Fast monitoring of species-specific peptide biomarkers using high-intensity-focused-ultrasound-assisted tryptic digestion and selected MS/MS ion monitoring

Article abstract of DOI:10.1021/ac200890w

A new strategy for the fast monitoring of peptide biomarkers is described. It is based on the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) and the monitoring of several peptides by selected MS/MS ion monitoring in a linear ion trap mass spectrometer. The performance of the method was established for the unequivocal identification of all commercial fish species belonging to the Merlucciidae family. Using a particular combination of only 11 peptides, resulting from the HIFU-assisted tryptic digestion of the thermostable proteins parvalbumins, the workflow allowed the unequivocal identification of these closely related fish species in any seafood product, including processed and precooked products, in less than 2 h. The present strategy constitutes the fastest method for peptide biomarker monitoring. Its application for food quality control provides to the authorities an effective and rapid method of food authentication and traceability to guarantee the quality and safety to the consumers.

Full text of DOI:10.1021/ac200890w

ARTICLE
pubs.acs.org/ac
Fast Monitoring of Species-Specific Peptide Biomarkers Using
High-Intensity-Focused-Ultrasound-Assisted Tryptic Digestion and
Selected MS/MS Ion Monitoring
Mꢀonica Carrera,*,† Benito Ca~nas,‡ Daniel Lꢀopez-Ferrer,§ Carmen Pi~neiro,† Jesꢀus Vꢀazquez,§ and
Josꢀe M. Gallardo†
†Marine Research Institute, Spanish National Research Council, Vigo, Pontevedra, Spain
‡Complutense University of Madrid, Madrid, Spain
§Severo Ochoa Molecular Biology Centre, Spanish National Research Council, Madrid, Spain
S Supporting Information
b
ABSTRACT: A new strategy for the fast monitoring of peptide
biomarkers is described. It is based on the use of accelerated in-
solution trypsin digestions under an ultrasonic field provided by
high-intensity focused ultrasound (HIFU) and the monitoring
of several peptides by selected MS/MS ion monitoring in a linear
ion trap mass spectrometer. The performance of the method was
established for the unequivocal identification of all commercial
fish species belonging to the Merlucciidae family. Using a
particular combination of only 11 peptides, resulting from the
HIFU-assisted tryptic digestion of the thermostable proteins
parvalbumins, the workflow allowed the unequivocal identification of these closely related fish species in any seafood product,
including processed and precooked products, in less than 2 h. The present strategy constitutes the fastest method for peptide
biomarker monitoring. Its application for food quality control provides to the authorities an effective and rapid method of food
authentication and traceability to guarantee the quality and safety to the consumers.
ast monitoring of biomarkers is essential to achieve a rapid
response and to make a precise decision in diverse life fields.
range.4 The capability of triple-quadrupole (TQ) instruments to
selectively isolate a precursor ion and the fragment ion(s) it
produces under collision-induced dissociation (CID) is exploited
for the experiments using SRM or MRM scan modes.5
F
It is crucial in clinical diagnosis, therapeutic supervision, envir-
onmental protection, and food quality control, among others.1,2
Currently, the most commonly used approach for the monitoring
of peptide/protein biomarkers is based on immunoassays, mainly
using ELISA and array techniques.1 The advantages of these
methods are their high specificity and sensitivity. Apart of being
time-consuming, a flaw of these techniques is that not always the
right antibody is available for each biomarker, making lengthy
and expensive work necessary to extend the battery of specific
antibodies for new standardized and affordable assays. Therefore,
the development of alternative and fast methodologies having
high reproducibility, sensitivity, and specificity is necessary.
The emerging targeted mass spectrometry (MS)-based pro-
teomics techniques can constitute an excellent alternative meth-
odology. When these selective and sensitive operating methods
are used, the MS analyzer is centered on analyzing only the
compound of interest by selected reaction monitoring (SRM) or
multiple reaction monitoring (MRM).3,4 Monitoring transitions
(suitable pairs of precursor and fragment ion m/z) constitute a
common assay to identify and quantify biomarkers and, by
inference, the change in the corresponding biological condition
being studied. This setup provides high analytical reproducibility,
a good signal-to-noise (S/N) ratio, and an increased dynamic
While SRM and MRM performed on a TQ are the most
sensitive scanning modes (low attomolar) with a broad dynamic
range (up to 5 orders of magnitude),6 their optimization for a
definite SRM/MRM assay is time-consuming. More importantly,
using these scanning procedures, complete MS/MS spectra are
not registered. The MS/MS spectrum of a molecule is of para-
mount importance to confirm the structure of the compound
detected. To solve this problem, new routines, such as MRM-
triggered MS/MS using hydrid quadrupole/linear ion trap mass
spectrometers, have been explored.7 In such assays, when a
significant signal for a specific MRM transition is detected, the
instrument switches the third quadrupole automatically to the
ion trap mode, collecting the full MS/MS spectrum.
