Tài liệu Báo cáo khoa học: Direct identification of hydrophobins and their processing in Trichoderma using intact-cell MALDI-TOF MS docx

Direct identification of hydrophobins and their processing
in Trichoderma using intact-cell MALDI-TOF MS
Torsten Neuhof
1
, Ralf Dieckmann
1,
*, Irina S. Druzhinina
2
, Christian P. Kubicek
2
,
Tiina Nakari-Seta
¨
la
¨
3
, Merja Penttila
¨
3
and Hans von Do
¨
hren
1
1 TU Berlin, Institut fu
¨
r Chemie, FG Biochemie und Molekulare Biologie, Berlin, Germany
2 FB Gentechnik und Angewandte Biochemie, Institut fu
¨
r Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, TU Wien,
Vienna, Austria
3 VTT Technical Research Centre of Finland, Espoo, Finland

Hydrophobins are small proteins thought to be ubi-
quitous in filamentous fungi. They are usually present
on the outer surfaces of cell walls of hyphae and coni-
dia. Here, they mediate interactions between the fun-
gus and the environment, such as surface recognition
during pathogenic interactions with plants, insects or
other fungi, and also in symbiosis. The size of hydro-
phobins ranges from approximately 75 to 400 amino
acid residues; they contain eight positionally conserved
cysteine residues, and can be divided into two classes
according to their hydropathy profiles and spacing
between the conserved cysteines [1].
The anamorphic fungal genus Trichoderma (Hypocre-
ales, Ascomycota) contains cosmopolitan soil-borne
fungi with economic importance as biocontrol agents
and producers of beneficial metabolites and enzymes. In
addition, Trichoderma spp. have recently been reported
to occur as endophytes, eliciting positive plant responses
against potential pathogens [2]. Hydrophobins are likely
to play a role in this process, and a hydrophobin gene
has in fact recently been isolated that leads to overpro-
duction of hydrophobins during endophytic interactions
between Trichoderma asperellum and cucumber roots
[3]. However, hydrophobins may also be involved in the
Keywords
fungal biomarker; hydrophobin; intact-cell
MS; MALDI-TOF MS; Trichoderma
Correspondence
H. von Do
¨

hren, TU Berlin, Institut fu
¨
r
Chemie, FG Biochemie und Molekulare
Biologie, Franklinstr. 29, 10587 Berlin,
Germany
Fax: +49 30 314 24783
Tel: +49 30 314 22697
E-mail:
*Present address
AnagnosTec, Gesellschaft fu
¨
r Analytische
Biochemie und Diagnostik mbH, Potsdam-
Golm, Germany
(Received 19 September 2006, revised 27
November 2006, accepted 6 December
2006)
doi:10.1111/j.1742-4658.2007.05636.x
Intact-cell MS (ICMS) was applied for the direct detection of hydropho-
bins in various species and strains of Hypocrea ⁄ Trichoderma. In both myce-
lia and spores, dominating peaks were identified as hydrophobins by
detecting mass shifts of 8 Da of reduced and unreduced forms, the analysis
of knockout mutants, and comparison with protein databases. Strain-speci-
fic processing was observed in the case of Hypocrea jecorina (anamorph
Trichoderma reesei). An analysis of 32 strains comprising 29 different spe-
cies of Trichoderma and Hypocrea showed hydrophobin patterns that were
specific at both at the species and isolate (subspecies) levels. The method
therefore permits rapid and direct detection of hydrophobin class II com-
positions and may also provide a means to identify Trichoderma (and other

fungal) species and strains from microgram amounts of biomass without
prior cultivation.
Abbreviations
HFB, hydrophobin; ICMS, intact-cell MALDI-TOF MS.
FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS 841
mechanism of mycoparasitism as well as the coloniza-
tion of decaying wood.
Our information about the roles of hydrophobins
in the physiology of Trichoderma, as well as in other
fungi, is mostly derived from reversed genetics. Little
is known about the occurrence and processing of the
individual hydrophobins on the fungal surface. As a
method for the rapid detection of hydrophobins
from a large number of small samples, we have
investigated the potential of intact-cell MALDI-TOF
MS (ICMS). Initial applications of ICMS on fila-
mentous fungi have demonstrated characteristic sets
of ions for strain identification in mycelia [4–8] and
spores [9–12]. We will show here that class II hydro-
phobins account for the main characteristic peaks of
Trichoderma, as the intact-cell extraction procedure
employing a solvent mixture of acetonitrile and
methanol is suitable for dissolving these cell wall
constituents.
Results
Identification of new Trichoderma hydrophobins
by EST search
In order to determine which hydrophobins could
potentially be detected in Hypocrea jecorina, we first
mined its genome database for members of class II

