MALDI-TOF mass spectroscopy of yeasts and filamentous fungi for research and diagnostics in the agricultural value chain

TLDR: The role of MALDI-TOF MS as a tool for species identification; in particular with respect to DNA-based identification methods is discussed, and the value of custom-made reference spectra for MalDI biotyping is highlighted.

Abstract: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has become a standard tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics laboratory. The method is continuously being improved, and new applications for distinguishing strains, identifying metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in patient samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research. This review focuses on MALDI-TOF MS approaches for the identification of yeasts and filamentous fungi. In contrast to bacterial diagnostics, MALDI biotyping of fungi is more challenging and less established. We thus start by discussing the role of MALDI-TOF MS as a tool for species identification; in particular with respect to DNA-based identification methods. The review then highlights the value of custom-made reference spectra for MALDI biotyping and points out recent advancements of MALDI-TOF MS, mainly from the field of clinical diagnostics that may be adopted and used for fungal diagnostic challenges. The overview ends with a summary of MALDI-TOF MS studies of yeasts and filamentous fungi of agricultural relevance.

Full text

Drissner and Freimoser
Chem. Biol. Technol. Agric. (2017) 4:13
DOI 10.1186/s40538-017-0095-7
REVIEW
MALDI-TOF mass spectroscopy
ofyeasts andlamentous fungi forresearch
anddiagnostics inthe agricultural value chain
David Drissner
1
and Florian M. Freimoser
2*
Abstract
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has
become a standard tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics
laboratory. The method is continuously being improved, and new applications for distinguishing strains, identify-
ing metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in patient
samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in
agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research.
This review focuses on MALDI-TOF MS approaches for the identification of yeasts and filamentous fungi. In contrast to
bacterial diagnostics, MALDI biotyping of fungi is more challenging and less established. We thus start by discussing
the role of MALDI-TOF MS as a tool for species identification; in particular with respect to DNA-based identification
methods. The review then highlights the value of custom-made reference spectra for MALDI biotyping and points out
recent advancements of MALDI-TOF MS, mainly from the field of clinical diagnostics that may be adopted and used
for fungal diagnostic challenges. The overview ends with a summary of MALDI-TOF MS studies of yeasts and filamen-
tous fungi of agricultural relevance.
Keywords: Agriculture, MALDI-TOF MS, Biotyping, Diagnostics, Filamentous fungi, Yeast
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(
http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/
publicdomain/zero/1.0/
) applies to the data made available in this article, unless otherwise stated.
Introduction
Matrix-assisted laser desorption ionization time-of-
flight mass spectrometry (MALDI-TOF MS; also called
MALDI biotyping) has become increasingly popular for
the identification of microorganisms and their functions
and is now approved for the routine identification of bac
-
teria and fungi in clinical diagnostic laboratories [
1–3].
It is the goal of this review to highlight recent techno
-
logical advancements of MALDI biotyping and to illus-
trate applications of this tool for research in agriculture
and the agricultural value chain. is outlook focuses
on MALDI-TOF MS applications in mycology, because
these are usually more demanding than bacterial identifi
-
cations [
4, 5] and will thus greatly benefit from the recent
advances in bacterial MALDI-TOF MS diagnostics. In
this context, it is important to note that MALDI-TOF
MS is not an alternative to DNA sequence-based species
identification, but rather a complementary method. In
order to make use and realize the potential of MALDI-
TOF MS it may thus be helpful bringing to mind the
advantages and disadvantages of this method; in particu
-
lar as compared to DNA-based assays and techniques.
MALDI biotyping andDNA-based identication
complement each other
e identification of fungi usually involves a DNA
sequence-based analysis that determines the identity of
a given isolate based on sequence similarity; often tak
-
ing advantage of the universal fungal barcode sequence of
the nuclear ribosomal internal transcribed spacer (ITS)
[
6]. Second and third generation sequencing techniques
now allow the sequencing of thousands or millions of
barcodes, or even entire genomes, in parallel and such
Open Access
*Correspondence: florian.freimoser@agroscope.admin.ch
2
Research Division Plant Protection, Agroscope, Schloss 1,
8820 Wädenswil, Switzerland
Full list of author information is available at the end of the article

Page 2 of 12
Drissner and Freimoser
Chem. Biol. Technol. Agric. (2017) 4:13
high-throughput technologies are available to virtually
every laboratory. ese barcode (amplicon) and metage
-
nome sequencing methods allow identifying hundreds or
thousands of species simultaneously and are thus power
-
ful tools to describe the composition of entire microbial
communities (Fig.