Selected MS/MS ion monitoring (SMIM) in an ion trap (IT)
mass spectrometer is another scanning mode that allows for a
sensitive monitoring of specific molecules, producing complete
structural information.8 The high scanning speed attainable in
Received: April 7, 2011
Accepted: May 31, 2011
Published: May 31, 2011
r
2011 American Chemical Society
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the IT allows for the production of MS/MS spectra in a fraction
of a second, registering the information given by the complete
spectra. High-confidence MS/MS spectra are recorded due to
the possibility of an averaging of the signal during acquisition.
The utility of this operating mode has been demonstrated
in several previously published studies,8À10 where the possibility
of a virtual plotting of all the transitions produced has been
discussed.
The main difficulty when LCÀMS is used to monitor bio-
markers arises from the complexity of the samples, which may
result in false positives. Although the chromatographic gradient
time may be properly manipulated to avoid this, lowering the
number of components in the sample, when it is possible, may
help to simplify the assays. Shorter analysis times are needed
when fewer components are present in the sample. Fast and easy
protein fractionation or purification steps conducted prior to
LCÀMS analysis make the analysis simpler and faster.11
Procedures to enhance the protease activity, such as the
application of microwaves,12 high pressure,13 or the energy pro-
duced by ultrasound,14 can accelerate the time-consuming
trypsin digestion. The application of only 1À2 min of high-
intensity focused ultrasound (HIFU) to in-solution tryptic
digestions has been reported to achieve an efficiency and
reproducibility similar to those obtained by traditional overnight
protocols.14À16
A new targeted MS-based strategy for the fast monitoring of
peptide biomarkers based on the combination of these meth-
odologies described is proposed and has been applied to the fast
authentication of all commercial species from the Merlucciidae
family. It is based on (a) the purification of parvalbumins
(PRVBs) by heat treatment (time 45 min), (b) their accelerated
tryptic digestion using HIFU (time 2 min), and (c) the monitor-
ing of 11 PRVB peptide biomarkers by SMIM in a linear ion trap
(LIT) mass spectrometer (time 60 min). Each step was indivi-
dually adjusted to minimize the time of analysis. With this new
and competitive strategy, the unequivocal identification of these
closely related fish species in any seafood products, including
processed and precooked, can be achieved in less than 2 h.
Table 1. Reference Species and Commercial Foodstuffs from
the Merlucciidae Family Considered in This Studya
Reference Species
species/subspecies
common name
origin
M. merluccius
M. capensis
European hake
Spanish coasts
South Africa
Cape hake
M. senegalensis
M. polli
Senegalense hake
Benguela hake
Deep-water Cape hake
Patagonian hake
Peruvian or Chilean hake
Austral hake
Northwest Africa
Northwest Africa
South Africa
M. paradoxus
M. hubbsi
South America
South America
South America
New Zealand coasts
North America
North America
New Zealand coasts
South America
M. gayi
M. australis polylepis
M. australis australis
M. productus
M. bilineariz
Ma. nov. nov.
Ma. nov. magellanicus
Austral hake
Pacific hake
Silver hake
Blue grenadier
Patagonian grenadier
Commercial Products
code
product presentation
declared species
CP1
CP2
CP3
CP4
CP5
CP6
CP7
CP8
CP9
CP10
raw fillets frozen
M. capensis or M. paradoxus
M. autralis
raw center cuts frozen
raw loins frozen
M. capensis or M. paradoxus
M. capensis or M. paradoxus
hake unidentified
raw center cuts frozen
raw center cuts frozen
battered precooked sticks frozen
battered precooked sticks frozen
battered precooked sticks frozen
battered precooked sticks frozen
battered precooked fillets frozen
hake unidentified
hake unidentified
hake unidentified
hake unidentified
hake unidentified
a Abbreviations: M., Merluccius genus; Ma. nov., Macruronus novaezelandiae.
Parvalbumin Purification. Sarcoplasmic protein extraction
was carried out by homogenizing 5 g of white muscle in 10 mL of
10 mM TrisÀHCl buffer, pH 7.2, supplemented with 5 mM
phenylmethylsulfonyl fluoride (PMSF), for 30 s in an Ultra-
Turrax device (IKA-Werke, Staufen, Germany). In the case of
battered precooked foodstuffs the casing was first removed. The
sarcoplasmic protein extracts were then centrifuged at 40000g
for 20 min at 4 °C (J221-M centrifuge; Beckman, Palo Alto, CA).
PRVBs were purified by taking advantage of their thermostability,
heating the sarcoplasmic extracts at 70 °C for 5 min.10 After
centrifugation at 40000g for 20 min (J221-M centrifuge), super-
natants composed mainly of PRVBs were quantified by the bicinch-
oninic acid (BCA) method (Sigma Chemical Co., St. Louis, MO).
Protein Digestion Using HIFU. PRVB supernatants were
subjected to HIFU-assisted trypsin digestion as previously
described.14 A total of 20 μg of heated extract was subjected to
in-solution digestion with 1 μg of trypsin without addition of
urea, dithiothreitol, or iodoacetamide (Promega, Madison, WI),
simultaneously applying HIFU. A high-intensity ultrasonic probe
with a 1 mm probe tip (Dr. Hielscher, Teltow, Germany) was set
to 50% amplitude and was used to perform the ultrafast digestion
for 1 min. Another 1 μg of trypsin was added again to the sample,
and the HIFU application was repeated for 1 min.
’ EXPERIMENTAL SECTION
Reference Species and Commercial Foodstuffs. All the
main commercial species from the Merlucciidae family were
employed in this study: eleven different hake species, including
two different subspecies from Merluccius australis and two
grenadier subspecies belonging to the Macruronus novaezelandiae
species (Table 1). Except for European hake, the specimens were
frozen on board at À30 °C, with special care in keeping their
morphological characteristics in good shape, and shipped by
plane to the laboratory for the analyses. The weight of every
specimen studied was in the range of 3À6 kg. At least 10 fish
belonging to each different species were subjected to taxonomi-
cal study according to their anatomical and morphological
features by an expert marine biologist and by genetic identifica-
tion using forensically informative nucleotide sequencing
(FINS) at the Food Biochemistry Laboratory of the Marine
Research Institute (Vigo, Pontevedra, Spain).17À19 Five correctly
identified individuals for each of the species were considered as
reference species. In addition, for the validation step using
commercial real samples, a total of 10 hake foodstuffs were
included in the work (Table 1). All samples were analyzed in
triplicate.
LCÀMS/MS Analysis. Peptide digests were acidified and an-
alyzed by LCÀESI-IT-MS/MS using a Surveyor LC system
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coupled to an LTQ LIT mass spectrometer (Thermo Fisher, San
Jose, CA). The peptide separation (1 μg) was performed on a
0.18 mm  150 mm BioBasic-18 RP column (ThermoHypersil-
Keystone) using 0.5% acetic acid in Milli-Q water and in 80%
acetonitrile (ACN) as mobile phases A and B, respectively. A 60 min
linear gradient from 5% to 40% B, at a flow rate of 1.5À1.7 μL/min,
was used. ESI parameters were as follows: spray voltage, 3.5 kV; N2
flow, 10 arbitrary units; capillary temperature, 200 °C. Peptides were
analyzed in positive mode from 400 to 1600 amu (three micro-
scans), followed by four data-dependent MS/MS scans (three
microscans), using an isolation width of 3 amu and a normalized
collision energy of 35%. Fragmented masses were set in dynamic
exclusion for 3 min after the second fragmentation event, and singly
charged ions were excluded from MS/MS analysis.
Selected MS/MS Ion Monitoring. SMIM analysis was per-
formed using a Surveyor LC system coupled to an LTQ LIT mass
spectrometer (Thermo Fisher, San Jose, CA), as described
previously8 with minor modifications. The peptide separation
(1 μg) was performed on a 0.18 mm  150 mm BioBasic-18 RP
column (ThermoHypersil-Keystone) using 0.5% acetic acid in
water and in 80% ACN as mobile phases A and B, respectively.
A 45 min linear gradient from 5% to 40% B, at a flow rate of 1.5À
1.7 μL/min, was used. ESI parameters were as described
previously. Peptides were detected in the positive ion mode
using SMIM.8 For this method, the MS instrument was pro-
grammed to perform continuous MS/MS scans (five micro-
scans) of doubly charged precursor ions from all candidate
peptide biomarkers along the complete chromatographic separa-
tion. The normalized collision energy was set to 35%, and a 3 amu
mass window was used to fragment selected parent ions.
Mass Spectrometry Data Processing. MS/MS spectra were
searched using SEQUEST (Bioworks 3.1 package, Thermo Fisher)
againsttheTeleosteiUniProt/TrEMBLdatabase(release2010_12;
158.545 entries), which also included their respective decoy
sequences. The following constraints were used for the searches:
semitryptic cleavage with up to two missed cleavage sites and
tolerances of 1.8 Da for precursor ions and 0.8 Da for MS/MS
fragment ions. The variable modifications allowed were methio-
nine oxidation (Mox), carbamidomethylation of Cys (C*), and
acetylation of the N-terminus of the protein (N-Acyl). The
database search results were subjected to statistical analysis with
the PeptideProphet algorithm (v.4.4).20 The false discovery rate
(FDR) was kept below 1%.
using HIFU (time 2 min), and (c) monitoring of 11 species-
specific PRVB peptide biomarkers by SMIM using an LIT mass
spectrometer (time 60 min). With this strategy, all relevant
commercial fish species belonging to the Merlucciidae family
can be unequivocally identified in any seafood product, including
precooked, in less than 2 h. The detailed results for each step and
the validation of this new fast monitoring strategy using un-
known hake commercial products are shown in the following
sections.
PRVB Purification and Enzymatic Digestion Accelerated
by HIFU. PRVBs, considered as the best protein biomarker for
the authentication of Merluciidae species,10,21 were purified from
the sarcoplasmic extracts, taking advantage of their thermo-
stability.10 Figure S-1 in the Supporting Information shows a
summary of the protein composition in the extracts before and
after the treatment with heat (70 °C for 5 min). The complete list
of proteins and peptides for both samples, identified by
LCÀMS/MS and Sequest search after a conventional overnight
trypsin digestion,22 are presented in the Supporting Information.
Protein composition of the original sarcoplasmic extracts re-
vealed more than 125 different proteins involved in 10 relevant
functional pathways (Table S-1 in the Supporting Information).
After treatment with heat, the majority of identified peptides
corresponded to PRVBs (77.87%) (Figure S-1). These results
demonstrated that the treatment with heat is a simple, fast,
and effective procedure to purify and enrich the samples in
only PRVBs.
Purified PRVBs were digested with trypsin using either the
conventional overnight procedure or the fast procedure acceler-
ated by HIFU. As reported previously,14 HIFU-assisted digestion
produced results comparable to those obtained by the conven-
tional overnight incubation methods, but in a fraction of time.
Moreover, the absence of urea in the digestion buffer prevented
undesired peptide side reactions, such as carbamylation of
N-termini and Lys residues, which may occur when HIFU is
applied in the presence of urea.23,24
The combination of a fast and easy protein purification
procedure (time 45 min) with the use of HIFU for the protein
digestion (ime 2 min) considerably simplified and reduced the
time needed for the sample preparation, reflected in the overall
time needed for monitoring.
Selection of the Species-Specific Peptide Biomarkers. The
next step in the proposed strategy consisted in selecting the
smaller number of species-specific peptides, which must be
monitored, to effectively identify all the species from the Merluc-
ciidae family. Parvalbumin peptide sequences with a high inter-
specific variability, obtained afterthe extensive de novo sequencing
of PRVBs previously published,10 were used for this purpose.
Eleven tryptic peptides were selected on the basis of the informa-
tion that their combined presence or absence could provide to
confidently identify all of the species under study (Table 2). A
Basic Local Alignment Search Tool (BLAST) search was per-
formed using the UniProtKB database to validate the uniqueness
of the peptide sequences selected. Four of them were present in
only one specific species and can be considered as a canonical
peptide for each of these Merlucciidae species (S-MER794,
S-MER612, S-MER721, S-MER973). The sequences of the rest
were shared by PRVBs from several Merlucciidae species or other
organisms. However, their use following a specific and systematic
combination avoids interferences and allows for a correct discri-
mination of all the hake species under study. Figure 2 shows the
flow diagram for the unambiguous systematic discrimination
The proteins identified in the original and heated sarcoplasmic
extracts were submitted to ingenuity pathway analysis (IPA;
Ingenuity Systems, Redwood City, CA). Only pathways scoring
Àlog(p) g 2, which have >99% confidence of not being
generated by chance, were selected.
For the SMIM mode, virtual chromatogram traces were plotted
and optimized using QualBrowser software (Thermo Fisher) to
show the selected transitions for each parent ion. In addition, MS/
MS spectra collected in the SMIM mode were used to validate the
peptide identities using SEQUEST as described before.
’ RESULTS AND DISCUSSION
Strategy for the Fast Monitoring of Species-Specific Pep-
tide Biomarkers. The strategy for the fast monitoring of peptide
biomarkers proposed in this work is summarized in Figure 1. This
strategy integrates three steps: (a) purification of thermostable
proteins (PRVBs) by short heat treatment followed by centrifu-
gation (time 45 min), (b) in-solution trypsin digestion accelerated
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Figure 1. Analytical scheme for the fast monitoring of the species-specific peptide biomarkers proposed in this work.
which can be achieved. According to this scheme, the presence/
absence of the peptide F-MER604 determines if any member from
the Merlucciidae family is present in the sample. Discrimination
between the genera Merluccius and Macruronus can be achieved by
the determination of the presence/absence of peptides G-MER517
and G-MAC524, respectively. Within the Merluccius genus, the
presence/absence of the peptide S-MER967 allows for the
classification of hake species into two groups according to their
geographic distribution: American hakes (M. hubbsi, M. gayi, M.
australis polylepis, M. australis australis, M. productus, or M.
bilineariz) or Euro-African hakes (M. merluccius, M. capensis, M.
senegalensis, M. polli, or M. paradoxus). Finally, as can be seen in
Figure 2, the combination of the presence/absence of other eight
peptides allows for the unambiguous identification of any specific
species from the Merlucciidae family.
Fast Identification of Hake Species Using SMIM. For each
of the reference hake species, PRVB peptide pools obtained from
the accelerated tryptic digestions were subjected to SMIM
analysis in an LIT mass spectrometer focusing the MS/MS
events on the corresponding precursor ions for the 11 peptides
selected. The selected m/z value for each precursor ion corre-
sponded to the predominant charge state, which was +2 for all of
them (Table 2). Figure S-2 in the Supporting Information details
the MS/MS spectra for each peptide. Once MS/MS spectra were
recorded, virtual chromatograms for all the different fragment
ions could be obtained. For each of the peptide markers, mass
transitions were noted according to the criteria of sensitivity and
selectivity. As the peptide mixture used is not too complex,
selectivity was not a matter of concern and the transitions chosen
in every case were in accordance with the maximum intensity
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Table 2. Peptide Biomarkers and Specific Transitions for the Identification of All Species from the Merlucciidae Familya
a m/z = mass/charge ratio. Key: S1, M. merluccius; S2, M. capensis; S3, M. senegalensis; S4, M. polli; S5, M. paradoxus; S6, M. hubbsi; S7, M. gayi; S8, M.
australis polylepis; S9, M. australis australis; S10, M. productus; S11, M. bilinearis; S12, Macruronus spp. A filled square denotes the presence of a peptide
biomarker and an empty square the absence of a peptide biomarker. bSparus aurata, Fundulus grandis, Fundulus similis, Fundulus heteroclitus,
Hypophthalmichthys nobilis, Paralichthys olivaceus, Theragra chalcogramma.
Figure 2. Flow diagram for a systematic discrimination of Merlucciidae species using only 11 specific tryptic peptides from PRVBs. Y denotes the
presence and N the absence of a particular peptide.
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Figure 3. Reference SMIM traces for each Merlucciidae species, plotting the corresponding canonical transition for each PRVB tryptic peptide
biomarker.
of the fragments, which mostly corresponded to y ions. There-
fore, the combination of highly sensitive transitions (precursor
m/z f fragment m/z) (Table 2), together with the use of simple
peptide mixtures (coming mainly from PRVBs), made possible
the representation of specific transitions with a high signal-to-
noise (S/N) ratio. Tracing these transitions for each peptide
biomarker, according to the flow diagram described in Figure 2,
made possible unequivocal identification of all the reference hake
species (Figure 3).
The results obtained matched precisely those obtained by
DNA analysis. However, the time needed for performing both
techniques was extremely different. At least 24 h was needed for a
genetic identification by FINS, while only less than 2 h was
necessary to complete the identification following the proposed
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Table 3. Results of the SMIM Assay of Commercial Hake
Productsa
present strategy constitutes the fastest method for peptide
biomarker monitoring by targeted proteomics described up to
now, whose application in the food quality control area provide
the authorities an effective, competitive, and rapid method of
food authentication and traceability that guarantees quality and
safety to the consumers.
species identified by
product
declared species
HIFU + SMIM, time <2 hb
CP1
CP2
CP3
CP4
CP5
CP6
CP7
CP8
CP9
CP10
M. capensis or M. paradoxus
M. australis
M. paradoxus
M. australis polylepis
M. paradoxus
M. capensis
M. capensis or M. paradoxus
M. capensis or M. paradoxus
hake unidentified
’ ASSOCIATED CONTENT
M. paradoxus
M. productus
S
Supporting Information. Additional information as
b
hake unidentified
noted in text. This material is available free of charge via the
Internet at http://pubs.acs.org.
hake unidentified
M. paradoxus
Macruronus spp.
Macruronus spp.
M. paradoxus
hake unidentified
’ AUTHOR INFORMATION
hake unidentified
hake unidentified
Corresponding Author
*Phone: +34 986 231930. Fax: +34 986 292762. E-mail: mcarrera@
iim.csic.es.
a M. indicates the Merluccius genus. b These results were validated by
genetics after 24 h of analysis.
strategy. To our knowledge, this is the fastest method to achieve
food species authentication.
’ ACKNOWLEDGMENT
Application to Commercial Sample Identification. To
validate this new strategy, 10 commercial hake products were
subjected to analysis for their authentication. Table 1 summarizes
the products that were tested, which had been previously
subjected to one or more processing treatments, even precooked.
Two main label categories were observed: labels with species
declared (four samples) and labels with no declared species,
which presented only a general commercial name (i.e., hake) (six
samples). Results for the identification of the commercial
samples using the designed strategy are shown in Table 3 and
in Figure S-3 in the Supporting Information. Samples with
complete declared labeling were found to be mostly correct
(CP1ÀCP4). However, some products with no declared species
were assigned to species belonging to the Macruronus genus
(CP8 and CP9). This genus presents a low commercial value and
is not as valued by the consumer.25 We have presented here a
strategy that allows the fast detection of mislabeling practices in
these fish products, and moreover, this work also shows that
the use of thermostable proteins allows the application of this
fast monitoring method to battered precooked products
(CP6ÀCP10).
We express our gratitude to Mrs. Lorena Barros for her
excellent technical assistance and to Freiremar SA and the
Centro Tecnolꢀogico del Mar (CETMAR) for their assistance
in the collection of the reference species used in this study. This
work was supported by the Comisiꢀon Interministerial de Ciencia
ꢀ
y Tecnologia (CICyT) (Project AGL2000-0440-P4-02).
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A new strategy for the fast monitoring of species-specific
peptide biomarkers for foodstuff authentication has been pro-
posed. The principle it is based on is the use of a fast purification
step of the target protein, the acceleration of in-solution protein
digestion by HIFU, and the monitoring of several peptides by
SMIM in a linear ion trap mass spectrometer. The use of the
SMIM mode of scanning allows for a more simple setting of the
procedures, giving the possibility of obtaining full MS/MS
information necessary for the validation of the structure of the
component, and allows the choice of the most adequate transi-
tions for each precursor ion, with attention to the criteria of
sensitivity and selectivity. Thus, in combination with a fast
purification and digestion step of the target protein, the most
sensitive transitions may be considered for all the markers.
With this new strategy, all relevant commercial fish species
belonging to the Merlucciidae family present in any seafood
product can be unequivocally identified in less than 2 h. The
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