hydrophobins. Besides the well-known HFB1 and
HFB2, the HFB3 hydrophobin has been identified by
cloning the corresponding gene [13] and further char-
acterization of the protein [14]. We here identified
HFB4, HFB5, and HFB6. In addition, two hydropho-
bin-encloding EST sequences were retrieved from
the TrichEST database ():
one encoding an ortholog of HFB3 from T. longibra-
chiatum (L22T11P141R12690, L14T53P137R01628,
L22T11P138R12431, and L22T11P137R12300), and
the other one encoding an ortholog of HFB1 of
T. atroviride (L12T11P119R10608). Their sequence
relationships and putative processing sites are illustra-
ted in the alignment given in Fig. 1.
ICMS analysis of Trichoderma
Several strains of Trichoderma were studied initially to
examine the effectiveness of ICMS as an analytical
method for distinguishing different species of Trichoder-
ma. A rapid analytical procedure based on ICMS was
established in order to characterize the low-molecular-
weight proteometric (up to 20 000 Da) and peptidomet-
ric (up to 2000 Da) profiles at the same time. Thirty-two
Trichoderma strains belonging to various species were
subcultivated on agar plates at an incubation tem-
perature of 25 °C and analyzed without further pre-
treatments as described in Experimental procedures.
Vegetative mycelia or spores were transferred from the
biomass growing on agar plates directly to the MALDI
sample plate and mixed with an acidic matrix in an
organic solvent mixture. An estimated 10

6
cells were
used per spot. Data obtained from triplicate samples
grown for different times confirmed that the MS analy-
ses were reproducible with respect to the characteristic
biomarkers obtained. Figure 2 shows typical MALDI-
TOF mass spectra of four strains of H. koningii, T. long-
ibrachiatum, H. virens and T. atroviride. The spectra
Fig. 1. Alignment of the six hydrophobin protein sequences of H. jecorina. Protein accession numbers and first amino acid of the protein
after predicted signal peptide cleavage are: HFB1 (P52754; Q17), HFB2 (S62621; A16), HFB3 (trire.GWV1.31.87.1, scaffold 31: 136 622–
136 948; A17), HFB4 (estExt_fgenesh1_pg.C_50116, scaffold 5: 390 006–390 493; D25), HFB5 (trire.GWV1.11.179.1, scaffold 11: 162 998–
163 354; A17), HFB6 (trire.GWV1.3.266.1, scaffold 3: 1 189 586–1 190 177; no predicted signal peptide).
Trichoderma hydrophobins T. Neuhof et al.
842 FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS
showed a characteristic set of mass peaks in the range
of 5–10 kDa, typically including two dominating peaks
at approximately m ⁄ z 7000. As mycelia and spores
largely remained intact, and the extraction solution
contained acetonitrile and methanol, the well-known
hydrophobins were suspected to be the signal source,
and some were assigned by database analysis.
Identification of the class II hydrophobins
produced by H. jecorina
In order to identify the already known hydrophobin
peaks and to validate the method described here, we
first performed a detailed analysis of H. jecorina ¼
T. reesei QM 9414. To date, two major hydrophobin
(HFB) proteins have been characterized in detail: the
97-residue HFB1-precursor with a molecular mass of
9874.32 Da is processed to a 75 amino acid peptide

with a molecular mass of 7540.58 Da, which is further
reduced by disulfide bond formation to 7532.58 Da
[15]. The 86 residue HFB2 precursor with a mass of
8766.28 Da is processed to a 71 amino acid peptide
with a calculated molecular mass of 7196.42 kDa, and
further reduced by disulfide formation to 7188.42 Da
[15]. Both hydrophobins were detected as [MH]
+
sig-
nals of the oxidized forms (Hfb1, m ⁄ z 7533; Hfb2,
m ⁄ z 7189). A minor peak of m ⁄ z 7041 presumably cor-
responds to the processed Hfb2 lacking the terminal
Phe (7041.24 Da) (Fig. 3).
The same peaks were observed in the spectra
obtained from isolated reference substances of HFB1
and HFB2 proteins (Fig. 3C). A second minor peak of
m ⁄ z 7229 correlates with oxidized HFB2 cleaved at
Ala13 lacking the N-terminal Phe. This tentative corre-
Fig. 2. Intact-cell MALDI-TOF mass spectra
of mycelia and spores of Trichoderma
strains. The masses 7347 and 7494 of
T. atroviride spores correlate with two proc-
essed products of the spore hydrophobin
SRH1 [16] cleaved at the N-terminal MQFSI-
VALFATGALA site and the C-terminal Phe,
respectively.
Fig. 3. Intact-cell MALDI-TOF mass spectra
of H. jecorina strain QM 9414 (D), and the
mutant strains QM 9414 Dhfb1 (B) and
QM 9414 Dhfb1Dhfb2 (A). HFB I and HFB II