1a) [7–9]. However, although mas
-
sive parallel DNA sequencing generates a wealth of data,
often more data are generated than needed and storage
of the data and extraction of the required information is
challenging and often limiting [
10]. In addition, prepara
-
tion and quality control of the template libraries for DNA
sequencing is time consuming and costly (Table
1). In
specific cases, for the repeated identification of a defined
number of species, it may thus be best not to gener
-
ate the largest amount of data, but rather to generate
exactly the amount of data needed in the fastest and most
economical way. It is in this realm, where MALDI biotyp
-
ing shines (Fig.
1a; Table1).
In contrast to DNA sequencing-based approaches,
MALDI-TOF MS uses whole cells or crude, acidic
extracts, and mass spectra for the identification of indi
-
vidual species [
2, 11]. e simple sample preparation,
short measurement times, easy preparation of reference
spectra, and low costs per sample are important advan
-
tages of MALDI-TOF MS, as compared to specific, DNA-
based assays that are often laborious to establish and
usually require costly reagents (Table
1) [1, 12]. MALDI-
TOF MS is also highly flexible and can be combined
with additional extraction or processing steps in order to
identify specific biomarkers, metabolites, or biochemical
functions. Because of these advantages, MALDI biotyp
-
ing has become a standard in routine clinical diagnostics
and MALDI-TOF MS devices have become readily acces
-
sible, also for researchers in other disciplines than clinical
diagnostics [
4, 13, 14]. is opens up new possibilities for
functional hypothesis-driven research.
Unless it is the goal to identify entire microbial com
-
munities, MALDI-TOF MS is the method of choice for
species identification, because it is fast, economical, and
many common bacteria and an increasing number of
fungi can be identified. In those cases where MALDI-
TOF MS does not reveal the identity of a particular
organism, classical Sanger sequencing or DNA-based
assays (e.g., qPCR or LAMP assays) are used for identi
-
fication (Fig.
1a; Table1). Once the organism is identified
based on such an assay, the generation of new MALDI-
TOF MS reference spectra will allow identifying this
species or isolate in the future (Fig.
1b). e iterative pro
-
cedure comprises DNA-based identification and MALDI-
TOF MS reference spectrum generation for each new
organism continually increases the number of species
that can be identified and thus the benefit of a particu
-
lar MALDI-TOF MS system (comprises robust software,
reliable algorithms, and databases). In particular in large
culturomics projects, an integration of MALDI-TOF MS
in the identification pipeline results in a comprehensive
database of reference spectra that allows identifying a
plethora of species and even challenges, or rather com
-
plements, DNA sequencing-based analyses of microbi-
omes [
15, 16].
Custom-made reference spectra improve
MALDI-TOF MS-based identication
DNA-based identification methods benefit from large,
public, DNA sequence repositories, and many openly
accessible or downloadable analysis tools. In contrast,
MALDI-TOF MS reference spectra, as well as analysis
tools, are usually proprietary, only commercially avail
-
able, and heavily focused on medical applications. Even
Many species /
community
MALDI
biotyping
Barcode / metagenome
NGS sequencing
qPCR, LAMP assay,
Sanger sequencing
Fungal identification
«problem»
1 or few species
Correct species / OTUs identified
MALDI
biotyping
Species
identified
Species NOT
identified
Create reference
spectrum
a
b
Fig. 1 MALDI biotyping and DNA-based identification complement
each other. a MALDI biotyping is the method of choice for the iden-
tification of a defined number of species. b Generating MALDI-TOF
MS reference spectra for fungal species identified by DNA-based
methods allows the rapid and economical detection of this species
by MALDI biotyping in the future

Page 3 of 12
Drissner and Freimoser
Chem. Biol. Technol. Agric. (2017) 4:13
organisms commonly found in environmental, agricul-
tural, or food samples are thus often not recognized by
standard, commercial MALDI-TOF MS systems. In addi
-
tion, MALDI-TOF MS spectra are not universal, such as
a DNA sequence, but depend on the crude cell extract or
the physiological state of the fungal cells that are applied
onto the target plate and used for recording mass spectra.
Consequently, the age and growth conditions of a micro
-
bial culture, as well as the settings of the ionizing laser,
flight tube, and mass detector, can influence a MALDI
biotyping experiment.
Many of these problems can be overcome by generat
-
ing custom-made, specific MALDI-TOF MS reference
spectra for the particular application in question [
5].
Since these reference spectra are generated with the same
MALD-TOF MS device, settings, and sample prepara
-
tion as the experimental samples, the scores obtained
with these references are usually higher as compared to
generic reference databases [
5, 17–19]. Custom-made
reference spectra thus allow ample flexibility with respect
to different sample preparation protocols and experimen
-
tal set-ups in a particular lab or for specific applications.
