibiotic molecules, modification products, the
component of bacterial cells, ribosomal methylation, muta-
tions, etc.
One important class of antimicrobial-resistant bacteria is β-
lactamase-producing bacteria that can hydrolyze β-lactam
antibiotics. MALDI-TOF MS has been used to detect the β-
lactamase activity.40 The degradation products after hydrolysis
show a difference in molecular mass compared to the native
antibiotic molecule, which can be represented by the peaks on
MALDI-TOF mass spectra (Figure 6). Freshly cultured
Figure 6. MALDI-TOF MS detection of β-lactam hydrolysis by
antimicrobial-resistant bacteria.
bacteria are washed by buffer, and the pellet after
centrifugation is resuspended in buffer containing β-lactam
and then incubated at 35 °C for 1−3 h. During incubation,
some β-lactam-type molecules, such as ampicillin and
piperacillin, can be degraded spontaneously. After incubation,
the supernatant is extracted and analyzed by MALDI-TOF MS.
The obtained peaks associated with β-lactam, corresponding
salts, and its degradation products are used to assess the β-
lactamase activity. Bacterial extracts can also be used for
antimicrobial resistance detection using MALDI-TOF MS.
Camara et al. in 2007 innovated the detection of a specific
lactamase peak in ampicillin-resistant E. coli strains. After
cocultivation of the resistant strain and ampicillin in Luria−
Bertani broth, protein extraction with a formic acid−isopropyl
alcohol−water solution, and sample spotting with SA as the
matrix, a β-lactamase peak at approximately 29 kDa was
detected by MALDI-TOF MS.41 The result was also confirmed
by the analysis based on sodium dodecyl sulfate−polyacryla-
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mide gel electrophoresis (SDS-PAGE) and liquid chromatog-
raphy mass spectrometry (LC-MS).41
Apart from the β-lactamase-producing bacteria, MALDI-
TOF MS has also been used to detect various antimicrobial-
resistance-associated proteins for the identification of many
different antimicrobial-resistant bacteria. In 2011, Lenka et al.
pointed out the possibility of SELDI-TOF MS in discriminat-
ing Escherichia coli strains carrying different resistance genes.42
In 2018, Zhu et al. utilized a TiO2-modified target plate as the
substrate for MALDI-TOF MS analysis of antimicrobial-
resistant bacteria.43 With the method, the mass range of
MALDI-TOF MS-based bacterial mass fingerprinting was
extended to 80 000 Da for the detection of antimicrobial-
resistance-associated proteins from intact bacteria. They
applied the method to the detection of extended-spectrum β-
lactamase-producing E. coli (ESBL-E. coli), multidrug-resistant
Pseudomonas aeruginosa (MDR-P. aeruginosa), and methicillin-
resistant Staphylococcus aureus (MRSA).
In view of the diverse drug resistance mechanism and the
varied sequences of antimicrobial-resistance-associated pro-
teins, only monitoring the hydrolysis of β-lactam and the
detection of known antimicrobial-resistance-associated pro-
teins cannot satisfy the clinical requirement of antimicrobial-
resistance detection. Wu et al. used LDI MS to assess the
bacterial viability and antimicrobial resistance by tracing the
redox of resazurin (RS) by viable bacteria.44 Bacterial cells
were incubated with various antibiotic drugs for a period and
then incubated them with RS. By monitoring the reduction of
RS by bacteria, the viability of the microbes after antibiotic
drug treatment could be obtained for the assessment of
antimicrobial resistance.
4.1.3. Limitations of Pathogen Identification by
MALDI-TOF MS. A major limitation of MALDI-TOF MS in
pathogen identification is the coverage of the spectral database,
which can only be experimentally acquired to date. The
coverage of microbial species in the reference database
determines the range of applications for pathogen identi-
fication by MALDI-TOF MS as spectral matching to the
database is the final and critical step for successful
identification. Many kinds of databases or software have
been developed by manufacturers and granted access for
clinical use, such as Bruker MALDI BioTyper, Shimadzu
SampleStations and AuraSolution, and BioMérieux Andromas
systems.45 In 2009, the Bruker IVD (in vitro diagnostic
products) MALDI Biotyper CA system obtained the CE mark
referring to the European IVD directive EC/98/79. In 2013,
the BioMérieux VITEK MS system received U.S. FDA 510(k)
de novo clearance. In the same year, the Bruker MALDI
Biotyper CA system was granted U.S. FDA clearance under
Section 510(k) for the identification of Gram-negative bacteria.
The MALDI Biotyper CA system includes a MALDI-TOF MS,
IVD-labeled reagents, a 48-spot target, supporting software,
and a microorganism reference library. In 2014, the China
Food and Drug Administration (CFDA) also approved the
access of the IVD MALDI Biotyper system into the Chinese
market as a medical device to identify microorganisms isolated
from human specimens. The reference library covering
thousands of microbial species is expanded continuously with
the appearance of new infection crises. In 2017, the U.S. FDA
authorized the expanded application of the BioMérieux VITEK
MS system in identifying mycobacteria, Nocardia, and molds
by granting 510(k) clearance. With the emerging Candida auris
(C. auris) pathogen causing bloodstream infections in
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hospitalized patients with high drug resistance, the U.S. FDA
approved the use of the Bruker MALDI Biotyper CA system in
C. auris identification in 2018, enlarging the licensed uses of
identifying clinically relevant bacteria or yeast species. As an
alternative to upgrading the database experimentally, there are
also methods developed to match experimental spectra to
public protein sequence database. Cheng et al. identified 10
genes to encode proteins most often observed by MALD-TOF
MS from bacteria. Using the 10 genes to annotate peaks on the
MALDI-TOF spectra of bacteria, genus-level identification
with an accuracy of 84.1% was achieved.46
In addition to the coverage of microbial species in the
reference database, there are some other limitations in the use
of MALDI-TOF MS-based systems as routine diagnostic tools
for bacteria or fungi identification. Purified single colonies
obtained by bacterial culture are required for MALDI-TOF MS
analysis to reach high accuracy. The overall positive rate of
bacterial culture from infected clinical samples is in general
low. To solve the limitation, specific sample pretreatment can
be adopted, including bacterial enrichment by magnetic
beads47 or microfluidic techniques48 to shorten or avoid
bacterial culture, developing new matrixes or surface enhance-
ment methods to obtain more molecular information43 for
higher identification accuracy, coupling with high-resolution
mass analyzers for higher specificity in identification, or
developing new algorithms or data analysis frameworks49 for
deep data mining to enable direct identification of bacterial
mixtures,50 reorganization of antimicrobial-resistant bacteria,
and virulence factors.
