Identification of harmless and pathogenic algae of the genus Prototheca by MALDI-MS.

Abstract

The only plants infectious for mammals, green algae from the genus Prototheca, are often overseen or mistaken for yeast in clinical diagnosis. To improve this diagnostical gap, a method was developed for fast and reliable identification of Prototheca. A collection of all currently recognized Prototheca species, most represented by several strains, were submitted to a simple extraction by 70% formic acid and ACN; the extracts were analyzed by means of MALDI-MS. Most of the peaks were found in the range from 4 to 20 kDa and showed a high reproducibility, not in absolute intensities, but in their peak pattern. The selection of measured peaks is mostly due to the technique of ionization in MALDI-MS, because proteins in the range up to 200 kDa were detected using gel electrophoresis. Some of the proteins were identified by peptide mass fingerprinting and MS(2) analysis and turned out to be ribosomal proteins or other highly abundant proteins such as ubiquitin. For the preparation of a heatmap, the intensities of the peaks were plotted and a cluster analysis was performed. From the peak-lists, a principal component analysis was conducted and a dendrogram was built. This dendrogram, based on MALDI spectra, was in fairly good agreement with a dendrogram based on sequence information from 18S DNA. As a result, pathogenic and nonpathogenic species from the genus Prototheca can be identified, with possible consequences for clinical diagnostics by MALDI-typing.

Full text

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RESEARCH ARTICLE
Identification of harmless and pathogenic algae
of the genus Prototheca by MALDI-MS
Martin von Bergen1?, Angelika Eidner2?, Frank Schmidt1, Jayaseelan Murugaiyan1,
Henry Wirth1,3, Hans Binder3, Thomas Maier4and Uwe Roesler2,5
1Department of Proteomics, UFZ – Helmholtz-Centre for Environmental Research, Leipzig, Germany
2Institute of Animal Hygiene and Veterinary Public Health, University of Leipzig, Leipzig, Germany
3Interdisciplinary Centre for Bioinformatics, University of Leipzig, Leipzig, Germany
4Bruker Daltonics, Bruker Daltonik GmbH, Leipzig, Germany
5Institute of Animal Hygiene and Environmental Health, Free University Berlin, Berlin, Germany
Received: November 29, 2007
Revised: November 22, 2008
Accepted: January 15, 2009
The only plants infectious for mammals, green algae from the genus Prototheca, are often
overseen or mistaken for yeast in clinical diagnosis. To improve this diagnostical gap, a
method was developed for fast and reliable identification of Prototheca. A collection of all
currently recognized Prototheca species, most represented by several strains, were submitted
to a simple extraction by 70% formic acid and ACN; the extracts were analyzed by means of
MALDI-MS. Most of the peaks were found in the range from 4 to 20kDa and showed a high
reproducibility, not in absolute intensities, but in their peak pattern. The selection of
measured peaks is mostly due to the technique of ionization in MALDI-MS, because proteins
in the range up to 200kDa were detected using gel electrophoresis. Some of the proteins were
identified by peptide mass fingerprinting and MS2analysis and turned out to be ribosomal
proteins or other highly abundant proteins such as ubiquitin. For the preparation of a
heatmap, the intensities of the peaks were plotted and a cluster analysis was performed. From
the peak-lists, a principal component analysis was conducted and a dendrogram was built.
This dendrogram, based on MALDI spectra, was in fairly good agreement with a dendrogram
based on sequence information from 18S DNA. As a result, pathogenic and nonpathogenic
species from the genus Prototheca can be identified, with possible consequences for clinical
diagnostics by MALDI-typing.
Keywords:
MALDI-typing / Prototheca
1Introduction
Colorless algae of the genus Prototheca within the Chlor-
ellaceae family are the only known plants that cause
infectionsinhumansand
status of Prototheca has been evolving in recent decades
and five species are currently assigned to this genus:
P. zopfii, P. wickerhamii, P. blaschkeae, P. stagnora and
P. ulmea [1–3]. Furthermore, P. zopfii is divided into two
animals.Thetaxonomic
genotypes (1 and 2) with different antigen patterns and
biochemical features [3, 4]
Numerous studies have reported a pathogenic potential
for P. wickerhamii and P. zopfii. The cases of human
protothecosis are predominantly caused by P. wickerhamii
and occur as local (predominantly cutaneous) and systemic
infections mainly in immune-compromised patients, e.g.
patients infected with HIV or treated with glucocorticoids
[5–8]. P. blaschkeae were isolated from some cases of
onychomycosis [3]. Canine protothecosis is caused by
P. wickerhamii and P. zopfii, and is characterized by similar
clinical symptoms as in humans [9].