For example, reference spectra for different cell densi
-
ties of a particular organism may allow an estimation of
cell densities [
20]. It is of course also possible to gener
-
ate reference spectra for fungi grown on different agar
plates, different physiological states, different extraction
protocols, or directly applied bacterial or yeast cells (as
opposed to crude extracts).
e integration of custom-made reference spectra in
a commercial MALDI-TOF MS system is only the first
step towards a truly open platform for MALDI-TOF
MS-based species identification. Although it is easy
to generate reference spectra, a comprehensive public
repository of MALDI-TOF MS reference spectra is lack
-
ing. Such a resource would greatly facilitate the exchange
of reference spectra between research groups and thus
allow identifying microorganisms for which no refer
-
ence spectra have been generated in a particular lab [
21].
Until now, only few MALDI-TOF MS spectra databases
for microbial identification are publicly available. e
FoodBIMS database comprises reference mass spectra
of food-borne bacteria [
22] (http://bioinformatica.isa.
cnr.it/Descr_Bact_Dbase.htm
). SpectraBank is a freely
accessible database that also comprises mainly bacterial
reference spectra [
23], and the free Spectra site hosts an
extended database of MALDI-TOF MS reference spec
-
tra for bacteria and fungi (which is made available by
the Public Health Agency of Sweden (Folkhälsomyn
-
digheten):
http://spectra.folkhalsomyndigheten.se/spec-
tra/welcome.action).
In addition to MALDI-TOF MS data repositories,
instrument- and provider-independent analysis tools
for microbial biotyping using mass spectra are urgently
needed. SPECLUST is an application to perform clus
-
ter analyses of MALDI-TOF MS spectra and was for
example used to separate the bacterium Ralstonia sola
-
nacearum into different species [
24, 25]. More recently,
Starostin et al. [
26] developed a tool to use geomet
-
ric distances between MALDI-TOF MS spectra rep-
resented in a multi-dimensional space to distinguish
closely related Bacillus strains. Mass-up is a comprehen
-
sive, open-source tool for the processing and analysis of
MALDI-TOF MS data [
27]. Besides classification, it has
also biomarker discovery, principle component analy
-
sis, and clustering functions implemented and was, for
example, used to fingerprint bacterial isolates or classify
peritoneal dialysis patients by mass spectrometry-based
Table 1 MALDI-TOF- andDNA sequencing-based approaches complement each other
Properties and comparison of MALDI biotyping and DNA sequencing-based approaches (of barcodes/PCR products or genomic DNA) methods for the identication
of fungi. References: [
100, 101]
The advantages of each method are shown in italics
MALDI biotyping qRT-PCR/lamp assay Sanger sequencing NGS sequencing
Starting material Crude extracts or whole cells DNA extract from mixed
sample
DNA extract from pure
sample
DNA extract from com-
munity
Premises Reference spectrum Specific assay developed Clone or PCR product avail-
able
None/PCR product
Equipment costs (machines) High Low-medium High High
Cost per sample Negligible Low-medium Medium High
Time for sample preparation Short Short-long Long Long
Time for analysis Short Medium Medium Medium-long
Data Mass spectrum of one
sample
Detection of one DNA frag-
ment
DNA sequence of 400–
1000 bp
In total 50 Mbp–1000 Gbp
Number of species/strains
detected
Usually 1 per sample 1 per assay 1 per sample Up to many thousands
Cultivation step required Usually yes No Usually yes No

Page 4 of 12
Drissner and Freimoser
Chem. Biol. Technol. Agric. (2017) 4:13
profiling [
28, 29]. For baseline correction, normaliza-
tion, peak detection, and matching, it uses open-source
packages such as MALDIquant [
30] and MassSpec Wave
-
let [
31], which are also available as separate R packages.
Finally, BIOSPEAN (
http://software.cr-hana.upol.cz/bio
-
spean/login.php
) is a web-based application and database
that was specifically developed for analyzing whole-cell
MALDI-TOF MS data and includes peak picking, genera
-
tion of MS databases, and data sharing among users [
32].
e public deposition of MALDI-TOF MS spectra,
together with information concerning culture condi
-
tions and experimental details is highly desirable and
could largely expand the potential of MALDI-TOF MS
for agricultural and food diagnostics, as well as ecologi
-
cal research. Standardized extraction buffers (an acidic
extraction using formic acid and acetonitrile seems often
used) and particularly matrix solutions (e.g., α-cyano-
4-hydroxycinnamic acid, HCCA) would much improve
comparability of results among different laboratories.