For the detection of antimicrobial resistance, it is difficult to
discriminate subspecies and strains that are drug resistant or
not, even though bacteria can be confidently identified at the
species level based on MALDI-TOF fingerprinting. Therefore,
antibiotic susceptibility testing (AST) needs to be applied after
MALDI-TOF identification. Besides, there is room for
improvement in the detection of the enzymes that degrade
antibiotics, detection of the commonly shared resistance
mechanism by constructing a protein fingerprint database of
antibiotic-resistant isolates, analysis of the modification of
target points, and quantitative analysis of antibiotics influx and
efflux.40 More efforts are needed to enable the extension of
MALDI TOF-based pathogen antimicrobial-resistance analysis
to routine clinics.
4.2. Disease Diagnosis by MALDI-TOF MS-Based
Peptidome and Proteome Profiling of Body Fluid
The results of the keyword co-occurrence analysis show that
MALDI MS has been widely developed for disease diagnosis.
As shown in the network diagram (Figure 3), blue keywords of
“cancer”, “breast cancer”, “ovarian cancer”, “Alzheimer”, and
“lung disease” can be seen, indicating that these diseases are
focused on very frequently in MALDI-related reviews and may
attract a high level of interest in the development of MALDI
MS-based clinical diagnostics. The keywords of “peptidomes”,
“serum”, and “plasma” in the blue section imply that proteins
and peptides in human body fluids, especially blood, are often
used as research objects in MALDI MS-based disease
diagnostic studies. The keyword of “model” in the blue section
indicates that disease diagnostic models are often established.
The keywords of “proteomics” and “lc-ms-ms” can also be
found in the blue section, based on which it can be found that
LC-MS/MS-based proteomics is commonly used for protein
identification together with MALDI MS.
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As the pathogenesis and phenotype of different diseases
vary, so do the diagnostic modalities. Here is a summary of
some common diagnostic options for different diseases. For a
physical test, doctors usually start by searching for visible
abnormalities such as enlarged organs that may indicate the
presence of disease. For laboratory tests, samples such as blood
and urine are usually collected and tested to help identify
abnormalities. Imaging tests allow doctors to examine internal
organs in a noninvasive way, including computed tomography
(CT), magnetic resonance imaging (MRI), positron emission
tomography (PET), ultrasound, and X-ray. In most cases, a
biopsy is the only way to definitively diagnose cancer. In view
of the long cycle, late course of disease, low sensitivity, and
strong invasiveness of many clinical diagnostic methods as well
as the complexity of the occurrence and development of the
diseases themselves, it is necessary to develop new detection
methods to detect diseases and help doctors make medical
decisions.
Anomalies of protein expression such as disruption,
mutation, and misfolding are closely linked to various diseases.
Some proteins change significantly as disease progresses,
resulting in abnormal levels of protein concentrations in the
blood. The changed proteins are then potential disease
biomarkers. For example, prostate cancer-specific antigen
(PSA) is used clinically as a serum marker for prostate cancer
and is recommended by the American Cancer Society for early
detection of cancer. However, PSA is also related to other
cancers; therefore, diagnosis based on a single protein marker
normally suffers from low specificity. Monitoring a panel of
distinctive proteins can greatly improve the sensitivity and
specificity of disease diagnosis and help doctors make more
accurate clinical decisions.
Searching for specific disease biomarkers has been tough
work all along. One widely used method is detecting and
quantifying specific biomolecules in the body fluids of patients
and controls, particularly accessible body fluids including
serum, urine, or saliva, which are sources of protein-rich
information.51 Of these, serum is the most commonly analyzed
specimen.52 In addition, in recent years, plasma exosomes have
been increasingly focused on in diagnostic studies of disease.
Exosomes are nanoscale cellular multivesicular vesicles that
encase intracellular substances such as lipids, metabolites,
proteins, and genetic materials.53 Secreted by all types of
human cells and circulating in the body, exosomes are
associated with many physiological and pathological processes.
Cancer-derived exosomes can contribute to the development
of cancer and have been shown to be a target for research in
cancer biology and clinical laboratories.54
Since the concentration of circulating biomolecules is highly
variable, it is essential to analyze the biomolecule information
in body fluids accurately with a reasonable sample grouping
and sample size. Before the appearance of biomass
spectrometry, there was no means to analyze proteins in a
rapid, sensitive, and high-throughput manner. The analysis and
identification of unknown proteins by traditional methods
need to isolate and purify the proteins first, which requires a
long experimental cycle. The invention of MALDI MS in the
late 1980s allowed the accurate measurement of endogenous
biomolecules in a large scale with high throughput. After
simple pretreatment, samples on target plates can be analyzed
by MALDI-TOF MS in less than 1 min per spot. By analyzing
protein changes at different stages of disease onset, protein
biomarkers can be identified at different times of the disease,
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which can not only provide important diagnostic indicators for
early disease diagnosis and detection but also serve as targets
for drug screening and guidance for drug discovery. Compared
to traditional biochemical assays, MALDI-TOF MS shows
higher sensitivity. It also has other characteristics such as low
cost and easy operation. With the introduction of MALDI-
TOF MS, the discovery of disease-related biomarkers was
largely facilitated by monitoring a panel of features, driving the
development of disease diagnosis and personalized treatment.
The main processes for developing disease diagnosis models
based on MALDI-TOF MS are usually similar. Clinical
samples from the case and control groups, generally human
body fluids (i.e., blood, urine, saliva, sweat, tissue fluid), were
collected and divided into a training group and a testing group.