indicate the processed hydrophobins.
Whereas the HFB I peak is missing in the
disruption mutant, the proportions of the
processed HFB2 hydrophobins have been
shifted. The double knockout (A) has no sig-
nificant mass peaks in this region. As a con-
trol spectrum, the purified hydrophobins are
shown in (C).
T. Neuhof et al. Trichoderma hydrophobins
FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS 843
lation of mass data is achieved by the calculation of
successive deletions of terminal amino acid residues of
all six hydrophobins providing this single match.
A rapid means of identifying peaks corresponding to
hydrophobins in MALDI-TOF mass spectra was elab-
orated, making use of the fact that hydrophobins con-
tain eight cysteines forming four disulfide bonds.
Reduction of the four disulfides with dithiothreitol
increased the masses of all peaks corresponding to
hydrophobins by 8 Da. Thus, the masses of processed
HFB1 and HFB2 show shifts from m ⁄ z 7533 to 7541
and from m ⁄ z 7189 to 7197, respectively (supplementary
Fig. S1). This characteristic feature of hydrophobins
could therefore be used as a diagnostic tool to identify
hydrophobin peaks in intact-cell MALDI-TOF spectra.
In order to prove that the peaks described above
originate from HFB1 and HFB2, hfb1D and
hfb1 ⁄ hfb2DD strains were also analyzed to confirm the
peak assignments: The double mutant hfb1
–

⁄ hfb2
–
did
not show HFB1 and HFB2 signals (Fig. 3A), whereas
Fig. 4. Intact-cell MALDI-TOF spectra of mycelia (A, C, E) and sporulating mycelia (B, D, F) of three strains of H. jecorina grown on malt
agar. The masses displayed have an error of about 0.1%, so peaks of 7232 (A), 7237 (E) and 7234 (F) represent similar peptides. Strain 618
mycelia (A) show a variety of peaks, in contrast to strains 665 and 937, shown in (C) and (E). However, there are only few similarities: 7232
and 7237 in (A) and (E), or 7509 and 7514 in (A) and (C). An obvious shift is the appearance of higher mass peaks upon sporulation, presum-
ably related to the only large hydrophobin of H. jecorina.
Trichoderma hydrophobins T. Neuhof et al.
844 FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS
in the knockout mutant hfbI
–
, only the respective mass
peak was missing (Fig. 3B).
Deviating post-translational processing
of hydrophobins in H. jecorina strains
To investigate strain diversity with respect to meta-
bolite production and low-mass proteomics by ICMS,
three phylogenetically described isolates of H. jecorina
were studied. As shown in Fig. 4, spectra of mycelia
and sporulating mycelia directly taken from the
plates after 1 or 3 days, respectively, differ in peak
compositions and intensities. Surprisingly, all spectra
differ with respect to strain QM 9414. Strain
CPK 618 mycelia show a prominent signal of
m ⁄ z 7232 (Fig. 4A), which disappears in the sporula-
tion process, with new signals of m ⁄ z 8859, 8802 and
7521 appearing (Fig. 4B). To obtain a preliminary
correlation of observed masses with hydrophobin