?These authors contributed equally to this work.
Correspondence: Dr. Martin von Bergen, Department of Proteo-
mics, UFZ – Helmholtz-Centre for Environmental Research,
Permoserstr. 15, D-04318 Leipzig, Germany
E-mail: Martin.vonbergen@ufz.de
Fax: 149-341-235-1786
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.clinical.proteomics-journal.com
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World-wide, P. zopfii has been identified to induce a
therapy-resistant inflammation of the mammary gland in
dairy cows, which may lead to severe economic losses in an
infected herd. Here, isolates assigned to ‘‘variant’’ or geno-
type 2 were reported to be the infectious agents [10–13]. On
the other hand, P. zopfii genotype 1 is also often isolated in
cattle barns, and has to be designated only as a milk
contaminant. Because of its epidemiological impact and
because of the broadly occurring resistance of the patho-
genic Prototheca isolates against antimycotics, the correct
identification of P. zopfii, P. blaschkeae and P. wickerhamii
isolates is of considerable importance in clinical micro-
biological laboratories.
The diagnosis of Prototheca spec. is still based upon the
time-consumingcultivation
medium, and on the additional investigation of lactophenol
cotton blue stained cells by light microscopy [14–16].
However, because of the Candida-like appearance of the
grown colonies and slow growth of most Prototheca strains,
these methods are uncertain. Comparative investigations
by means of Fourier-transformed infrared spectroscopy
showed distinct differences between all Prototheca species,
including P. blaschkeae, the former ‘‘variant 3’’ of P. zopfii.
However, the discrimination of the two P. zopfii genotypes
(the former variants 1 and 2 [17]) remains difficult. P. zopfii
genotypes can currently be differentiated by sequence analy-
sis of the 18S rDNA, or by diagnostic PCR or RFLP [3].
Although this method is the most accurate available up to
now, this test is not capable of differentiating Prototheca sp.
from yeast and the different strains of Prototheca from each
other in a single run.
This diagnostical gap can be overcome by using MALDI-
TOF MS spectra for identification. The possibility of iden-
tifying microorganisms by a combination of MALDI-TOF
MS spectroscopy and advanced statistical analysis was
first reported about a decade ago [18–20]. In principle,
samples are obtained by simple extraction and then
measured by MALDI-TOF MS (for review see [21]). The used
range of 3–20kDa excludes smaller metabolites. The
obtained peaks, however, do not necessarily represent
proteins. On the one hand, MALDI-TOF MS strongly favors
proteins and peptides smaller than 20kDa with some
dependence upon the matrix used [22]. In reference to
the term ‘‘proteome,’’ the collection of proteinagous mole-
cules falling in this molecular range are referred to as the
peptidome [23]. These molecules are well suited for both
reverse phase chromatography and MALDI-TOF MS,
which were widely used to generate peptidome-wide
approaches for analytical and diagnostical purposes [24]. On
the other hand, the population of smaller peptides can be
dominated by irreproducible variations of breakdown
products, so that for biomarker discovery more stable
proteins are preferred [25].
For identification of microorganisms on the basis of
extracted peptides, these have to be of intermediate or high
abundance in order to obtain reliable results. Furthermore,
onSabouraud-dextrose-agar
they have to be expressed constitutively, because even slight
variance in growing conditions can have gross effects on
protein expression patterns in microorganisms [26, 27]. One
group of peptides that fulfil all these criteria are the ribo-
somal proteins such as the 56 ribosomal proteins of E.coli,
which ranges from 4364 to 29727Da [28] and are constitu-
tively expressed with quite high abundance. The suitability
of ribosomal proteins for identification has been reported
recently [21, 29].
The identification of microorganisms requires compila-
tion of a database with the reference spectra of known
species as a first step. There are commercially available
databases that currently comprise spectra of approx. 1500
different species [30]. In this approach, a number of spectra
from one species are applied to the BioTyperTMsoftware
where the discriminating peaks are extracted. There is also
academic freeware such as MS-Screener [31] and EXPAN-
DER [32] available, which can be used for the evaluation of
the data and are suitable for the purpose of identifying
microorganisms.