Pretreatment was performed for all samples (i.e., desalin-
ization, enrichment, purification) followed by the analysis of
MALDI-TOF MS. It should be noted that matrix selection and
spotting methods need to be selected according to the target
mass range, actual sample peaks, and even results of the pre-
experimental analysis. Appropriate machine-learning methods
(e.g., partial least-squares discriminant analysis, logistic
regression, support vector machine, random forest, etc.) are
used to extract one or more sets of MALDI distinctive peaks
between the case and the control groups in the training
samples. The proteomic methods based on LC-MS/MS can be
used to identify the selected MALDI-TOF MS distinctive
peaks as proteins. The step of feature extraction can be
considered as “disease biomarker discovery”. With the selected
biomarkers, a model for diagnosing the disease can be
established based on specific machine-learning methods. The
established model can be assessed among testing samples in
terms of precision, accuracy, sensitivity, specificity, and an area
under receiver operating characteristic curve (AUC). A
receiver operating characteristic (ROC) curve, discussing the
measure of AUC, is a plot of the sensitivity versus 1�the
specificity of a diagnostic test. It provides a useful summary of
the overall diagnostic accuracy of the test.55 On the basis of
these evaluation indicators, a MALDI-TOF-based diagnosis
method with the relative best performance for the prediction of
specific diseases can be determined. On the other hand, when
known protein biomarkers exist, it is also possible to extract
and enrich the biomarkers using chemical or biological affinity
methods followed by MALDI-TOF MS analysis. Here, in this
section, we introduce the representative application examples
of MALDI-TOF MS in the diagnosis of cancer, infectious
disease, and neurodegenerative disease.
4.2.1. Cancer Diagnosis and Prognosis. Cancer
diagnosis and prognosis accounts for a large proportion of
MALDI-based clinical disease diagnosis. Take ovarian cancer
as an example. Many studies have reported the use of MALDI-
TOF MS in distinguishing ovarian cancer (OC) from healthy
controls. Cancer antigen 125 (CA125) is one of the two
biomarkers approved by the FDA for the diagnosis of
recurrence and treatment response in ovarian cancer.56
CA125 alone can predict OC up to 9 months before diagnosis.
However, not all early-stage tumors produce CA125. It might
be regulated by other benign gynecological diseases as well.
Other biomarkers are required for early OC diagnosis.57
Timms et al. found that the combined use of CA125 and two
MALDI-TOF MS feature peaks, identified as connective tissue-
activating peptide III and platelet factor 4, could detect OC
about 6 months earlier than using CA125 alone.58 Addition of
the cancer progression or tumor development-related serum
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peptides can advance the diagnosis of OC with a high degree
of confidence.58 Adoption of specific sample pretreatment
methods can improve the MALDI-TOF MS-based diagnosis of
OC. One study reported that analysis of ions with low mass
from serum performed better in differentiating ovarian cancer
from controls after removing high levels of proteins and
peptides.59 More discussion on small molecule studies by
MALDI-TOF MS is presented in section 4.4. There are studies
extracting the low-abundance proteins or peptides through
enrichment technology such as magnetic beads before
MALDI-TOF MS analysis.60 Periyasamy et al. suggested that
solid-phase extraction before MALDI-TOF MS analysis can
improve the sensitivity of a diagnosis model to differentiate
serous adenocarcinoma (a common type of epithelial OC) and
healthy controls.61 Swiatly et al. proposed that the combined
usage of iTRAQ (isobaric Tags for Relative and Absolute
Quantification)-based quantitative proteomic analysis and
MALDI-TOF MS can improve the differentiation of benign
and malignant tumors in OC.62
MALDI-TOF MS has also been applied to the early
diagnosis and prognosis of many other cancers, such as
prostate cancer,60,63 liver cancer,64 and multiple myeloma.16,65
In 2019, Long et al. developed a MALDI-TOF MS-based
methodology to diagnose multiple myeloma by detecting
Bence−Jones proteins in human urine samples.16 The study
included 21 positive urine samples and 27 negative urine
samples. The urine proteins were first enriched by macro-
porous ordered silica foams (MOSF) and then analyzed by
MALDI-TOF MS. In the study, a rapid method with high
sensitivity (95.24%) and specificity (100%) was obtained for
the diagnosis of multiple myeloma, which outperformed
immunofixation electrophoresis-based66 and immunonephel-
ometry-based methods.67 In 2020, Sun et al. applied the
technology of MALDI-TOF MS serum peptide fingerprinting
to prediagnose prostate cancer (PCa) involving 100 PCa
patients and 47 non-PCa controls (20 healthy people and 27
inflammatory hyperplasia people) (Figure 7b).60 First, the
authors enriched the target low molecular wight proteins or
Figure 7. Synthesis of HICNPs, and schematic diagram of prostate
cancer prediction based on MALDI-TOF MS fingerprinting of human
serum proteins/peptides. Reprinted with permission from ref 60.
Copyright 2020 Elsevier.
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Figure 8. Scheme of establishing a diagnostic model for rapid screening of COVID-19 patients by MALDI-TOF MS analysis of human serum.
Reprinted with permission from ref 74. Copyright 2021 American Chemical Society.
peptides using synthetic hydrophilic interaction chromatog-
raphy nanoparticles (HICNPs) to attenuate interference in
human serum (Figure 7a). These functionalized mesoporous
silica materials with a large pore surface area, highly ordered
pore structure, homogeneous mesoporous hydrophilic effect,
and some chemical and mechanical stability can be used as
good substrates for selective protein/peptide adsorption. After
coincubation with HICNPs magnetic beads, washing, and
elution, the serum MALDI-TOF MS signals of the samples
were simplified with more valuable fingerprint-like patterns.
Machine learning was eventually used to establish a diagnostic
model of PCa. Recently, MALDI-TOF MS fingerprinting was
further applied to discover lipid PCa biomarkers in urine
samples from 121 PCa patients and 18 healthy people.63 In this
study, the diagnostic accuracy ranged from 83.3% to 100.0%. It
is worth noting that although the example cited here is being
developed to diagnose PCa cancer, it could be subsumed in
section 4.4 in this review because urinary lipids belong to small
molecule metabolites. The MALDI-based cancer diagnosis in
section 4.2 mostly focuses on biological macromolecules such
as proteins and peptides. The main difference between the
analytical protocols for large molecules (e.g., m/z 2000−
20 000) and that of small molecules (e.g., smaller than m/z
1000) is the choice of the target analytical mass range and
hence the suitable matrices or materials to assist the ionization.