data, we again calculated from the available sequence
data sets of masses for each hydrophobin, succes-
sively subtracting terminal residues and introducing
disulfide bonds. The m ⁄ z 7232 peptide could thus be
tentatively assigned to the hydrophobin HFB3 in the
Fig. 4. (Continued).
T. Neuhof et al. Trichoderma hydrophobins
FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS 845
oxidized form, with cleavage of the N-terminal pep-
tide at Ala31 and the C-terminal dipeptide at
Pro102. The same mass is also observed in strain
CPK 937 (Fig. 4E,F) as a prominent signal in myce-
lia that almost disappears in the sporulation process.
Peaks of m ⁄ z 8858 and 8863 were also observed in
sporulating strains CPK 618 and CPK 665, respect-
ively (Fig. 4B,D). These correspond in mass to HFB5
with an N-terminal cleavage at Ala8 including oxida-
tion (8861 Da).
Comparison of the mycelial compositions of strains
CPK 665 (Fig. 4C) and CPK 937 (Fig. 4E) shows four
major peaks, all differing in mass, but none of them
corresponds to the QM 9414 strain (Fig. 3). The main
peak of CPK 665, m ⁄ z 7147, correlates with HFB1
with four disulfide bonds, cleaved N-terminally at
Arg22 and at the C-terminal tetrapeptide. In
QM 9414, HFB1I is not processed C-terminally. The
m ⁄ z 6999 peak, the second prominent peak of strain
CPK 665, can be assigned to Hfb2 C-terminally
cleaved at Lys66 and oxidized. This peak is not present
in any other strain investigated.

Although they are rather speculative, the interpreted
masses agree with verified cleavage sites observed for
HFB1 and HFB2 and known sites for signal peptidas-
es and Kex2-type peptidases (Table 1). Verification of
these assessments by tryptic digestion and sequencing
is in progress.
Hydrophobin patterns in other T. atroviride and
T. longibrachiatum strains
T. atroviride
A hydrophobin gene (srh1) encoding a class II hydro-
phobin with phylogenetic similarity to H. jecorina
HFB2 (I. S. Druzhinina and C. P. Kubicek, unpub-
lished results) has been found in T. atroviride (therein
named ‘T. harzianum’ [16]). The main components of
the sporulating mycelia of the same strain (T. atrovi-
ride P1) could indeed be assigned to this hydrophobin,
assuming similar post-translational processing as for
the H. jecorina HFB2 (Fig. 2, top spectrum). The
peaks at m ⁄ z 7499 and 7352 correspond to the proc-
essed spore hydrophobin SRH1 with the cleaved signal
sequence MQFSIVALFATGALA and an additional
C-terminal Phe cleavage, respectively, including loss of
8 Da for the disulfide bonds. A minor peak at
m ⁄ z 7741 could be tentatively correlated with the
SRH1 hydrophobin with N-terminal cleavage of
MQFSIVA, C-terminal processing following the two
Glu residues of AAAQGTF, and four disulfide bonds.
Interestingly, these peaks could not be detected in
vegetative mycelia of T. atroviride P1 (Fig. 2), which
displayed a similar peak pattern, but with slightly dif-

ferent masses of 7181, 7339 and 7739 Da. A database
search in TrichoEST for the presence of other T. atro-
viride hydrophobins led to the identification of an
HFB1-like protein, which, after N-terminal processing
(MKFFTAAALFAAVAIA), C-terminal processing
(AVGA) and disulfide bond formation, has a mass of
7743 Da.
T. longibrachiatum
The main mass peak of T. longibrachiatum of m ⁄ z 7242
was assigned to an HFB3-type of hydrophobin identified
by searching the TrichoEST database. The 10 235 Da
precursor peptide would have been cleaved at the unique
Arg site MQFLAVAALLFTAAFAAPSSEAHGL
RRR,
comprising 3 Arg residues in sequence (underlined), and
this would have been followed by the formation of four
disulfide bonds, leading to a mass of 7241 Da.
ICMS of class II hydrophobins can distinguish
Trichoderma strains at the subspecies level
The results described above for H. jecorina show that
even under carefully controlled culture conditions,
different strains of this taxon displayed different MS
fingerprints. Because of the ease and speed of the
analysis, we investigated whether the hydrophobin
class II pattern of Trichoderma could be used in
strain diagnosis at the species level. To this end, we
selected isolates for 29 different species of Hypo-
crea ⁄ Trichoderma and compared their hydrophobin
molecular masses. Table 2 shows that, indeed, all of
the species tested exhibited a unique combination of