The combination of MALDI-measurement of whole-cell
lysates or extracts with PCA and cluster algorithms has been
used for the identification of a wide range of microorgan-
isms, covering Gram-negative and Gram-positive bacteria,
fungi and cyanobacteria [33–39]. Here a method is presented
which aims at identifying green algae of the genus Proto-
theca that can occur as infectious pathogens in animals and
humans. To support diagnostic applications we developed
an experimental protocol and built a database, which
allowed identification of isolates of Prototheca in environ-
mental and medical samples.
2Materials and methods
2.1Chemicals
All chemicals and solvents used were of pro analysis quality
and purchased from Sigma (Taufkirchen, Germany) or
Merck (Darmstadt, Germany). High purity water was
produced by an Ultra Clear UV plus system from SG GmbH
(Barsb. uttel, Germany).
The MALDI matrix solution was prepared by dissolving
6mg CHCA in 50% ACN/2.5% TFA in H2O.
2.2Culturing of algae strains
A total of 19 strains, representing 7 algae species (5 Proto-
theca spec. and 2 Chlorella spec.) and 2 genotypes of Proto-
theca zopfii were used in this study (Table 1). All strains were
either type, reference, or other well-characterized isolates
that had previously been identified by sequence and
biochemical analysis.
All strains were maintained on Sabouraud dextrose
agar (Merck) plates, excepting C. vulgaris, which was
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Table 1. Used Prototheca species, strains, and isolates
TaxaStrains
Designation Source/clinical signs
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 1
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca zopfii Genotype 2
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca blaschkeae
Prototheca wickerhamii
Prototheca wickerhamii
Prototheca wickerhamii
Prototheca wickerhamii
Prototheca wickerhamii
Prototheca wickerhamii
Prototheca wickerhamii
Prototheca wickerhamii
Prototheca wickerhamii
Prototheca stagnora
Prototheca stagnora
Prototheca ulmea
PZ I-1E, R, a)
PZ I-2E, R, a)
SAG 2063T, E, a) b)
Hey1E, a)
Hey 5E, a)
Hey 7E, a)
Hey 9E, a)
Hey 10E, a)
Hey 12E, a)
Hey 13E, a)
Hey 15E, a)
POT 1C, a)
Gron 1E, a)
SAG 263-7a) b) c) d)
SAG 263-10a) b) c) d)
SAG 2021T, C, a) b)
PZ II-2R, C, a)
PZ II-3R, C, a)
InsBC, a)
POT 2C, a)
POT 3C, a)
POT 4C, a)
DAWC, a)
Bras 1AC, a)
Bras 8C, a)
Bras 14C, a)
LAG 1C, a)
SAG 263-3C, a) b) d)
SAG 263-4C, a) b) d)
SAG 263-1a) b) d)
PZ III-1R, E, a)
PZ III-2R, E, a)
SAG 2064T, C, a) b)
PZ III-4E, a)
PZ III-5E, a)
PZ III-6E, a)
PZ III-7E, a)
PZ III-8E, a)
Au 66C, a)
Au 67C, a)
Au 70C, a)
LAG 10C, a)
ATCC 16529T, E, c)
CBS 157.74C, e)
CBS 344.82C, e)
RW 1C, a)
RW 2E, a)
RW 2aE, a)
RW 3E, a)
ATCC 30395C, c)
BuWC, a)
ATCC 16528T, E, c)
UTEX 1442E, d)
ATCC 50112T, E, c)
Pig manure, Germany
Pig manure, Germany
Pig manure, Germany
Pig feces, Germany
Pig feces, Germany
Pig feces, Germany
Pig feces, Germany
Pig feces, Germany
Pig feces, Germany
Pig feces, Germany
Pig feces, Germany
Bovine mastitis, Germany
Pig feces, Germany
Environment (?)
Sap from wounded Robinia pseudoacacia
Bovine mastitis, Germany
Bovine mastitis, Germany
Bovine mastitis, Germany
Human systemic infection (HIV assoc.), Austria
Bovine mastitis, Germany
Bovine mastitis, Germany
Bovine mastitis, Germany
Rectal swab from dog with chronic colitis, USA
Bovine mastitis, Brazil
Bovine mastitis, Brazil
dog with hemorrhagic enterocolitis, Brazil
Bovine mastitis, Belgium
Human intestine
Human intestine
Sap from wounded tree
Cattle manure, Germany
Cattle manure, Germany
Human onychomycosis, Germany
Cattle manure, Germany
Cattle manure, Germany
Cattle manure, Germany
Cattle manure, Germany
Cattle manure, Germany
Bovine mastitis, Germany
Bovine mastitis, Germany
Bovine mastitis, Germany
Bovine mastitis, Belgium
Human sewage
Human systemic infection
Human dermatitis
Human dermatitis, Germany
Environment, Germany
Environment, Germany
Environment, Germany
Human palmar lesions
Human gastroenteritis
Sludge, Lebanon
Digested sludge, USA
Sap from wounded Ulmus americana, USA
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cultured on Bold’s Basal Medium. The Prototheca strains
were incubated at 251C in the dark. The photosynthetic
algae A. protothecoides and C. vulgaris were cultured at room
temperature under light exposure.
2.3Preparation of protein extracts
A standard protocol, in which colonies were directly applied
to a MALDI-target [30], was modified by introducing a
washing step. In brief, isolated colonies from agar plates
were suspended in 300mL water followed by the addition of
900mL absolute ethanol. After centrifugation at 12000rpm
for 2min the supernatant was removed. The pellet was
washed three times by cycles of resuspension and centrifu-
gation in 1mL water. The final cell pellet was dissolved in
50mL of 70% formic acid followed by the addition of 50mL
ACN and thorough mixing. The suspension was centrifuged
at 12000rpm in a bench-top centrifuge for 5min at room
temperature, then 0.5mL of the clear supernatant was
spotted in duplicates onto the MALDI target (MTP
AnchorChip, Bruker Daltonik, Bremen, Germany). After air
drying, each sample was overlaid with 1.5mL of CHCA
matrix solution and allowed to dry for several minutes
before the MALDI-TOF MS measurement.
2.4 SDS-PAGE
For 1-D SDS-PAGE [40], 50mg protein were precipitated
with a final concentration of 10% trichloroacetic acid and
washed twice with acetone [41]. The final pellets were
redissolved in 10mL sample buffer, heated for 5min at 601C
and applied to the gel (4% acrylamide concentration in
stacking and 12% acrylamide concentration in separating
gel); the protein was visualized by silver staining [42]. For
separation of peptides, 30mg of protein extract was applied
to a peptide gel (4% acrylamide in stacking, 10% acrylamide
in spacer gel and 16.5% in separating gel) and stained by
Coomassie Blue.
2.5 MALDI-MS for biotyping and protein
identification by nano-LC-ESI-MS
A standard protocol, in which colonies were directly applied
to a MALDI-target [30], was modified by introducing a
washing step. In brief, isolated colonies from agar plates
were suspended in 300mL water followed by the addition of
900mL absolute ethanol. After centrifugation at 12000rpm
for 2min the supernatant was removed. The pellet was
washed three times by cycles of resuspension and centrifu-
gation in 1mL water. The final cell pellet was dissolved in
50mL of 70% formic acid followed by the addition of 50mL
ACN and thorough mixing. The suspension was centrifuged
at 12000rpm in a bench-top centrifuge for 5min at room
temperature, then 0.5mL of the clear supernatant were
mixed with DHB and spotted in duplicates onto a MALDI
target (MTP ground steel, Bruker Daltonik).
Protein bands of interest were cut from polyacrylamide
gels and digested overnight using trypsin (Sigma, Germany)
as described elsewhere [42]. The cleaved peptides were
eluted, concentrated by vacuum centrifugation and then
separated by RP nano-LC (LC1100 series, Agilent Technol-
ogies, Paolo Alto, California; column: Zorbax 300SB-C18,
3.5mm,150?0.075mm2;
0–60% ACN). The peptides were identified by on-line
MS/MS (LC/MSD TRAP XCT mass spectrometer, Agilent
Technologies) [43]. Subsequently, a database search was
conducted using the MS/MS ion search (MASCOT, http://
www.matrixscience.com) against all fungal entries of
NCBInr (GenBank) with the following parameters: trpysin
digestion, up to one missed cleavage, fixed modifications:
carbamidomethyl (C), and with the following variable
modifications: oxidation (M), peptide tol.: 71.2Da, MS/MS
tol.: 70.6Da and peptide charge: 11, 12 and 13.
eluate:0.1%formicacid,
2.6Data analysis
Peaks were detected from the raw mass spectra using the
centriod algorithm from FlexAnalysis 2.4 with S/N56
Table 1. Continued
TaxaStrains
DesignationSource/clinical signs
Chlorella protothecoides
Chlorella protothecoides
Chlorella protothecoides
Chlorella saccharophila var. ellipsoidea
ATCC 30407E, c)
UTEX 249E, d)
SAG 211-10aE, b)
SAG 211-1aT, E, b)
Sap from wounded Populus alba
Environment
Sap from wounded Ulmus sp.
Freshwater
T, Type strain; R, Reference strain; C, Clinical isolate; E, Environmental isolate.
a) Culture Collection of the Institute of Animal Hygiene and Veterinary Public Health of the University Leipzig, Leipzig, Germany.
b) Culture Collection of Algae at the University of Go ¨ttingen (SAG), Go ¨ttingen, Germany.
c) American Type Culture Collection (ATCC), Manassas, USA.
d) The Culture Collection of Algae at The University of Texas (UTEX), Austin, USA.
e) Centraalbureau voor Schimmelcultures (CBS), Baarn, Neederlands.
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(Bruker Daltonics) where only the highest 100 peaks were
labeled to normalize the spectra. The generated peak lists
were exported and processed using the MS-Screener
program (Version 1.0.1) [31]. To aggregate the peak lists into
matrices for subsequent generation of heatmaps the
Expander [32] software was used; this software is based on
cluster analysis using PAST [44].
Hierarchical agglomerative clustering was performed
based upon mass spectra binned with an accuracy
of 3.0Da and preprocessing using quantile normalization.
The distance matrix between the spectra was calculated
using the average linkage metrics based on Pearson
correlation coefficients. The hierarchical agglomerative
algorithm successively merges the closest (most similar)
objects (spectra or groups of spectra). The final number
of clusters was visualized as a color-coded heatmap
(see Supporting Information). The algorithm was imple-
mented in C11 (program code is available on-line at
http://izbifs.izbi.uni-leipzig.de/?wirth/Prototheca.zip).
For comparison between MALDI-MS based biotyping
and genomic similarity measures, the 18S rDNA sequences
of the algae strains investigated were analyzed as recently
described [3]. In particular, we sequenced the 18S rDNA of
the Prototheca strains studied, then performed multiple
sequence alignment using CLUSTAL W [45] with the
MegAlignTMmodule of the LASERGENEssoftware package
(DNASTAR, Madison, WI, USA). The available 18S rDNA
reference sequences (GenBank) of some of the investigated
algae strains were included under the accession numbers
indicated in the tree (Fig. 4A). Finally, the aligned sequences
were analyzed by the neighbur-joining method [46] using the
TREECON&software without distance estimation.
3 Results
3.1Reproducible production and measurement of
protein extracts from algae strains
Any identification of pathogenic organisms based on MALDI-
spectra requires a simple and robust sample preparation and
reliable spectra. For sample preparation we added one simple
washing step to a well-established protocol [30]. To test the
reliability we analyzed the sources of variance by conducting
technical and biological replicates. The technical replicates
showed only minor deviations, whereas the biological replicates
exhibited larger variances especially in terms of peak intensities.
To reduce the technical variances, each measurement was
performed in four replicates as standard procedure. The peak
intensities obtained (e.g. the peaks at 6590.32, 8012.78 and
8806.49 m/z) vary by about 25% between the replicates. These
variances are only relevant if the peak intensity is of utmost
importance for classification; they can be neglected if different
species show completely different peaks. In the cross-species
comparison (Fig. 1) the differences in the peak pattern as such
in the spectra become obvious. The spectra obtained from
P. blaschkeae (SAG 2064; Fig. 3), P. zopfii Genotype 1 (SAG
2063; Fig. 1C) and P. zopfii Genotype 2 (SAG 2021; Fig. 1D)
differed slightly in intensity, but more importantly they showed
clear differences in the peak patterns. Beside the peaks detected
in MALDI-MS, which almost certainly represent proteins, a
great number of proteins with higher molecular weights were
also shown to be present in the extracts by 1-D SDS-PAGE (data
not shown).
3.2The extraction protocol yielded a wide range of
proteins; identification of proteins in the range
from 8 to 20kDa revealed highly abundant
ribosomal proteins
To validate the influence of the extraction method on the
composition of proteins, samples were subjected to 1-D
PAGE, suitable for separation of proteins from 10 to
200kDa (Fig. 2). The gel was stained by silver because of the
low protein content. The bands obtained range from below
10 to about 120kDa, whereas the MALDI-spectra show a
much narrower peak region (3–20kDa) owing to the selec-
tivity of the ionization process in MALDI-MS, which favors
smaller peptides. Some of the sample extracts were applied
to a peptide gel to assign the observed peaks (Fig. 4B). The
bands of interest, with an apparent molecular weight of
10–20kDa, were cut off and analyzed using mass spectro-
metry to identify the respective peptides
The eight most abundant proteins detected in the range of
8–20kDa provide evidence for ribosomal proteins (L30, S15,
S14, S11, S16, L21 and L12) and ubiquitin (Table 2). All
assignments were performed on the basis of cross-species
comparisons because of the lack of a sequenced Prototheca
species. Ostrecoccus tauri seems to be the most closely related
species with three hits in the MASCOT search, followed by
Chlamydomonas incerta. The quality of this identification
method is sufficient to claim unambiguous identification only
for the first six proteins, due to the lack of sequence infor-
mation about nearest phylogenetic relatives. For the ribosomal
proteins L21 and L12, the measurement of only one peptide
qualifies these identifications as likely, because the MASCOT
search was performed against the complete NCBI database.
3.3Differentially expressed proteins allow
discrimination of Prototheca species
Based on the 18S rDNA phylogenetic tree, the quality of the
protein-based phylogenetic trees was validated. 18S rDNA
sequence data for all Prototheca and Chlorella strains inves-
tigated were available from GenBank or were achieved and
presented in a recent study [3]. In the 18S rDNA-based
phylogeny, the strains of the two genotypes of P. zopfii as
well as of P. blaschkeae and P. wickerhamii forms consistent
individual clades and the investigated strains show a high
degree of homology within their genotype or species.
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X Data
0
1e+4
2e+4
3e+4
4e+4
5e+4
X Data
0
5e+4
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3e+4
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5e+4
0
5e+4
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2e+4
3e+4
4e+4
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5e+4
1e+4
2e+4
3e+4
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Intensity
0
1e+4
2e+4
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m/z
0
1e+4
2e+4
3e+4
4e+4
5e+4
A
B
C
D
E
F
G
H
4000 6000 8000 10000 12000
Figure 1. Comparison of spectra of different species. Repre-
sentative MS-spectra in the range from 3.5 to 12kDa from
different strains as follows: (A) Prototheca wickerhamii; (B)
Prototheca stagnora; (C) Prototheca wickerhamii; (D) Chlorella
protothecoides; (E) Prototheca zopfii Genotype 2; (F) Prototheca
zopfii Genotype 1; (G) Prototheca blaschkeae and (H) Prototheca
ulmea.
5050
50
4040
40
3030
30
2525
25
1010
10
6060
60
7070
70
85 85
85
100100
100
120120
120
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200
150150
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MW
in kDain kDa
in kDa
III-3-1III-3-2III-3-3
III-3-4
III-3-5
6
5
4
3
222
40
30 30 30
8
6
5
4
33
10 10 10
151515
202020
252525
50
40
40
777
111
III-3-1III-3-2
III-3-3
III-3-4III-3-5
MW
MW
III-3-1III-3-2III-3-3
III-3-4
III-3-5
8
6
5
4
50 50
III-3-1III-3-2
III-3-3
III-3-4III-3-5
MW
in kDa
8
III-3-1III-3-2
III-3-3
III-3-4III-3-5
A
B
Figure 2. Separation of protein extracts by SDS-PAGE. Protein
extracts from Prototheca blaschkeae used for MALDI-analysis
were applied to 10% (A) and 16.5% (B) acrylamide concentration
in the separation gel. The gels were stained with silver (A) and
with Coomassie Blue (B). The bands indicated with arrows were
cut and the proteins in them identified.
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For the preparation of the protein-based phylogenetic
tree, first a heatmap of the presence and intensity of the
peaks was generated (Fig. 3), based upon which the clus-
tering by Pearson correlation was performed and compared
with the DNA-based phylogenetic tree (Fig. 4).
This showed obvious features such as the high consis-
tency of the clustering within the different species. The
various spectra from one strain normally show a deviance of
less than 20%. The spectra of the biological replicates varied
from 0.06% (Goez6), typically 0.10% (Ulmea), up to 0.21%
(CBS1), with an average of 0.11% over all samples.
Another general aspect is the perfect clustering of spectra
belonging to a group of Prototheca species represented by
more than three strains. This rule applies for the species
P. wickerhamii, P. blaschkeae, P. zopfii Genotype 2 and
P. zopfii Genotype 1. Within these species the deviance
ranges from 0.70% (P. zopfii Genotype 2) via 0.82% (P. zopfii
Genotype 1) to 0.86% (P. wickerhamii and P. blaschkeae), but
these groups are clearly separated from each other and form
coherent clusters. The picture is different for the two species
C. protothecoides and P. stagnora. The spectra of these fall
into two groups, from which one group of C. protothecoides
shows a slightly higher similarity with the P. zopfii branch
(node at 92%) and the other for the P. wickerhamii branch
(node at 97% variance). On the basis of DNA sequences
there is a higher similarity toward the P. wickerhamii branch
of the phylogenetic tree.
A similar inconsistency was found for P. stagnora that
falls into two clusters: one is an outlier-group in relation to
most of the species, as also found on the basis of DNA
Figure 3. Heatmap of MS-spectra. The spectra are binned with an accuracy of 3.0Da and the intensity is plotted in terms of colours,
whereby red represents high and green low intensity.
780
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sequences, and the other showed 0.86 variance toward the
P. blaschkeae cluster. The spectra of the first group stem
from strain UTEX 1442 was isolated from digested sludge in
the USA, whereas the other strain (ATC 16528) was isolated
from sludge in Lebanon.
4Discussion
4.1Extension of MALDI-typing beyond bacteria
MALDI-typing was developed for the identification of bacteria
(for review see [47]) but it seems likely that the method can be
extended beyond the kingdom of eubacteria. Other studies
have shown the suitability of this method for the phylum of
cyanobacteria [35] or dermatophytes [48]. Here we have
adapted the method to single cell algae from the kingdom of
green plants. The main morphological difference to bacteria
is obviously not their size, but the composition of the cell wall
and cell membrane. Therefore, it is likely that initial failures
to obtain meaningful spectra can be assigned to these
features of algae cells. The standard sample preparation
procedure is applicable for nearly all kinds of microorganisms
similar to Gram-positive and Gram-negative bacteria and
yeast [30]. Limitations were sometimes observed for organ-
isms with very heavy, robust cell walls, such as certain fila-
mentous fungi. Beside the problem of opening the cells and
releasing the proteins of interest, cell surface modifications,
such as lipids or carbo-hydrates, can also interfere with the
MALDI measurement. In most cases, additional washing
steps (either with 70% ethanol or with pure water) before the
celldisruptionare sufficient
substances. For very robust organisms, an additional boiling
step before adding ethanol can improve MALDI measure-
ments significantly. The results presented here open the path
for using MALDI-typing for the identification of higher
organisms such as plants and animals.
to removetroublesome
4.2Comparison between classifications based on
nucleotides and proteins
The ‘‘gold standard’’ in the identification of microorganisms
has shifted in the past few decades from the morphological
and physiological analysis to a nucleotide-based one since
more and more sequence information and amplification
techniques became available. Recently, the so-called bar-
coding approach has started to generate a more complete
picture of evolutionary relationships between genera and
species. Therefore, a set of genes was selected that has been
sequenced in a great number of species from all taxa [49]. In
this approach the 18S-rDNA genes are of utmost impor-
tance, although also the sequences from other proteins such
as cytochrome oxidase were also used. However, sequencing
is not a screening technique, so most often specific primers
are used for PCR or RFLP analysis. These techniques have
P. zopfii GT2, SAG 2021
P. zopfii GT2, PZ II-2
P. blaschkeae, SAG 2064
P. wickerhamii, CBS 344.82
P. wickerhamii, ATCC 16529
P. wickerhamii, CBS 157.74
P. stagnora, ATCC 16528
P. ulmea, ATCC 16529
Aux. protothecoides, ATCC 30407
Ch. vulgaris, SAG 211-1b
P. zopfii GT1, PZ I-1
P. zopfii GT1, PZ I-2
P. zopfii GT1, SAG 2063
P. zopfii GT2, PZ II-3
P. blaschkeae, PZ III-1
P. blaschkeae, PZ III-2
A
B
Figure 4. Comparison of a gene-based phylogenetic tree with a
protein-based phylogenetic tree. The phylogenic tree based on
18SRNA (A) is shown in comparison to a phylogeny based on
MS-spectra (B).
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the potential for application as screening procedures, but
reduce the amount of information from the sequence.
Resources are limited, and although the technique of in-
depth sequencing has been extensively developed, it is still
too costly for such application, leaving a gap and a need for
fast and reliable techniques for species identification. The
technique of MALDI-typing has yielded valuable results for
bacteria and the comparison to the ‘‘gold standard’’ of RNA-
gene phylogeny showed that this holds also true for the
identification of Prototheca species.
In principle, the informational content of a nucleotide
sequence is threefold higher than that of the corresponding
protein and, in addition, the protein can exist in multiple
modifications not coded genomically. The development of
mass spectrometers has increased the resolution space from
about 50 peaks in the range from 400 to 2000Da to that
measured in this study by nearly two orders of magnitude by
using nano-LC coupled instruments. Consequently, shotgun
approaches are already capable of detecting more than 3000
proteins in a single run [50] and will probably also come into
common use for species identification. Recent studies have
shown the capacity of shotgun mass mapping to enable clas-
sification down to the strain level in the case of Lactobacillus
[51]. This wealth of informational content will improve the
resolution and reliability of MS-based identification. Beside
the nontargeted approach of using the complete proteome
species, identification can also make use of focussing on
specific classes of proteins, e.g. membrane proteins [52] that
are of special importance in pathogenic bacteria. The problem
of condition-dependent protein expression is circumvented by
taking most into account constitutively expressed proteins,
and in addition by preparing a data set that contains master
spectra derived from several reference spectra.
4.3Differentiation and identification of pathogenic
and nonpathogenic algae species
Based on the 18S rDNA phylogenetic tree, the quality of
the protein-basedphylogenetic approach
MALDI-MS was proven. The cladogram based upon
MALDI-MS results resembled mostly the one based on 18S
rDNA analysis. The inconsistencies, e.g. spectra of P. stag-
nora and C. protothecoides each falling into two different
clusters, might be explained by the different heritage of the
strains. In the case of P. stagnora one was isolated from
digested sludge in the USA, the other from sludge in
Lebanon. The different sources might have caused a
different protein expression pattern (‘‘Biotypes’’) that
remained even under changed cultivation conditions.
Another possible reason is the missing 18S rDNA sequence
data of the two outlier-group strains of P. stagnora (strain
UTEX 1442) and C. protothecoides (strain SAG 211-10a).
Therefore, it seems to be possible that these two strains are
currently not assigned to their correct taxon.
However,apart from minor
DNA-sequence-based phylogenetic tree, the performed
MALDI-MS analysis of intact proteins has been proven to
allow a fast and accurate identification of the currently
described pathogenic Prototheca species and genotypes (e.g.
P. wickerhamii, P. blaschkeae and P. zopfii genotype 2) as well
as of some closely related green algae species.
basedupon
deviances from the
4.4 Conclusions
The described MALDI-MS analysis combined with unsu-
pervised clustering provides a novel valuable tool for
Table 2. Identified proteins
Spot
No.
Swiss-Prot
Acc. No.
Entry nameProteinScore Mr
pI Seq.
cov. %
Pept.
mat. No.
1
2
P42739
Q7XYA1
UBIQ_ACECL
Q7XYA1_GRIJA
Ubiquitin (Acetabularia cliftonii, green algae)
Ribosome protein L30 (Griffithsia japonica, red
algae)
40S ribosomal protein S15 (Chlamydomonas
incerta, green algae)
40S ribosomal protein S14 (Chlamydomonas
incerta, green algae)
40S ribosomal protein S11 (Dunaliella tertiolecta,
green algae)
40S ribosomal protein S16 (ISS) (Ostreococcus
tauri, green algae)
Q00U75_OSTTA 60s ribosomal protein L21 (ISS) (Ostreococcus
tauri, green algae)
Q01BX3_OSTTA putative 60S ribosomal protein L12 (ISS)
(Ostreococcus tauri, green algae)
778536
12410
6.56 36%
9.75 10%
2
3 111
3 Q1WLZ3 Q1WLZ3_CHLIN6414936 10.05 13%2
4 P46295RS14_CHLRE 122 16349 10.31 15%2
5 P42756 RS11_DUNTE8517912 10.43 9%4
6Q018M7
Q018M7_OSTTA
10918066 10.43 10%2
7 Q00U75 6119441 10.79 4%1
8Q01BX3 75 24761 11.11 6%1
The proteins identified from the gel in Fig. 2 are summarized by listing the Swiss-Prot accession number, the uniprot accession number,
the protein identification, and the MASCOT-MOWSE score as well as the molecular weight, pI, sequence coverage and number of
peptides used for identifications. Except for the samples 7 and 8, for all other proteins at least two peptides were found; also for samples 7
and 8 significant MOWSE scores were achieved.
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detecting Prototheca species. In comparison to the classical
microbiological tests this method is faster and offers a
higher reliability, if performed by trained personnel. More-
over, MALDI-MS analysis involves lower costs and effort
than the currently used genomic approaches (sequencing,
PCR, RFLP) and might thereby support the clinical diag-
nosis of Prototheca infections.
We thank Michaela Risch for excellent technical assistance.
The authors have declared no conflict of interest.
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