On the mature foundation of MALDI-TOF MS finger-
printing technology, many other samples have been used for
biomarker discovery and cancer diagnosis, especially exosomes
isolated from body fluids.65,68−70 In 2019, Zhu et al. used
MALDI-TOF MS to analyze exosomes extracted from serum
of melanoma patients and healthy donors and demonstrated
that the mass fingerprinting of bloodstream-circulating
exosomes can be used for cancer diagnosis and monitoring.69
In 2021, Han et al. used MALDI-TOF MS to analyze serum
exosomes from 12 healthy donors, 20 osteosarcoma patients
without lung metastasis, and 20 osteosarcoma patients with
lung metastasis. It was found that the MALDI-TOF MS-based
serum exosome mass fingerprinting can not only identify
osteosarcoma but also differentiate osteosarcoma patients with
lung metastasis from those without lung metastasis.68 Seven
protein biomarkers of osteosarcoma with lung metastasis were
identified, including immunoglobulin lambda variable 2-23
(IGLV2-23), immunoglobulin lambda variable 4-3 (IGLV4-3),
immunoglobulin lambda variable 1-51 (IGLV1-51), immuno-
globulin kappa variable 3-15 (IGKV3-15), immunoglobulin
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heavy variable 4-4 (IGHV4-4), immunoglobulin lambda
variable 4-60 (IGLV4-60), and hemoglobin subunit alpha
(HBA1). In 2021, cells collected from the surface of suspected
skin areas via sterile adhesive sampling discs were used by Zhu
et al. to detect melanoma and predict skin disorder
progression.71 Noninvasive sampling is another advantage of
this study considering the benign method of obtaining cells
from the skin surface.
4.2.2. Infectious Disease Diagnosis. In addition to the
direct identification of a pathogen, which is detailed in section
4.1, MALDI-TOF MS-based profiling of human body fluids
was also used for the diagnosis of infectious diseases.
Mycobacterium tuberculosis (MTB) is a common “lung disease”
and can also be described as an infectious disease. Initial
methods for diagnosing MTB are usually based on micro-
biologic techniques such as acid-fast bacillus smear microscopy
and MTB culture. These methods require a long analysis time
and are not sensitive or specific enough.72 Liu et al. developed
a rapid diagnosis of active MTB infections by monitoring
serum CFP-10 (TDAATLAQEAGNFER; m/z 1593.75) and
ESAT-6 (WDATATELNNALQNLAR; m/z 1900.95). These
biomarkers could theoretically be used to diagnose all MTB
infections based on MALDI-TOF MS. The analysis time was
shortened to 4 h with high sensitivity and specificity.73 In
addition, techniques can be combined to aid MALDI-TOF MS
analysis for the diagnosis of MTB infection, including
microwave irradiation for CFP-10 and ESAT-6 digestion,
stable isotope-labeled internal standard peptides for CFP-10,
and ESAT-6 quantification and antibody-conjugated nanodisks
for target peptide enrichment and MALDI signal enhance-
ment.73
Since 2019, a novel coronavirus infectious disease COVID-
19 caused by Severe Acute Respiratory Syndrome Coronavirus
2 (SARS-CoV-2) has become massively prevalent worldwide.
The enormous demand for disease diagnosis has put enormous
pressure on traditional molecular detection methods such as
reverse transcription (RT) polymerase chain reaction (PCR).75
Apart from viral nucleic acids, viral proteins, antibodies specific
to the virus, and host-induced molecular changes can also be
used for the diagnosis of COVID-19 or progression
mornitoring.76,77 In 2020, Nachtigall et al. developed a novel
method to detect SARS-CoV-2 infection based on MALDI-
TOF MS analysis of nasal swab samples using 362 samples
(211 RT-PCR positive samples and 151 RT-PCR negative
samples), achieving an identification accuracy of 93.9%.75 In
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2021, Yan et al. used MALDI-TOF MS to analyze the serum of
COVID-19 patients and controls to collect the serum
peptides/proteins mass fingerprinting involving 146 COVID-
19 patients, 73 non-COVID-19 patients with similar clinical
symptoms, and 46 healthy controls (Figure 8). The samples
were collected in very early 2020, reflecting the immune
response to the very original SARS-CoV-2 strains in mainland
China. Various machine-learning methods were applied for
feature selection from the MALDI-TOF mass spectra of serum
peptidome, and different classification models were built for
the diagnosis of COVID-19. Eventually, a rapid diagnosis
model was established to detect COVID-19 infections with
high accuracy (99%), sensitivity (98%), and specificity
(100%).74 Fifteen serum protein/peptide biomarkers were
identified for COVID-19.
4.2.3. Neurodegenerative Disease Diagnosis. Alz-
heimer’s disease (AD) is a neurodegenerative disease. In the
early phase of discovering AD biomarkers, CSF, a proximal
fluid, was often studied. As a review summarized, the most
characteristic AD biomarkers in CSF are β-amyloid (βA), tau
protein, and phospho-tau.78 As early as 1993, the first study of
targeted βA proteomics detected multiple βA isoforms in CSF
using MALDI-TOF MS.79 On the basis of initial efforts to
explore markers, more accessible fluids, particularly blood,
were focused on reducing the invasiveness of sampling.78 In
2018, Nakamura et al. found a new composite biomarker, βA
precursor protein (APP) 669-711/Aß42 and Aß40/Aß42
ratios in plasma, to predict positive or negative brain βA
based on MALDI-TOF MS analysis, illustrating the high
performance of plasma biomarkers in brain βA burden
prediction and AD diagnosis.80 Last year, Shimadzu Japan
released a MALDI-based Amyloid MS CL system for testing
the levels of amyloid peptides in blood that are associated with
AD. The product was licensed for manufacture and sale by the
Japanese Ministry of Health, Labor, and Welfare in December
2020.
4.2.4. Limitations of Disease Diagnosis by MALDI-
TOF MS-Based Peptidome and Proteome Profiling.
Although many advantages of MALDI MS, such as high
throughput, short time analysis, easy operation, and low cost,
have been repeatedly reported in the literature for different
disease diagnostic applications, there are still some common
application drawbacks of the technique. Unfavorable repeat-
ability is a major issue to the clinical spread of MALDI-TOF
MS, limiting the application of the technique when considering
quantitative analysis. The issue is mainly caused by the random
procedure of matrix−analyte cocrystal formation and the
random sampling by a laser on the sample spot. Various
techniques have been adopted to enhance the repeatability of
MALDI-TOF MS in view of quantification, including the
development of a new matrix than can form a homogeneous
crystal on the target plate, new sample and matrix deposition
methods, optimization of internal standards, ionization process
optimization, and postdata acquisition processing.78 In future
work, the focus should be on validation of a MALDI-TOF MS-
based diagnosis model in many different centers over a long
period. Besides, there is still uncertainty in the results of
protein identification based on proteomics techniques for
MALDI MS signature peaks. Because of the complex
composition of clinical samples, the differences between the
experimental techniques, such as pretreatment, mass analyzer,
ion separation, and detection, and artificially introduced
differences, such as data processing and protein matching
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rules, the identification of potential protein markers needs to
be further confirmed by other methods like biological
experiments, etc.