peaks with unique molecular masses. It is therefore
interesting to note that even phylogenetically closely
related species (such as T. hamatum and T. asperel-
lum,orT. harzianum and T. fulvum [17], or T. fas-
ciculatum and T. strictipile, which were recently
revised to be actually the same species [18]), could be
clearly separated. This is in accordance with the data
on H. jecorina shown above, and implies that hydro-
phobin fingerprints can in fact distinguish isolates at
the subspecies level. All spectra are compiled in sup-
plementary Fig. S2.
Discussion
Hydrophobin patterns
Genome sequencing of filamentous fungi has revealed
the presence of multiple hydrophobin genes in filamen-
tous fungi. We here report the the sequences of four
Trichoderma hydrophobins T. Neuhof et al.
846 FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS
new type II hydrophobins for H. jecorina, in addition
to the known HFB1 and HFB2. Likewise, we identi-
fied new hydrophobins in T. atroviride in addition to
the known sporulation-specific one, and in T. longibra-
chiatum. Direct MS analysis of mycelia in differing
physiologic states provides evidence for differential
expression of these genes in relation to the morpho-
logic state. However, there is no clear match of the
observed mass peaks to the predicted propeptides
expected to originate from cleavage of signal peptides.
Instead, further processing has been observed, as has
been demonstrated before from N-terminal sequence

analysis of H. jecorina HFB1 and HFB2. Hydrophobin
patterns even suggest strain-specific multiple cleavages
of propepides.
Hydrophobin processing
The export of most type II hydrophobins involves both
signal peptide cleavage and maturation of the propep-
Table 1. Confirmed and predicted hydrophobin cleavage sites.
Strain Hydrophobin Mass N-terminal processing C-terminal cleavage Comments
H. jecorina
QM 9414
HFB1 7533 EDR ⁄ SNG – Non-Kex2 type
protease processing,
shown by N-terminal
sequencing
HFB2 7189 ALA ⁄ AVC – Signal protease at A15,
shown by N-terminal
sequencing
HFB2 7042 ALA ⁄ AVC – F Unknown protease,
predicted C-terminal
cleavage
HFB2 7229 TSA ⁄ LAA – F Signal protease at A13,
predicted C-terminal
cleavage
H. jecorina
strains 618 and
937
HFB3 7232 ANA ⁄ FCP PIP ⁄ AI Predicted sites of signal
peptidase and
predicted C-terminal
cleavage by unknown

protease
HFB4 8862 TVA ⁄ LFI – Predicted cleavage
within signal peptide
H. jecorina
strain 665
HFB1 7147 EDR ⁄ SNG CQT ⁄ AVG Confirmed and
predicted non-Kex2
sites
HFBII 6999 ALA ⁄ AVC CQK ⁄ AIG Confirmed signal
peptide site, predicted
non-Kex2 site
T. atroviride P1 SRH1 7499 ALA ⁄ SVS – Predicted signal
peptide cleavage
SRH1 7352 ALA ⁄ SVS – F Predicted signal
peptide cleavage,
predicted C-terminal
cleavage
SRH1 7741 IVA ⁄ LFA EE ⁄ AQG Predicted alternative
signal peptide
cleavage,
predicted C-terminal
cleavage
HFB1 7743 AIA ⁄ GPV CQT ⁄ AVG Predicted signal
peptide cleavage,
predicted C-terminal
cleavage
T. longibrachiatum HFB3 7242 RRR ⁄ DQA – Predicted Kex2 site
cleavage
T. Neuhof et al. Trichoderma hydrophobins
FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS 847

tides. A considerable amount of information is avail-
able on eukaryotic signal peptidase specificities, and
cleavage site predictions can be performed using,
for example, signalp ( />SignalP/) version 3.0 [19]). Owing to the limited
amount of information available on eukaryotic pro-
teins, an evaluation of version 2 reported a 78% accu-
racy [20]. In this study, a selection of verified and
predicted sites for hydrophobins is reported (Table 2).
On the basis of confirmed structural data of H. jecorina
HFB1, HFB2 and HFB3, we here predict the presence
and cleavage patterns of additional hydrophobins.
These data are based on calculated masses, which need
to be confirmed by other experiments, such as isolation
and sequencing work, or indirect studies employing
proteinase knockouts or proteinase inhibition. This
may indeed be speculative, but provides useful hypo-
thetical data based on: (a) selective extraction leading
to a limited set of no more than six small proteins; (b)
known cleavage sites of signal peptidases and fungal
Kex2-like proteinases; and (c) actual structural studies
of H. jecorina HFB1 and HFB2.
In H. jecorina, maturation of HFB1, HFB2 and
HFB3 includes signal protease processing that removes
peptides 15 and 16 amino acids in length. This is fol-
lowed by propeptide cleavage by a non-Kex2 protein-
ase in the case of HFB1, cleaving at a monobasic site,
and by Kex2 cleavage in the case of HFB3 [21],
Table 2. Biomarker masses of various Hypocrea ⁄ Trichoderma strains detected by ICMS. CBS, Centraalbureau voor Schimmelcultures, the
Netherlands; DAOM, Eastern Cereal and Oilseed Research Centre, Canada; ATTC, American Type Strain Culture Collection, USA. The iden-
tity of all strains was verified by sequencing internal transcribed spacer (ITS)1 and ITS2 and (if necessary) the long intron of tef1, and analyz-