However, since different types of cells and organisms
require different extraction buffers and matrices for the
best MALDI-TOF MS spectra, including this information
in the reference spectra and in biotyping experiments
seems necessary.
Clinical MALDI-TOF MS applications benet fungal
diagnosis andresearch
e low sample preparation costs and fast measurement
time, in addition to its accuracy, are highly attractive
properties of MALDI-TOF MS; in particular for clini
-
cal applications. MALDI biotyping of clinically relevant
microorganisms is thus more advanced than of other
microbes and new methods are first introduced and
tested with clinical samples. Consequently, the majority
of MALDI-TOF analyses of fungi so far have dealt with
clinical isolates [
33]. In particular, the rapid and eco
-
nomical identification of Candida species is an impor-
tant medical application [
34–39], but filamentous fungi
present in clinical samples have been studied by MALDI
biotyping as well [
40–43]. Overall, MALDI-TOF MS is
an accurate, reliable, and rapid method for the identifi
-
cation of human pathogenic fungi, and new applications
for clinically relevant yeasts and filamentous fungi are
continuously being developed [
43–45]. A comparison
of two commercially available systems, VITEK MS (bio
-
Mérieux) and MALDI Biotyper (Bruker Daltonics) with
their associated databases, has shown similar identifica
-
tion efficiencies of clinically relevant yeasts for the two
systems [
46, 47], while other studies found differences
between biotyping systems [
48, 49]. e identification
power is improved by including in-house generated refer
-
ence spectra in the databases [
17, 43, 46] (also see above).
In addition, it was shown that the sample preparation
method and quality of the database are crucial for accu-
rate identification [
38]. In general, identification rates
for yeasts are above 90% and higher when using acidic
extracts, as compared to direct transfer of whole cells
[
38, 50]. In many cases, clinical diagnostics of human
pathogenic bacteria and fungi drives the development of
new MALDI biotyping approaches that will also benefit
fungal diagnosis and research in other areas. It therefore
seems worthwhile discussing new MALDI-TOF MS tech
-
niques that are mainly used in the clinical setting in order
to highlight the potential and outline opportunities for
MALDI-TOF MS of yeasts and filamentous fungi.
Shortening the cultivation time forfaster MALDI-TOF MS
identication
In clinical diagnostics, as in agricultural and food safety
diagnostic, time-to-result and cost are the most impor
-
tant criteria for selecting analytical methods and may,
in the most extreme situation, literally be a matter of
life or death. e cultivation step, protein extraction,
and pure cultures, in many cases required for a success
-
ful MALDI-TOF MS identification, may thus prevent
this method from being used [
51]. erefore, efforts are
undertaken and considerable progress has been made to
address these shortcomings. For the diagnosis of bacte
-
rial pathogens responsible for bloodstream infections the
mean incubation time to identify Gram-positive bacteria
was 5.9h, which dropped to 3.1h if a crude acidic extract
was prepared [
52]. In other studies, 97 to 69.5% of bac
-
teria were correctly identified after a short incubation of
positive blood cultures on solid media of only 3 to 5 h
[
53–56], which enabled identification on the same day as
the positive blood sample was detected. By increasing the
sample concentration (by reducing the sample spot size),
creating reference spectra for different cell densities, and
immunoaffinity enrichment of bacteria, it was possible to
detect as few as 10 to 100 bacterial cells after a blood cul
-
ture time of only 4h [
20]. ese examples illustrate the
vastly shortened time-to-result of clinical MALDI-TOF
MS applications: the duration of the cultivation step has
been reduced to just a few hours and is thus in the same
range as DNA amplification steps. In other studies of
positive urine or blood samples, or of cerebrospinal fluid,
the cultivation step was omitted entirely, and pathogenic
bacteria and yeasts were directly identified [
57–59].
MALDI-TOF MS applications beyondspecies identication
MALDI-TOF MS is not only used for taxonomic iden-
tification, but also able to draw functional conclusions
about clinically relevant properties of a particular isolate
[
60]. Since MALDI-TOF MS is, in principle, able to iden
-
tify any ionizable compound [
61], it can detect antibiotic
resistance (e.g., via the identification of specific proteins

Page 5 of 12
Drissner and Freimoser
Chem. Biol. Technol. Agric. (2017) 4:13
or degradation products of antibiotics), but has also been
used to reveal recombinant proteins, plasmid insertions
in bacteria, or other biomarkers and diagnostic peptides
in bacteria [
62–66].