4.3. Analysis of Nucleic Acids with MALDI-TOF MS
Keyword “nucleic acid” can be found in Figure 3. From Figures
3 and 4, it can be found that the application of MALDI-TOF
MS in the analysis of “nucleic acid” was developed earlier than
the other themes but with fewer continuous efforts in the
following years compared to many other applications of
MALDI-TOF MS, e.g., pathogen identification and cancer
diagnosis. Nevertheless, nucleic acid analysis is indeed one of
the most successful applications of MALDI-TOF MS in the
clinical scene. DNA and RNA are two common classes of
nucleic acids. DNA can store the genetic information and play
key roles in maintaining the proper function of all living
organisms. RNA is similar to DNA in structure but is single-
stranded and with uracil (U) as one of the four bases instead of
thymine (T) in DNA. A major class of RNA is the mRNA that
can direct the synthesis of proteins based on genetic
information. For DNA and RNA, the sequence of their
nucleotides determines their uniqueness. During tumor-
igeneses, breaks and recombination at the genomic level
often occur. When two genes are broken in half and
misaligned, it is possible to form a new gene fragment,
which is known as a fusion gene. In most cases, fusion genes
can lead to the production of abnormal sequences or functional
proteins or to the dysregulation of the expression of certain
genes, which can cause or promote the development of tumors.
With the development of matrices that can assist the
ionization of DNA and RNA, the technology of MALDI-TOF
MS has been applied to the analysis of nucleic acids. In 1990, it
was first reported that MALDI-TOF MS can be applied to the
analysis of oligonucleotides.81 Subsequently, various matrices
(e.g., 2,4,6-trihydroxyacetophenone82 and glycerol83) and
matrix additives (e.g., various sugars84 and spermine85) were
developed to enhance the ionization of nucleic acids. Since the
common matrices in nucleic acid analysis can generate large
crystals, which would hinder the ionization efficiency and
reproducibility, a sample preparation method was developed to
miniaturize sample spot sizes to improve the homogeneity of
matrix−analyte crystals and hence the reproducibility of mass
spectra.86 As nucleic acid samples are normally in the presence
of high concentrations of salts and salts can inference the
ionization of analytes during MALDI, cation exchange resins
are normally used to remove salts in nucleic acid samples.87
MALDI-TOF MS is often combined with PCR for nucleic acid
analysis. The main workflow is composed of gene locus
selection, primer design, and quality control, PCR (primer
extension and base-specific cleavage) amplification, Shrimp
Alkaline Phosphatase (SAP) reaction and purification, single-
base extension, sampling, desalting, and MALDI-TOF MS
analysis. On the basis of the method, a commercial system,
MassARRAY, was developed by Agena Bioscience.
4.3.1. Screening of SNP, DNA Methylation, and
Inherited Genetic Diseases. The screening of nucleic acid
fragments associated with distinct genotypes or mutants by
MALDI-TOF MS has been developed mainly for single-
nucleotide polymorphism (SNP) genotyping, DNA methyl-
ation identification, and inherited genetic disease screening.
Single-nucleotide polymorphism (SNP) often occurs in human
DNA and is the most common type of genetic variation in
human beings. The presence of SNP can be used to predict the
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personal risk of developing specific diseases or track the

inheritance of disease genes within families. The screening of
SNP can be directly realized with the PCR-MALDI-TOF MS
workflow using a commercial solution of MassARRAY (Agena
Bioscience, California, United States). Kenji et al. applied
MALDI-TOF MS to screen SNPs and demonstrated ethnic
differences in coronary artery disease-associated SNPs among
two healthy Israeli populations (Ashkenazi Jews and Yemenite
Jews) based on 15 SNPs determined from 14 candidate
genes.88
DNA methylation is a potential biomarker for several
cancers with a close association with tumorigenesis, develop-
ment, and cell carcinogenesis. MassCLEAVETM chemical
methods (Sequenom, California, United States) based on the
MassARRAY system (Agena Bioscience, California, United
States) have shown high sensitivity in the detection of
methylated DNA, allowing high-resolution quantitative meth-
ylation analysis by base-specific cleavage on bisulfite-converted
DNA. Ehrich et al. used the method to quantify methylation
differences between normal and neoplastic lung cancer tissue
samples.89 Gao et al. detected a higher level of methylation
status in gastric cancer tissues than normal tissues by
comparing the hypermethylation levels of Nell-1 in tumor
tissue, paraneoplastic tissue, and normal tissue from gastric
cancer patients.90
The molecular diagnosis of inherited genetic diseases or
genetic disorders was also developed based on MALDI-TOF
MS genotyping. In 2011, Lambros et al. established a novel
method to identify recurrent fusion genes in cancers using the
MassARRAY platform (Figure 9).91 The increasing number of
genomes undergoing massively parallel sequencing will
undoubtedly require scalable platforms to validate the fusion
genes identified. This study shows the feasibility of it.