ing the sequences by
TRICHOKEY [28] and TRICHOBLAST [29].
Species m ⁄ zm⁄ zm⁄ zm⁄ zm⁄ zm⁄ zm⁄ zm⁄ zm⁄ z
H. tawa CBS 246.63 7014 7214 7718 7895
H. semiorbis CBS 244.63 7024 7570
H. hunua CBS 238.63 7186 7269 7432 7594 7727
H. gelatinosa ATCC 7476 7167 7496
H. citrina CBS 977.69 7171 7440
H. aureoviridis CBS 254.63 7214 7309 7473 7611
T. strigosum CBS 348.93 7129 7490 7634
T. tomentosum CBS 349.93 7171 7399 7501
T. strictipilis CBS 347.93 7155 7346 7490
T. longipile CBS 340.93 7257 7313 7517
T. fasciculatum CBS 118.72
a
7120 7286
H. minutispora CBS 342.93 7282 7459
T. pubescens DAOM 162162 7255 7618
T. viride ATCC 28020 7141 7603
T. stromaticum CBS 101875 7093
T. flavofuscum CBS 248.59
b
7176 7307
T. citrinoviride IMI 232088 7021 7293 7584 7748
T. brevicompactum CBS 109720 7141
T. hamatum CBS 393.92 7055 7342 7616
T. asperellum CBS 358.97 7156 7368 7522
T. croceum DAOM 167068
c
7104 7188 7284

T. fulvum DAOM 175924 7333 7646
T. oblongisporum CBS 343.93 7240 7380 7470
H. lixii CBS 273.78 7223 7429 7835
T. longibrachiatum CBS 816.68 7242
d
7484
H. jecorina QM 9414 7042
e
7189
e
7229
e
7533
f
T. spirale CBS 346.93 7202 7481 7721
H. virens CBS 249.59 7183
H. koningii CBS 460.96 7073 7456
T. atroviride ATCC 74058 7186 7344 7744
g
T. atroviride (spores) 7352
h
7499
h
7526 7739
i
a
T. fasciculatum is a synonym of H. strictipilis.
b
T. flavofuscum is a synonym of H. virens.
c

T. croceum is a synonym of T. polysporum
(¼H. pachybasioides).
d
Identified as HFB3 type with processing at the Arg site: MQFLAVAALLFTAAFAAPSSEAHGLRRR.
e
Identified as
processed HFB2 (see Fig. 2).
f
Identified as processed HFB1 (see Fig. 2).
g
Identified as processed HFB1-type hydrophobin: GPVEVRTGGG-
SICPDGLFSNPQCCDTQLLGIIGLGCEVPSQTPRDGADFKNICAKTGDQALCCVLPIAGQDLLCQA.
h
Identified as processed SRH1 (see Fig. 1).
i
Identified as processed SRH1: LASVSVCPNGLYSNPQCCGANVLGVAALDCHTPRVDVLTGPIFQAVCAAEGGKQPLCCVVPVAGQDLLCEE.
Trichoderma hydrophobins T. Neuhof et al.
848 FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS
whereas HFB2 is not cleaved further in the N-teminal
region. Mass spectra provide evidence for a C-terminal
Phe cleavage for HFB2.
The ICMS spectra presented here provide evidence
for similar cleavage patterns of hydrophobins HFBIII
of H. jecorina and T. longibrachiatum, and SRH1 and
HFB1 of T. atroviride, involving signal peptides, Kex2-
type processing, and C-terminal amino acid cleavage
(Table 2). In addition, the recorded masses provide
evidence for alternative processing reactions. One reac-
tion concerns alternative signal peptide cleavage sites
at Ala13 for H. jecorina HFB2 and at Ala7 for T. atr-