Recently, MALDI-TOF MS has been employed to rap
-
idly and simultaneously detect and identify the Alter-
naria mycotoxins alternariol, monomethyl ether, and
tentoxin in cereal grains [
67]. It may be promising adapt
-
ing this method for the identification of mycotoxins in
further food products in the near future. e identifi
-
cation of clinically relevant anaerobic bacteria has also
greatly benefited from MALDI-TOF MS approaches. is
slow growing and fastidious group of pathogens has been
mainly diagnosed by biochemical tests, but MALDI-TOF
MS approaches have successfully identified such anaero
-
bic pathogens and could delineate antibiotic-resistant
and antibiotic-susceptible strains within the same species
[
68–70]. In the case of pathogenic yeasts, the sibling spe
-
cies of the Cryptococcus gattii/Cryptococcus neoformans
complex, which cannot be discriminated by routine bio
-
chemical techniques, have been distinguished by MALDI
biotyping [
71]. Similarly, MALDI-TOF MS could reliably
separate closely related members of the genus Saccha
-
romyces (S. arboricola, S. bayanus, S. cariocanus, S. cer-
evisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and S.
pastorianus) [
72]. Finally, MALDI-TOF MS more rapidly
identified reduced susceptibility to caspofungin or tria
-
zoles in Candida and Aspergillus species, as compared
to classical determination of the minimum inhibitory
concentration [
39, 73, 74]. With yeast cells from positive
blood cultures and prepared by using the Sepsityper kit
(Bruker Daltonics), direct MALDI-TOF MS identified
the Candida species in 62.5% of the samples and anti
-
fungals susceptibility results were obtained for 72.7% of
the blood samples [
35]. Alterations in protein profiles of
fungi following the exposure to antifungal compounds
could be similarly used to monitor fungicide resistance in
agriculturally relevant species.
Sample processing can broaden the use ofMALDI
biotyping
As for the highly sensitive species identification, dis-
cussed above, sample processing (e.g., tryptic digestion,
acidic/organic extraction, nano-liquid chromatography)
can greatly increase the sensitivity and specificity of a
MALDI-TOF MS method and thus enable subspecies
identification [
66, 75]. A further possibility is the func
-
tional modification of the MALDI target plates them-
selves, which was for example performed with antibodies
for direct immunoaffinity MALDI-TOF MS of hapto
-
globin or by dioxide coating in order to enrich phospho-
peptides [
76, 77]. In order to increase the number of mass
peaks that may be used as biomarkers or the sensitivity of
a MALDI-TOF MS analysis, samples have been success-
fully treated with detergent, sonication, corona plasma
discharge, or heat [
78–80]. In another study, target bacte
-
ria were separated and enriched without prior cultivation
by using magnetic nanobeads that were functionalized
with specific antibodies [
20]. A rapid sample pretreat
-
ment consisting of removal of interfering blood cells and
centrifugation and washing steps, which can also be per
-
formed with a commercially available kit (Sepsityper), is
crucial for obtaining high identification rates of micro
-
organisms, including yeasts, directly in positive blood
cultures [
81, 82]. ese examples highlight that sample
pretreatments are promising tools that may also benefit
applications in agricultural diagnostics.
MALDI-TOF MS ofpolymicrobial samples andinfected
material
A standard MALDI biotyping experiment requires pure
cultures of the microbial species to be identified. How
-
ever, the identification of microorganisms from mixed
or complex samples derived, for example, from patients,
the environment, or infected plant tissue or food by
MALDI-TOF MS is an important goal. So far, only a few
reports have demonstrated the identification of bacteria
and microalgae in mixtures by using biomarkers or cor
-
relation coefficients [
83–85]. ese studies have shown
that ion suppression can affect the detection of specific
masses of one or the other microorganism and thus com
-
plicate species identification in mixed samples [
83, 84,
86]. On the other hand, novel mass peaks only observed
in the mixed sample were discovered and may serve as
biomarkers for mixtures or contaminations [
83, 86]. For
samples that contain more than two organisms, species
identification based on biomarkers that invariably indi
-
cate the presence of a particular species performed better
than correlation-based methods [
84].
Fungal MALDI biotyping asa tool foragricultural
diagnostics andresearch
MALDI biotyping of yeasts and filamentous fungi is
more difficult than bacterial identifications, because the
former result in less mass peaks and fewer reference
spectra are available [
4, 5]. In contrast to clinically rele
-
vant fungi, only a limited number of fungal plant patho-
gens, postharvest diseases, or food contaminants have
been detected by MALDI biotyping. e non-medical
applications of MALDI-TOF MS for fungal diagnostics
and research that we are aware of are summarized in
Table
2. ese studies document that in particular the
genera Fusarium, Trichoderma, and Saccharomyces are
being used as models for the development of MALDI-
TOF MS applications and for assessing the potential of
this technology. In contrast to clinical studies, naturally