However, its false-positive rate is unsatisfactory, which remains
to be solved. In 2016, Tian et al. established a method to
detect the multiplex mutations in lung cancer cell lines with
Figure 9. Illustration of the MALDI-TOF MS-based detection of
high sensitivity (100%) and specificity (96.3%) using the recurrent fusion genes. PCR amplification of an amplicon from the
MassARRAY platform.92 The authors pointed out the breakpoint region (a) was followed by several steps of sample
limitation that the developed method relied on a group of preparation and hybridization with custom-designed extension
preselected sites, so that it cannot comprehensively detect the primers (b). Only in the presence of a fusion gene was a PCR
highly random mutational patterns in tumor suppressors.92 product generated for the extension reaction to take place. Samples
MALDI-TOF MS-based nucleic acid analysis has also been were spotted onto a SpectroChip after cation removal (c) and then
applied in genetic diseases, such as trisomy 21 (Down analyzed by the MALDI-TOF MS system (d). If the predicted alleles
syndrome), a genetic disorder caused by the presence of all were detected by the probe combination, a fusion gene was present
or part of the third copy of chromosome 21,93 genetic deafness (red arrow), whereas if peaks were only seen representing unextended
primers (black arrow) with no alleles detected, no fusion gene was
inherited from parental generation with genetic and
present (e). Reprinted with permission from ref 91. Copyright 2011
chromosomal abnormalities,94 and familial adenomatous Springer Nature.
polyposis (FAP) caused by APC germline mutations, an
autosomal dominant colorectal cancer susceptibility syn-
drome.95 used in clinical settings to improve SARS-CoV-2 nucleic acids
4.3.2. Screening of SARS-CoV-2 Variants. MALDI-TOF testing efficiency. At the end of 2021, Zhao et al. developed a
MS-based nucleic acid detection can also be used for pathogen novel strategy of a MALDI TOF-based multiplex PCR mini-
detection. In view of the recent COVID-19 pandemic and the sequencing technique to identify SARS-CoV-2 variants,
continuous evolution of SARS-CoV-2 variants, we present here including SARS-CoV-2 and the variants of B.1.1.7 (Alpha),
the application of MALDI-TOF MS-based nucleic acid analysis B.1.351 (Beta), B.1.429 (Epsilon), B.1.526 (Iota), P.1
in the identification of SARS-CoV-2 virus strains. In 2020, (Gamma), and B.1.617.2 (Delta).97 RNA was extracted from
Wang et al. applied PCR-MALDI-TOF MS to detect SARS- clinical oropharyngeal swabs. Genes containing targets of SNPs
CoV-2 nucleic acids.96 In their study, sputum and pharyngeal were amplified using multiplex PCR. SNP sites were extended
swab samples collected from multicenters were analyzed. The using an extension mass probe. MALDI-TOF MS was
open reading frame 1ab (ORF1ab) gene and nucleocapsid performed to identify the m/z of the extended mass probe,
protein (N) gene of SARS-CoV-2 were the focus. The study based on which SARS-CoV-2 variants can be detected. Nine
revealed that SARS-CoV-2 detection by PCR-MALDI-TOF mutant types of SARS-CoV-2 variants at seven mutation sites
MS had high accuracy, sensitivity, and specificity and could be in the spike receptor binding domain (HV6970del, N501Y,
396 https://doi.org/10.1021/acsmeasuresciau.2c00019
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ACS Measurement Science Au
Figure 10. Summary of nanoparticles as matrices for small molecule analysis by MALDI-TOF MS: DHB, 2,5-dihydroxybenzoic acid; 9AA, 9-
aminoacridine; DAN, 1,5-diaminonaphthalene. DHB and DAN were used for positive-ion mode, and 9AA and DAN were used for negative-ion
mode. Reprinted with permission from ref 103. Copyright 2016 American Chemical Society.
K417N, P681H, D614G, E484 K, L452R, E484Q, and P681R)
were detected, and high specificity and an accuracy of 100%
were achieved among 20 clinical verification samples.97 In
2022, Han et al. reported a Y-structure-induced rolling loop
amplification method combined with MALDI-TOF MS for
nucleic acid detection of SARS-CoV-2. The method enabled
the simultaneous detection of the SARS-CoV-2 N gene and
orf1ab gene in a single reaction tube within 30 min at 55 °C
with high specificity compared to the SARS-CoV, MERS, and
bat-SL-CoVZC45 coronaviruses.98 The MassARRAY system
achieved the CE-IVD mark in Europe for the qualitative
detection of SARS-CoV-2 nucleic acids. Compared with the
real-time PCR test, the gold standard of SARS-CoV-2 early
diagnosis approved by the WHO, PCR-MALDI-TOF MS-
based assay had superior performance in the discrimination of
SARS-CoV-2 variants.99
4.4. Analysis of Small Molecules by MALDI-TOF MS and Its
Potential in Clinical Analysis
In the purple section of Figure 3 associated with “small
molecules analysis”, the keyword of “matrix” is highly
prominent. Most of its nearby keywords, including “porous
silicon”, “carbon nanotubes”, “affinity probes”, and ‘metal-
organic frameworks”, are related to the matrix or materials.
Therefore, the focus of laser desorption ionization (LDI)-
based small molecule analysis is on the development of suitable
matrices for a long time. In the yellow section of Figure 3, the
keyword “metabolites” can be classified as small molecules.
“Metabolites” can be regarded as endogenous compounds such
as amino acids, lipids, sugars, short peptides, alcohols, organic
acids, etc.100 Usually metabolites can be found in organelle
cells, organs, biological fluids, and organisms.100
MALDI-TOF MS has been widely applied for analyzing
many kinds of large molecules as described above. The
application of it for the analysis of small molecules is mostly
limited by the selection of a suitable matrix.101 Conventional
matrices used for the analysis of large molecules are often
unsuitable for the analysis of low molecular weight compounds
(m/z < 1000 Da) mainly due to the matrix background
interference, ion suppression, and nonuniformity of crystal-
lization by traditional organic matrices. For the analysis of
small molecules, many inorganic materials have been
397
pubs.acs.org/measureau Review
developed to assist the ionization process, such as nano-
particles (NPs) and nanotubes. Compared to an organic
matrix, inorganic nanomaterials can provide a clean ground in
the low-mass region and homogeneous surface for good
reproducibility. The original use of inorganic materials as the
matrix for LDI MS analysis was in 1988 when Koichi Tanaka
used cobalt powder to desorb and ionize proteins.2 With the
success of nanostructure-based surface ionization and advances
in nanomaterial synthesis techniques, the use of NPs for LDI
MS analysis flourished around 2010.102 For a long time, gold
and silver NPs synthesized by different methods were the most
widely adopted matrices for LDI MS-based small molecule
analysis.103 In the last two decades, a wide variety of NPs have
emerged, including metal oxides (TiO2104), carbon-based NPs
(colloidal graphite105), metal NPs (e.g., platinum106), and
semiconductor quantum dots (HgTe107), and many of them
have been used to assist ionization initiated by a laser. In 2021,
a kind of carbon nanomaterial matrix graphdiyne was
developed for the analysis of small molecules such as amino
acids, fatty acids, and peptides.108 It can enhance the
desorption and ionization efficiency with a low background
that has been used for discovering fatty acid biomarkers for
liver cancer diagnosis.108 A multishelled hollow Cr2O3 sphere
material was developed to enhance ionization with strong
photoresponse and to stabilize the effective LDI of metabolites.
Its high sensitivity and selectivity were demonstrated in
discriminating schizophrenia patients from healthy controls.109
An advanced mesoporous material PdPtAu was developed to
extract metabolites and assist in LDI. Its high sensitivity and
selectivity were demonstrated in differentiating gastric cancer
cases from normal controls.110 In addition, cation induction
and fragmentation of small metabolites and successful control
of metabolite fragmentation with the help of nanomaterials
were reported to enhance metabolite identification by
enlarging atomic/fragmentation coverage, which can help
simulate the different physiological or pathological processes
of specific diseases and explore more metabolic pathways.111
There are many types of matrices for the analysis of various
small molecules, and the selection mainly depends on the
target analytes. A review published in 2016 demonstrates the
different efficiency of NPs for LDI MS analysis of a wide range
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ACS Measurement Science Au pubs.acs.org/measureau Review

Figure 11. (a) Schematic workflow for the extraction of plasma metabolic fingerprints by vanadium core−shell nanorod-assisted laser desorption/
ionization mass spectrometry (LDI MS). Only Na+- and K+-adducted metabolite ions can be detected with the coexistence of high concentrations
of peptides and proteins, consuming only 500 nL of native plasma. (b) Schematic outline for the diabetic retinopathy (DR) differentiation from
non-DR (NDR) diabetic control and progression evaluation by machine learning of plasma molecular fingerprints. Reprinted with permission from
ref 115. Copyright 2020 John Wiley & Sons, Inc.

of small molecules with the addition of two widely used
organic matrixes in positive and negative modes (Figure
10).103 A thermal desorption model was established in the
study with the normalization by the strongest ion signal for
each analyte. The model can provide significant reference
information for the selection of NPs in the analysis of small
molecules based on MALDI-TOF MS. In 2021, Kulkarni et al.
summarized the recently published (from 2016 to 2021)
materials-based matrices for small molecule analysis in the
biomedical field.112 Three types of matrices (inorganic,
organic, and hybrid) and 32 materials were included for the
analysis of low molecular weight compounds, especially amino
acids, lipids, and metabolites.
The application of MALDI-TOF MS in small molecule
analysis can be divided into three main directions: tissue
fingerprinting, biofluid profiling, and cellular typing. For tissue
fingerprinting, LDI-based tissue fingerprinting allowed the in
situ analysis of relative molecular concentrations in organ
tissues. For instance, Zhou et al. used graphene oxide (GO) to
analyze small molecules in mouse brain tissue. The authors
also used the method to characterize the difference in the
spatial molecular distribution between surviving and necrotic
tumor regions of breast cancer mice.113 Palermo et al. used
fluorinated gold NPs (f-AuNPs) to analyze the metabolites in
mouse colon comprehensively. LDI MS analysis based on f-
AuNPs requires low laser energy to induce the release of
fluorocarbon chains with a low background signal and sensitive
detection of sample molecules, avoiding metabolite endoge-
nous fragmentation and maintaining the integrity of molecular
ions.114 Considering the contribution in terms of signal
enhancement and biocompatibility, Kulkarni et al. proposed
that LDI MS will be a promising tool for in vivo monitoring of
biological tissue dynamically.112
MALDI-TOF MS is also used to analyze the low molecular
weight compounds in biofluids for biomarker discovery and
disease diagnosis.112 Vedarethinam et al. developed vanadium
core−shell nanorods to profile molecular variation in the
398
plasma of diabetic retinopathy patients, based on which a
disease diagnosis model of diabetic retinopathy was established
with high sensitivity (94%) and specificity (90%) (Figure
11).115 In the presence of high concentrations of co-occurring
peptides and proteins, only Na+- and K+-coupled metabolites
can be detected using the vanadium core−shell nanorods.
More recently, the same group also reported the high
performance of nanoparticle-enhanced laser desorption/
ionization mass spectrometry (NPELDI-MS) in differentiating
between breast and nonbreast cancers with an area under the
receiver operating characteristic curve of 0.948.116 Wang et al.
assembled gold nanoparticle arrays at the liquid−liquid
interface to assist LDI MS with high desorption efficacy and
reproducibility in quantification (RSD < 5%) that can be used
to quantify glucose in the cerebrospinal fluid, allowing rapid
identification of patients with brain infections.117 The LDI MS-
based small molecule applications in biofluid profiling have
similarities with section 4.2 of this review. Both are about
biomarker discovery and disease diagnosis, but here we focus
on low molecular weight compounds, while section 4.2 mainly
concerns macromolecules (i.e., proteins and peptides).
Cell metabolism involves the dynamic changes of low
molecular weight compounds associated with the intrinsic and
extrinsic states of cells.118 Since individual cells have their
unique metabolic behaviors, the cell metabolites detected by
MALDI-TOF MS can be used for cell typing. For example,
Zhang et al. used TiO2-assisted LDI MS to profile the
metabolites of intact bacterial cells to detect the strains with
antimicrobial resistance. 119 Walker et al. detected 24
metabolites in single yeast cells using LDI MS.120
As mentioned above, the main obstacle that limits the
application of MALDI in the analysis of small molecules is the
selection of the matrix. Although great efforts have been made
by researchers to develop suitable matrices, more efforts are
necessary to promote the extension of LDI-based techniques to
clinical use for the analysis of small molecules or metabolites.
For this purpose, more studies should be focused on
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 Table 1. Summary of Clinical Applications of MALDI-TOF MS
application types common analytes principal methods main limitations potential solutions
pathogen (1) bacterial, yeasts, and proteins or polypeptides (1) sample profiling coverage of spectral database is limited (1) upgrade databases including expand
identification fungi identification35 species or strain coverage30 and improve
current spectral quality
(2) pattern matching high levels of interfering components and low concentrations of (2) enrich bacteria by magnetic beads,47
target pathogens in clinical samples resulted in a low positive test microfluidic techniques,48 etc.
rate
(3) develop matrixes or signal enlargement
methods43
(2) antimicrobial resist- antibiotic molecules, modification products, (1) sample profiling low accuracy in bacterial antimicrobial-resistance detection directly develop algorithms or data analysis
ance detection40 component of bacterial cells, ribosomal from MALDI-TOF MS profiling frameworks49 for deep data mining50
ACS Measurement Science Au

methylation, mutations, etc.

(2) feature extraction
(3) model establishment
and assessment
disease diag- (1) cancer diagnosis and proteins and peptides (1) biofluid profiling unfavorable repeatability in quantitative analysis limits clinical (1) develop matrix, deposition methods, and
nosis by prognosis65 application signal enlargement methods73
peptidome
(2) infectious disease74 (2) feature extraction (2) improve internal standard, ionization
and/or biomarker dis- process, and postdata acquisition
covery processing78
(3) neurodegenerative (3) model establishment (3) develop multicenter clinical validation
disease diagnosis80 and assessment work over a long period
nucleic acids (1) single-nucleotide DNA and RNA (1) gene locus selection, developed method relies on a group of preselected sites, so that it determining the target sites before
analysis polymorphism (SNP) primer design, and cannot comprehensively detect the highly random mutational MALDI-TOF MS analysis is necessary

399
genotyping88 quality control patterns in tumor suppressors
(2) DNA methylation (2) PCR amplification
identification89
(3) inherited genetic (3) single-base extension
disease screening93
(4) model establishment
and assessment
small mole- (1) tissue amino acids, lipids, sugars, short peptides, (1) biofluid profiling lack of suitable matrix for different kinds of small molecules develop matrix to enhance the detection
cules analy- fingerprinting114 alcohols, organic acids, etc. sensitivity of specific types of molecules103
pubs.acs.org/measureau

sis
(2) biofluid profiling116 (2) feature extraction
and/or biomarker dis-
covery
(3) cellular typing119 (3) model establishment
and assessment
Review

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ACS Measurement Science Au pubs.acs.org/measureau Review

developing matrices that can enhance the detection sensitivity 200000, China; orcid.org/0000-0002-6233-8459;
of specific types of molecules, show reliable quantification Email: liang_qiao@fudan.edu.cn
performance among multicenters, and remain consistent from
batch-to-batch for massive production. Authors
Dandan Li − Department of Chemistry and Shanghai
5. CONCLUSIONS Stomatological Hospital, Fudan University, Shanghai
200000, China; orcid.org/0000-0003-1495-4297
Through keyword co-occurrence analysis of more than 2000
Jia Yi − Department of Chemistry and Shanghai
previously published reviews with “MALDI” as the keyword in
Stomatological Hospital, Fudan University, Shanghai
the Web of Science database, the four most important MALDI
200000, China
clinical applications, “pathogen identification”, “disease diag-
Guobin Han − Department of Chemistry and Shanghai
nosis”, “nucleic acids analysis”, and “small molecules analysis”,
Stomatological Hospital, Fudan University, Shanghai
are summarized. On the basis of the dynamic changes of
200000, China
research focus on MALDI, an overlay visualization of the
keywords with publication years is also presented. Around Complete contact information is available at:
2010, MALDI was mainly used for the analysis of nucleic acids https://pubs.acs.org/10.1021/acsmeasuresciau.2c00019
with few related extension topics and late-stage research.
Considering that MALDI-based nucleic acid analysis was Author Contributions
developed earlier, the technology is relatively mature. The D.L. wrote the draft of the manuscript. J.Y., G.H., and L.Q.
decade from 2011 to 2021 has seen a focus on MALDI-TOF revised and finalized the manuscript.
MS-based research in “disease diagnosis “ and “small molecules Funding
analysis”, with the former having a relatively wider range of
clinical applications than the latter that mostly focuses on This work was supported by the National Natural Science
matrix development. The most significant applications of Foundation of China (NSFC, 22022401, 22074022, and
MALDI in disease diagnosis or prognosis mainly include 21934001) and the Ministry of Science and Technology of
“breast cancer”, “ovarian cancer”, “Alzheimer”, and “lung China (2020YFF0304502).
disease”. For the technology of MALDI-TOF MS-based Notes
disease diagnostics, the difficulties in transferring it to the The authors declare no competing financial interest.
clinic include biological verification of the biomarkers and
validation of the established methods by multicenter trials.
Since 2015, the theme of “pathogen identification” has
attracted considerable attention. MALDI-TOF MS-based
■ VOCABULARY
MALDI-TOF, MALDI (matrix-assisted laser desorption/
ionization) is an ionization method that uses laser energy to
bacterial identification mostly focuses on techniques for the
generate ions from molecules with minimal fragmentation
isolation and purification of pathogens from clinical samples,
with the assistance of a matrix that can absorb the laser
the expansion of spectral libraries, and the upgrading of
energy; TOF (time-of-flight) is a common high-throughput
software. As technology advances, many MALDI-based
and middle−high-resolution mass analyzer to couple with
microbial identification databases and systems have been
MALDI
licensed and put into clinical use. Nevertheless, it is still
bloodstream infections, bloodstream infections are infec-
necessary to develop MALDI-TOF MS-based antimicrobial-
tious diseases defined by the presence of viable pathogenic
resistance analysis for comprehensive clinical microbiology
microorganisms in the bloodstream
characterization. The important applications of MALDI in
keyword co-occurrence, keyword co-occurrence is a cluster
clinical research, including specific application categories,
method to display the keywords with the strongest citation
common analytes, main methods, limitations and solutions,
bursts, i.e., using bibliometric and visualization methods
have been summarized in Table 1.
SNP genotyping, SNP (single-nucleotide polymorphism)
In summary, the development of clinical applications of
genotyping is the measurement of genetic variations of
MALDI-TOF MS has been dominated by methodological
SNPs between members of a species
innovations, from sample pretreatment (e.g., desalting, sample
DNA methylation, DNA methylation is a biological process
spotting, matrix selection, etc.) and development of highly
by which methyl groups are added to DNA molecules
sensitive methods (e.g., enrichment, signal amplification, etc.)
VOSviewer, VOSviewer is software for the construction of
to data analysis (e.g., data process, algorithm development,
relationships between the structure, the evolution, and the
etc.). Besides, there could be some innovations at the front end
collaboration of knowledge domains
of the study design, such as delineating clinical staging and
typing, as well as selecting sample types and molecular mass
analysis ranges. Upon completion of a study, multidimensional
validation may be considered to improve the reliability of the
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