oviride SRH1. The predicted sites are compatible with
the specificity profiles of signal peptidases, whereas
their length is less than the average 15–40 residues.
The second type of reaction is the cleavage of C-ter-
minal peptides at Ala sites, e.g. at sites + 2 from the
last conserved Cys, at Lys-Ala positions, at Glu-Ala
positions, or at Thr-Ala positions. These predictions
need confirmation by further analysis.
Comparing ICMS of bacteria and fungi
ICMS provides a rapid means for distinguishing bac-
teria, spores, viruses and fungi [4–12]. It has the
advantages of being very rapid, using small samples
(subcolony amounts) and requiring minimal sample
preparation. Bacterial intact-cell MALDI-TOF spectra
in the range 2–20 kDa are dominated by a set of ri-
bosomal proteins as highly abundant intracellular
constituents [22]. This set of about 10–30 defined
masses permits the identification at the species and
subspecies ⁄ strain level. We here show that in filamen-
tous fungi, hydrophobins are the dominating protein
masses, whereas ribosomal proteins have not been
identified. This result can be ascribed to the unique
solubility properties of these hydrophobic proteins.
Although there are several hydrophobin genes pre-
sent, only a some of these might be expressed under
differing physiologic conditions. Thus, it has been
reported that the expression of hydrophobins is
dependent on the morphologic state of T. atroviride
(erroneously described as T. harzianum [16]). It has
also been shown that expression and localization of

specific hydrophobins in Cladosporium fulvum is
dependent on the stages of the plant infection pro-
cess, the hydrophobins being either retained on coni-
dia and aerial structures or being excreted [23]. In
Magnaporthe grisea, it has been demonstrated that
the formation of disulfide linkages is required for
secretion and cell wall localization [24].
Indeed, we have shown here that sporulating and
nonsporulating mycelia of several species differ in
hydrophobin composition. Unexpectedly, the patterns
observed indicate diverse cleavage reactions of the
respective prepropeptides. These patterns are unlikely
to be proteolytic artefacts of extraction, as proteases
are unlikely to be active in methanol ⁄ acetonitrile mix-
tures. As MALDI-TOF MS involves an especially gen-
tle ionization process, cleavages of peptide bonds are
generally not observed. Differences in hydrophobin
processing are thus interpreted as being dependent on
the presence and concentrations of specific proteinases
acting on the respective propeptides.
Hydrophobins as biomarkers for ICMS
of filamentous fungi
Hydrophobins are proposed to be suitable ICMS bio-
markers for the following reasons: (a) fungi contain a
set of hydrophobin genes, generally with developmen-
tally regulated expression; (b) the hydrophobic pep-
tides can be selectively dissolved and rapidly analyzed
by MALDI-TOF MS; (c) hydrophobin patterns are
diverse, due to post-translational processing; (d) the
presence of the characteristic four disulfide bonds can

be easily demonstrated by reduction; (e) owing to their
fairly small size, sequence information for the hydro-
phobins can be obtained by MS; (f) the respective
genes are accessible by standard PCR methods; and
(g) the variability of hydrophobins is fairly significant,
and exceeds the similarities of other biomarkers pro-
posed, e.g. ubiquitins. The sensitivity of detection
employing the MALDI-TOF technique can be expec-
ted to allow the identification of bacterial peptides
with  100 microbial cells.
Experimental procedures
Reagents and standards
2,5-Dihydroxybenzoic acid from Anagnostec (Potsdam,
Germany) was used as the matrix for MALDI-TOF experi-
ments. Trifluoroacetic acid, ethanol, acetonitrile and meth-
anol from Merck (Darmstadt, Germany) were used as
solvents. The reference hydrophobins HFBI and HFBII
were purified by two-phase separation and RP-HPLC puri-
fication as described previously [25].
Microbial strains and cultivations
The strains of H. jecorina used were: the wild-type strains
QM 6a, TUB F-1038 (CPK 618), TUB F-733 (CPK 665)
and CBS 498.97 (CPK 937) (all described in [26]); the cellu-
lase moderately overproducing mutant strain QM 9414
(ATTC 26921) [27]; and QM 9414 Dhfb1 (VTT D-99724
T. Neuhof et al. Trichoderma hydrophobins
FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS 849
[28]) and QM 9414 Dhfb1Dhfb2 (VTT D-99725 [25]). Other
Trichoderma spp. strains used in this study are listed in
Table 1.

Hypocrea jecorina QM 9414 and its mutants were cultiva-
ted in liquid cultures on microtiter plates (200 lL volume)
for 4 days in buffered minimal medium [15] complemented
with 3% glucose and 0.2% peptone. The other strains of
H. jecorina, as well as strains of other Trichoderma spp.,
were cultivated on malt extract agar (3%) at 25 °C.
Extraction and preparation of mycelia
for MALDI-TOF analysis
A few micrograms of fungal mycelia were suspended in
acetonitrile ⁄ methanol ⁄ water (1 : 1 : 1), and 1 lL of the sus-
pension was directly spotted onto target wells of a 100-posi-
tion sample plate and immediately mixed with 1 lLof
matrix solution [10 mgÆmL
)1
2,5-dihydroxybenzoic acid in
acetonitrile ⁄ methanol ⁄ water (1 : 1 : 1) and 0.3% trifluoro-
acetic acid]. The sample matrix mixture was allowed to air
dry prior to analysis. Alternatively, freeze-dried mycelium
obtained from shaken cultures or fungi grown on plates
was homogenized in 60% ethanol and centrifuged at
13 000 g using a Beckman Microfuge 11 (Beckman Coulter,
Unterscheissheim, Germany). One microliter of the protein
solution was spotted on a MALDI target plate and mixed
with matrix.
Reduction of disulfide bonds
For reduction of proteins containing disulfide bonds, cells
were suspended in 60% methanol, vortexed, and centri-
fuged at 13 000 g using a Beckman Microfuge 11, and the
supernatant was concentrated to dryness. The residual was
redissolved in 50 mm Tris ⁄ HCl (pH 8) and 1 mm dithio-

threitol and incubated for 1 h at room temperature.
Microbial characterization by MALDI-TOF MS
analysis
MS measurements were performed on a VOYAGER DE-
PRO TOF mass spectrometer from Applied Biosystems
(Foster City, CA, USA). Mass spectra were acquired in lin-
ear delayed extraction mode using an acceleration voltage
of 20 kV and a low mass gate of 1500 Da. For desorption
of the components, a nitrogen laser beam (k ¼ 337 nm)
was focused on the template. The laser power was set to
just above the threshold of ionization. Spectra for individ-
ual specimens were compiled, and results were averaged
from at least 100 shots taken across the width of the speci-
men for m ⁄ z values of 2000–20 000. In the linear mode, the
resolution was 2000, and the mass accuracy was
1000 p.p.m. (0.1%). Calibration was done with the H. jeco-
rina HFB1 protein with the 7533 Da calculated average
atomic mass. All masses determined correspond to average
atomic masses.
MS analysis of low-molecular-mass peptides
Measurements were performed in the delayed extraction
mode, allowing the determination of monoisotopic mass
values. A low mass gate of 800 Da improved the measure-
ment by filtering out the most intensive matrix ions. The
mass spectrometer was used in the positive ion detection
and reflector mode.
Database search and alignments ) H. jecorina
hydrophobins
The H. jecorina genome ( />trire1.home.html) was screened for hydrophobin-encoding
genes by using the tblastn (protein versus translated nuc-

leotide) program. We used the hydrophobin class II protein
sequences of other fungal species as queries to search the
H. jecorina genome. Then, all putative hydrophobins, inclu-
ding the newly identified hydrophobin from H. jecorina,
were used to identify further proteins with similar domains,
and finally all hypothetical proteins encoding hydrophobins
from the annotated genomes of the Broad Institute (http://
www.broad.mit.edu/), Neurospora crassa, Gibberella zeae
(Fusarium graminearum) and Magnaporthe griseae, were
also used.
Calculations of monoisotopic molecular masses of hydro-
phobins was performed with the expasy proteomics server
( or the peptide mass calculator
( To
correlate observed mass peak data, masses of various
N- and C-terminally processed and oxidized peptides were
calculated and compared.
Acknowledgements
This work was supported by a fellowship from the Deut-
sche Forschungsgemeinschaft (Do270 ⁄ 10) and by the
Fifth Framework program (Quality of Life and Man-
agement of Living Resources; Project EUROFUNG 2;
QLK3-1999-00729) of the European Community. The
T. reesei genome sequencing project was funded by the
Department of Energy. The authors thank M. Salohei-
mo for helpful comments and discussions.
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Supplementary material
The following supplementary material is available
online:
Doc. S1. Sequence data of hydrophobins.
Fig. S1. Dithiothreitol reduction of H. jecorina QM
9414 hydrophobins.
Fig. S2. Intact-cell MALDI-TOF spectra (6000–
10 000 m ⁄ z)ofTrichoderma ⁄ Hypocrea strains. All indi-
cated masses have been identified as hydrophobins by
an m ⁄ z shift of 8 upon dithiothreitol reduction.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-
ponding author for the article.
Trichoderma hydrophobins T. Neuhof et al.
852 FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS