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Proteomics 2008, 8, 20352044

2035

DOI 10.1002/pmic.200700944

RESEARCH ARTICLE

Comparison of two anoxia models in rainbow trout


cells by a 2-DE and MS/MS-based proteome approach
Tune Wulff1, 2, Else K. Hoffmann1, Peter Roepstorff3 and Flemming Jessen2
1

Department of Biochemistry, Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark


Danish Institute for Fisheries Research, Technical University of Denmark, Kgs. Lyngby, Denmark
3
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
2

In the literature, a variety of ways have been used to obtain anoxia, and most often results are
compared between studies without taking into consideration how anoxia has been obtained.
Here, we provide a comprehensive study of two types of anoxia, using a proteomics approach to
compare changes in protein expression. The two investigated situations were 30 min of chemical
anoxia (10 mM NaN3) followed by reoxygenation overnight (CR) and 2 h of N2-induced anoxia
(achieved by flushing with N2) followed by reoxygenation overnight (NR), after which samples
were resolved by 2-DE. Forty-five protein spots changed their abundance in response to CR and
35 protein spots changed their abundance in response to NR, but only six proteins changed their
abundance in response to both stimuli. By the means of MS/MS, 40 protein spots were identified
including proteins involved in processes like cell protection and protein synthesis. It was also
revealed that the level of a number of keratins was down-regulated. This study therefore provides
a valuable comparison of two different anoxia models and shows that great care should be taken
when comparing the effects of anoxia in studies that have used different types and durations of
anoxia.

Received: October 5, 2007


Revised: January 2, 2008
Accepted: January 25, 2008

Keywords:
2-DE / Anoxia / MS/MS / Rainbow trout / Reoxygenation

Introduction

Anoxia is a very severe cellular stressor and is a consequence


of pathophysiological conditions like stroke, cancer, or
ischemic cardiovascular diseases. Just as important as the
duration time of anoxia are the effects of the following
Correspondence: Dr. Tune Wulff, Danish Institute for Fisheries
Research, Technical University of Denmark, Sltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark
E-mail: tuw@difres.dk
Fax: 145-4588-4774
Abbreviations: ATP, adenosine triphosphate; CR, 30 min of chemical anoxia (10 mM NaN3) followed by reoxygenation overnight;
HIF, hypoxia-inducible factor; IR, ischemia/reperfusion; MW, molecular weight; NR, 2 h of N2-induced anoxia (achieved by flushing with N2) followed by reoxygenation overnight; Prdx, peroxiredoxin; RTHDF, rainbow trout hypodermal fibroblasts

2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

reperfusion. From studies on ischemia/reperfusion (IR), a


large number of effects on the exposed organism have been
detected [1, 2]. Long period of ischemia followed by reperfusion often induces apoptosis or necrosis [3, 4], while a short
and not lethal period of ischemia followed by reperfusion in
most tissues results in an initial response within the first
hours (04 h) and a late response that normally is seen after
approximately 1224 h [57].
Immediately after reperfusion the response will normally be mediated by PTM of the involved proteins [8]. The
late response has been examined in a number of studies in
cells subjected to an initial anoxic exposure followed by 24-h
reperfusion. This is due to the observation that around 24 h
after reperfusion the cells seem to exert a second late response that often includes synthesis of new proteins [5].
Several proteins, like cyclooxygenase 2 (COX 2), superoxide
dismutase and heat shock proteins, have been proposed to be
involved in this late response following reperfusion [9, 10].
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T. Wulff et al.

One of the main effectors during anoxia and reperfusion are


reactive oxygen species (ROS), which can covalently modify
proteins and lipid macromolecules leading to cell damage
[11], and the cells will therefore try to diminish the damaging
effects of ROS.
In fish, hypoxia is a common feature of the aquatic environment [12], and with growing emphasis on the benefits of
eating fish [13, 14] a better understanding of what happens
after harvesting of seafood is increasingly important. Furthermore, as rainbow trout is considered a hypoxia-sensitive
species [15, 16], results obtained in trout might also apply to
mammalian cells. Studies on rainbow trout hypodermal
fibroblasts (RTHDF) cells have proven them capable of sustaining long periods (up to 24 h) of anoxia [17].
Even though anoxia and reoxygenation are very important aspects with regard to fish, only a limited number of
studies have addressed the question of anoxia followed by
reoxygenation in fish [1820]. Studies using 2-DE to elucidate changes in overall protein expression following anoxia
and long-term reperfusion in fish do not exist. In contrast, 2DE has successfully been used in a number of studies to
examine differences in protein expression in fish in response
to a wide variety of different stimuli [2125]. Not all experiments are readily done in vivo, therefore, a number of different models have been used to obtain or simulate anoxia: (i)
chemical anoxia obtained by addition of azide [26, 27] or
cyanide [28] or (ii) exchanging oxygen with various combinations of gases [29]. The extensive use of different anoxia
models raises the questions of how the different models
compare to the situation in vivo, and also if results obtained
in different models can be readily compared to each other. In
the literature, studies have used a combination of the different anoxia models [3032]. The use of more than one way of
obtaining anoxia in one single study is advantageous when it
aids the process of elucidating the problem of interest.
However, a point, which does not always receive the necessary attention, is whether the cellular and physiological
responses to differently obtained anoxia are actually comparable. If not, this is a major concern that calls for great care
when making overall conclusions based on data obtained by
the use of different anoxia models. It is intuitively evident
that chemical anoxia obtained by addition of azide operates
differently than anoxia obtained by depletion of oxygen by
N2. Chemical anoxia directly targets the oxidative phosphorylation in the mitochondria whereas oxygen depletion also
affects other oxygen dependent cellular pathways.
Many changes occur during anoxia and reoxygenation,
such as changes in cellular metabolism, lowered adenosine
triphosphate (ATP) levels and production of ROS. Of these,
changes in cellular metabolism and ATP levels are most
likely to be similar in the two investigated models whereas
the production of ROS and in particular the O2 levels are very
different in the two models.
Thus, the present study was initiated to compare changes
in protein expression pattern induced by two different anoxia
models and subsequent reoxygenation by proteomics (2-DE
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2008, 8, 20352044

and MS/MS) in order to demonstrate the differences in cellular response between two different anoxia models and to
establish a better basis for drawing conclusions based on
data from different models. In addition, this study provides
valuable information about the effects of 30 min of chemical
anoxia followed by reoxygenation and 2 h of N2-induced
anoxia followed by reoxygenation on protein expression in a
cell line from the commercially important species, rainbow
trout.

Materials and methods

2.1 Materials
All general chemicals were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO). Glycerol
(87%), SDS, Pharmalyte 46.5 and Pharmalyte 58 were
from Amersham Biosciences (Uppsala, Sweden). Mark12
unstained low-molecular weight protein (LMW) standard
was purchased form Invitrogen; Life Technologies (Carlsbad,
CA).
2.2 Cell culture
RTHDF [26] were grown in atmospheric air at 217C in a Leibovitz L-15 medium, supplemented with 15% v/v FBS,
penicillin (100 units/mL) and streptomycin (100 mg/mL), as
described previously [27]. Cells were passaged every 7 days
(only passages 1540 were used), using a trypsin solution for
cell detachment made by dissolving 0.1% w/v trypsin
(Sigma) and 1 mM disodium-EDTA in PBS (137 mM NaCl,
2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4). All
reagents for cell culturing were used at 57C.
2.3 N2 anoxia/chemical anoxia
Experiments were performed in 75-cm2 culture flask with
cells grown to approximately 80% confluence. One day
prior to the start of the experiment cells were given fresh
medium.
The 2-h of N2-induced anoxia followed by reoxygenation
overnight (NR), was obtained by flushing with N2 and in
order to obtain instant anoxia (see Allen et al. [33] for discussion) the Leibovitz L-15 medium were flushed with nitrogen
prior to the start of experiment. To avoid reoxygenation of the
medium while applying it to the cells, this step was performed under continuous flushing of N2, and to keep the
flasks anoxic for the duration of the experiments they were
sealed with air-proof locks. During experiments, the O2 concentration never exceeded 0.1%.
The 30-min chemical anoxia followed by reoxygenation
overnight (CR) was induced by submitting cells to a Leibovitz L-15 medium supplemented with 10 mM NaN3.
Azide inhibits the cytochrome c oxidase complex IV, and
thereby blocks oxidative phosphorylation [34]. To simulate
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Proteomics 2008, 8, 20352044

the rapid reoxygenation that follows reperfusion the anoxic


period was followed by washouts first twice with 10 mL
PBS and then changing to normal Leibovitz L-15 medium.
Controls were submitted to the same changes of medium
as CR and NR and were incubated under normoxic conditions for an equivalent time. All cells were kept at 217C.
2.4 Sample preparation
Cells were harvested onto ice and all centrifugation steps
were conducted at 47C. Prior to harvesting, cells were washed
twice with 10 mL ice-cold PBS and then collected in 20 mL
ice-cold PBS by trypsination. Cells were pelleted by centrifugation at 18506g for 5 min and the supernatant was
removed. The pellet was washed in 1.5 mL of ice-cold PBS,
recollected by centrifugation at 7006g for 45 s, and stored at
807C. The pellet was completely dissolved in 100 mL of solution A (8 M urea, 2 M thiourea, 50 mM DTT, 1.5%
CHAPS, 2% Pharmalyte 46.8, 2% Pharmalyte 58, 10 mM
Trizmabase and 0.1% SDS). To remove any undissolved
material, the solution was centrifuged at 20 0006g for
15 min and the supernatant was collected. Protein concentrations were determined using Petersons simplification
[35] of the Lowry method [36].

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2.6 Staining and image analysis


The gels were silver stained according to a protocol modified
from Shevchenko et al. [37]. In short, the fixated gels were
rinsed for 10 min in H2O and then sensitized in a solution
containing 0.5 M Na-acetate and 0.125% glutardialdehyd for
30 min. Gels were then washed twice in H2O for 10 min each
and then incubated for 20 min in silver solution (24 mM
AgNO3, 9 mM NaOH, and 0.14% v/v NH3). After a 5-min
wash in H2O, gels were developed in 750 mM citric acid and
0.037% v/v formaldehyde, and the staining reaction was
stopped in 30% v/v ethanol and 7% v/v acetic acid. Silverstained gels were digitized using a 420 OE (PDI Bio-Rad,
Hercules, CA) flatbed scanner with a pixel size of 84.7684.7
pixels and the software PDQuest 7.1.1 Discovery Series (BioRad). A number of anchor spots were manually defined after
which matching of spots between different gels was performed automatically by PDQuest. The automated matching
of the gels was checked manually and corrected when needed. The quantity of each spot was calculated during spot
detection and Gaussian fitting measured as OD/(0.1 cm2).
The OD/(0.1 cm2) of each spot in a gel was divided by the
OD/(0.1 cm2) of all matched and unsaturated spots (peak
intensity is within the linear range of the scanner) in the gel
and a value as ppm was given to ensure a normalization of
the spots in a gel and to ensure a uniform quantification.

2.5 2-DE
2.7 MALDI-MS/MS
Proteins were separated according to charge on an 18-cm 1-D
Immobiline drystrip (Amersham Biosciences) with a linear
pI gradient of 47. Application of sample was performed by
re-swelling overnight of the drystrip with 100 mg of protein in
350 mL of solution A with Orange G as dye. To achieve
separation of the proteins in the first dimension, drystrips
were submitted to 60 000 V?h., after which they were stored
at 2807C.
Proteins were separated according to size in the second
dimension on a 12% w/v SDS-PAGE gel. Ten gels
(19623 cm) were run simultaneously on Hoefer DALT system (Amersham Biosciences) at 157C with a maximum of
40 mA/gel. Prior to 2-D PAGE, drystrips were reduced for
20 min in 10 mL equilibration buffer (6M urea, 50 mM TrisHCl, pH 8.8, 30% v/v glycerol, 2% w/v SDS) with 1% w/v
DTT, followed by 20 min of alkylation in 10 mL equilibration
buffer with 4.5% w/v iodoacetamide. Agarose (0.5% w/v) in
25 mM Tris-base, 192 mM glycine and 0.1% w/v SDS was
used for sealing the drystrips to the SDS gels. Gels were
stopped when the Coomassie front of the sealing solution
reached the end of the gels, after which they were immediately fixated for 30 min in 11.5% (w/v TCA and 3.45% w/v
5-sulfosalicylic acid. The gels were washed three to four
times (1 h of each wash in 40% v/v ethanol and 10% v/v
acetic acid) under constant shaking, after which they were
stored in the washing solution. Thorough wash of the gels
before silver staining greatly improved the quality of the
images.
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

To identify proteins of interest higher amounts of protein


(5002000 mg) were separated on additional 2-D gels that
were silver stained using a protocol without glutardialdehyde
adapted from Shevchenko et al. [38].
First, proteins were excised from the gels by a spot picker
(The Gel Company, San Francisco, CA, USA). Exercised spots
were washed in 50% ACN followed by a wash in H2O, and
subsequently they were dried. The dried spots were in-gel
digested with trypsin (Porcine sequencing grade modified
trypsin, Promega, Southampton, UK), and extracted in 0.1%
TFA, followed by desalting and concentration on microcolumns with POROS R2 resin (Applied Biosystems, Foster
City, CA). The columns were packed in a GELoader tip
(Eppendorf AG, Hamburg, Germany) as described previously
[38]. Finally, peptides were eluted from the microcolumns
with 0.8 mL matrix solution (5 mg/mL CHCA in 70% v/v
CH3CN and 0.1% TFA) directly onto the MALDI target.
Data acquisition was performed manually on an Applied
Biosystems 4700 Proteomics Analyser with TOF/TOF
optics (Applied Biosystems, Darmstadt, Germany) in positive
reflector mode. Depending on the sample analyzed, laser
intensity and number of laser shots were varied to obtain
optimal spectra. Only MS/MS spectra were used for identification of peptides and proteins, and MS spectra in the manual setup were therefore only used to select peaks for MS/MS
analysis. MS/MS spectra were retrieved for all MS peaks
other than trypsin or human keratin and with intensities that
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T. Wulff et al.

made it possible to produce an acceptable MS/MS spectrum.


Peaks originating from human keratin or trypsin added for
digestion of peptides were identified and eliminated using
the software PeakErazor (http://www.welcome.to/GPMAW)
[40]. MS/MS spectra were converted into peak lists using
Data Explorer (v.4.4, Applied Biosystems, Framingham, MA)
with default parameters. All spectra from single MALDI-spot
were merged into a single MASCOT generic file using an inhouse made PERL script (courtesy of Jakob Bunkenborg,
University Southern Denmark).
2.8 Protein identification and database searching
Protein identification was conducted using the search engine
MASCOT release 2.1 (www.matrixscience.com) [41] on MS/
MS spectra obtained from each individual spot. Searches were
performed against the NCBInr database including all entries
(number of sequences 3 423 372 with 2843 of these being from
rainbow trout). All protein identifications with a significant
score (p,0.05) were manually evaluated to reduce the risk of
false positives (taking into account, e.g. the presence or absence
of likely intense fragment ions). All MASCOT scores reported
for the MALDI-MS/MS search results are listed in Tables 1, 2
and 3. Results were obtained using a fragment ion mass tolerance of 0.5 Da and a precursor mass accuracy of 70 ppm, and
the following parameters: missed cleavages, 1; allowed modifications: carboxamidomethyl on cysteine as a fixed modification and oxidation of methionine as partial modification.
A number of spots were also manually de novo sequenced
assisted by Data Explorer (v.4.4, Applied Biosystems) and
AminoCalc (Protana A/S), while MSBlast (http://dove.emblheidelberg.de/Blast2/msblast.html) [42] was used to search
public databases, and GPMAW was used (http://
www.gpmaw.com [40]) for all sequence handling and storage.

Results

3.1 2-DE
Protein expression in the RTHDF cell line was investigated
after exposure to two different anoxic stimuli, i.e. 2 h of N2induced anoxia followed by reoxygenation and 30 min of
NaN3-induced chemical anoxia followed by reoxygenation.
After separation on 2-D gels (Fig. 1), 2200 different spots
were visualized by silver staining and around 800 of these
were represented in all 15 gels. The obtained images showed
a clear separation of the different proteins, except for a minor
streaking in the left (acidic) part of the gels (Fig. 1). This was
most likely due to salts and low protein solubility, which are
well known to interfere with focusing of proteins in the first
dimension [43]. However, the observed streaking was tolerable, as it only rendered a very limited number of proteins
undetectable (less than 2%). The poor resolution at pI values
below 4.54.7 made a proper alignment of the different spots
difficult and none of these spots (Fig. 1) were included in the
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2008, 8, 20352044

statistical analyses. The 2200 spots, therefore, covered a pI


range of 4.57 and a molecular weight (MW) of 14.4
116 kDa. Any up- or down-regulation in protein expression
was determined using a t-test (value ,0.05 was considered
significant), by comparing the level of intensity of the individual spot of the control samples to the level of intensity of
the corresponding spot from either CR or NR treated cells.
Forty-five protein spots changed their abundance in response
to CR (Fig. 1A) and of these 15 were up-regulated and 30
down-regulated. In response to 2 h of N2- induced anoxia
followed by reoxygenation overnight, 35 protein spots
changed their abundance (Fig. 1B) with nine of these being
up-regulated and 26 down-regulated. The overlap between
proteins that changed significantly in response to both of the
anoxia models constituted only six protein spots (Fig. 1C).
3.2 MS/MS
For identification of the 75 proteins affected by NR or CR in
the MS/MS mode, peptide sequences of a length of 523
amino acids were obtained upon tryptic digestion and 25
peptides were used to identify the different proteins. When
performing analyses of the MS/MS spectra, only one protein
was positively identified in each spot, but five proteins were
found in two separate spots. These five proteins changed
abundance in the same direction in both spots, which
excluded the possibility that any up-regulation of the protein
in one spot was counteracted by down-regulation in the other
spot. This resulted in a positive identification of 35 different
proteins in 40 (Tables 1, 2 and 3) of the 75 differently
expressed spots, which is equivalent to a success rate of more
than 50%. Only a limited number of protein sequences are
available from Oncorhynchus mykiss, and therefore only 21
MS/MS spectra of the 115 spectra used for identification of
the 35 proteins were matched to the sequences from this
species (a list of all sequences can be seen in the Supporting
Information). Of the remaining 28 identifications, only six
proteins were identified based on protein sequences from
organisms other than fish. When identification of a spot was
unsuccessful, it was in most cases due to very low levels of
protein present in the samples, which led to problems with
human keratin blunting the signal and thus making it
impossible to obtain useful spectra. Of the differently
expressed proteins that were identified, 23 responded to CR
(Table 1), 14 responded to NR (Table 2), and three responded
to both CR and NR (Table 3). Comparison of the experimental and theoretical MW (Tables 13) revealed as expected
that some of the proteins were truncated, because their
experimental MW was lower than expected from the theoretical MW, even though in other cases the experimental MW
exceeded the theoretical value. In total, 27 of the 35 identified
proteins had a MW within the 6 20% limit of the experimental value suggested by Wilkins et al. [43]. A comparison of
the estimated and the theoretical pI values showed that more
than half of the 40 identified protein spots had a experimental
pI value that was not within 6 0.25 U limits window of the
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Table 1. Identification of rainbow trout proteins whose abundance only changed in response to 30 min of chemical anoxia (10 mM NaN3)
followed by reoxygenation overnight. Proteins were identified by MALDI-MS/MS and cross-species matching with database
sequencesa)

Spot
no.
1
2c)
3
4
5c)
6c)
7
8
9
10
11
12
13
14
15
16
17
18c)
19
20
21
22
23

Description (species)

Accession
no.

Total
score

Seq.
cov.

MWe; pIe

MWth; pIth

Regulation

Calpain regulatory subunit


(Oncorhynchus mykiss)
Ubiquitin carboxyl-terminal hydrolase
isozyme L3 (Mus musculus) (4e-98)d)
K18,Simple type I Keratin
(Oncorhynchus mykiss)
Keratin 18 (Danio rerio)
Rho GDP dissociation inhibitor (GDI) alpha
(Danio rerio)
Rho GDP dissociation inhibitor (GDI) alpha
(Danio rerio)
Cathepsin D (Oncorhynchus mykiss)
K18, simple type I keratin
(Oncorhynchus mykiss)
Eukaryotic translation initiation (e-119)d)
factor 3, subunit 5 (Mus musculus)
EF hand domain containing 2
(Rattus norvegicus)
Sfrs1 protein (Danio rerio)
Serine-threonine kinase receptor associated
protein (Carassius auratus gibelio)
Carnitine deficiency-associated gene
expressed in ventricle 3 (Danio rerio)
Peroxiredoxin 6 (Pseudopleuronectes
americanus)
Annexin A3 (Homo sapiens)( 3e-85)d)
V-type ATPase B subunit
(Oncorhynchus mykiss)
Keratin 4 (Danio rerio)
Mitochondrial aldehyde dehydro- genase
2 family (Danio rerio)(0.0)d)
Cold inducible RNA binding protein
(Danio rerio)
Sept2 protein (Danio rerio)
Asparaginyl-tRNA synthetase
(Homo sapiens)
Hsp27 (Oncorhynchus mykiss)
Caspase 3B (Salmo salar)

Q8JHU8

153

21

28.3;4.77

24.4;4.93

Up

Q9JKB1

79

(4)

31.0;4.79

26.2;4.88

Up

O57607

110

(2)

20

41.5;4.18

48.8;5.39

Down

Q6P042
Q802W6

208e) (4)
139 (3)

7
21

44.0;4.63
29.1;5.02

48.6;5.53
23.2;5.00

Down
Up

Q802W6

119

(4)

15

33.3;4.93

23.2;5.00

Up

P87370
O57607

214
241

(3)
(3)

24
15

42.0;4.85
43.9;4.93

43.4;5.61
48.8;5.39

Down
Down

Q6IRT4

86

(3)

34.0;5.17

30.1;5.36

Down

Q4FZY0

100

(2)

12

33.3;5.26

26.8;5.42

Up

Q6PH14
Q98U02

111
232

(2)
(3)

24
12

35.5;5.15
36.8;5.19

20.7;7.74
36.1;5.49

Down
Down

Q7T370

80

(2)

10

37.3;5.25

26.2;5.27

Up

Q6PBK9

95

(2)

28.4;5.68

25.2;6.12

Up

P12429
Q9W6M4

87
157

(2)
(2)

31

40.6;5.46
58.3;5.53

36.2;5.20
56.1;5.46

Down
Down

Q9PV92
Q6TH48

120
140

(3)
(4)

58.1;5.72
58.1;5.79

54.3;5.42
56.5;6.06

Down
Down

BC048027

175e) (3)

16

21.6;5.98

19.2;9.29

Up

AAI07984
AAH01687

109
85

(3)
(2)

13
4

47.5;6.06
68.1;6.08

45.5;5.87
63.7;5.90

Down
Down

Q4F877
Q4ZHV0

191
88

(3)
(2)

25
11

32.3;6.28
33.9;6.53

25.1;7.90
31.4;5.97

Down
Down

(3)b)

a) Accession numbers are given according to TrEMBL and Swiss-Prot. MWe and MWth are experimental and theoretical molecular weights,
respectively, while pIe and pIth are experimental and theoretical isoelectrical values, respectively. Protein reported is the protein given
the highest score except when a protein sequence from Oncorhynchus mykiss appeared further down in the list with a significant score,
in that case, the rainbow trout sequence is reported instead. Multiple protein entries representing homologs from different species were
often found with equivalent scores; these are not reported.
b) Number of sequences used for identification.
c) The identification was based on an error tolerant search on MASCOT.
d) Protein identification resulted in the identification of unnamed protein. The obtained sequence was then used for a blast search (e-value
is given for chosen protein) by the NCBI BLAST2 service (http://expasy.org/tools/blast/) against the complete database, and an annotated homolog were chosen as the representative.
e) The reported MS blast scores are from the result obtained by blast searching using as query a combination of the peptide sequences
obtained by de novo sequencing (spectra are available in the Supporting Information).

2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Proteomics 2008, 8, 20352044

Table 2. Identification of rainbow trout proteins whose abundance only changed in response to 2 h of N2-induced anoxia followed by
reoxygenation overnight. Proteins were identified by MALDI-MS/MS and cross-species matching with database sequencesa)

Spot
no.

Description (species)

Accession
no.

Total
score

Seq.
cov.

MWe; pIe

MWth; pIth Regulation

24
25c)
26

Glyoxalase 1 (Danio rerio)


Proteasome subunit alpha type 3 (Homo sapiens)
Simple type II keratin K8a (S1)
(Oncorhynchus mykiss)
Chaperone protein GP96 (Danio rerio)
Guanosine diphosphate (GDP) dissociation
inhibitor 2 (Mus musculus)
Psmd8 protein (Rattus norvegicus)(e-113)d)
Capping protein alpha 2 (Monodelphis domestica)
Asparaginyl-tRNA synthetase (Homo sapiens)
Mvp protein (Danio rerio)
Pkm2 protein (Danio rerio)
Phosphoglycerate mutase 1 (Danio rerio)
ARP1 actin-related protein 1 homolog B
(Danio rerio)
Dihydrolipoamide dehydrogenase (Danio rerio)
Eukaryotic translation elongation factor 2
(Xenopus tropicalis)

Q6P696
P25788
Q90YH3

182 (5)b)
156 (3)
271 (5)

28
23

26.1;5.00
32.4;5.09
57.4;4.97

20.4;5.23
27.9;5.19
59.2;5.29

Up
Down
Down

Q7T3L3
Q61598

100 (2)
216 (4)

13
22

42.0;4.94
56.5;5.33

63.6;4.68
51.0;5.14

Down
Down

Q3B8P5
Q2QL99
AAH01687
Q7ZUB4
Q6NXD1
Q7SZR4
NP_998537e)

150
122
131
172
121
151
218

(2)
(2)
(2)
(3)
(3)
(2)
(3)

16
8
13
14
19
24

29.9;5.64
37.6;5.59
71.5;5.72
116;5.99
64.2;6.50
31.6;6.76
48.0;6.64

32.3;6.47
33.;5.55
63.8;5.90
95.1;5.48
58.6;6.36
29.1;6.19
42.5;6.19

Up
Up
Down
Down
Down
Up
Down

Q6TNU6
Q6P3N8

75 (2)
124 (2)

6
11

63.5;6.80
115;6.65

54.1;7.61
96.6;6.41

Down
Down

27
28
29
30
31
32
33
34
35
36
37

a)
b)
c)
d)

See legend to Table 1 for further information.


Number of sequences used for identification.
The identification was based on an error tolerant search on MASCOT.
Protein identification resulted in the identification of unnamed protein. The obtained sequence was then used for a blast search (e-value
is given for chosen protein) by the NCBI BLAST2 service (http://expasy.org/tools/blast/) against the complete database, and an annotated homolog were chosen as the representative.
e) The accession number is from NCBI.

Table 3. Identification of rainbow trout proteins whose abundance changed both in response to 2 h of N2-induced anoxia followed by
reoxygenation overnight and 30 min of chemical anoxia (10mM NaN3) followed by reoxygenation overnight. Proteins were identified by MALDI-MS/MS and cross-species matching with database sequences

Spot
no.

Description (species)

Accession
no.

Total
score

Seq
cov.

38
39
40

Ribosomal protein large P0-like protein (Sparus aurata)


Sfrs1 protein (Danio rerio)
Acidic ribosomal phosphoprotein (Pagrus major)

AAT44430
Q502R8
Q6Y203

177 (3)a) 13
207 (4) 51
324 (4) 28

MWe; pIe MWth; pIth Regulation

38.0;5.28 34.1;5.70 Down


36.1;5.29 29.0;7.74 Down
36.5;5.39 23.5;8.49 Down

a) Number of sequences used for identification. See legend in Table 1 for further information.

theoretical value defined by Wilkins et al. [44]. It should be


noted that most of the theoretical values were based on proteins originating from species other than rainbow trout,
rendering the exact MW and pI values of these proteins
unknown.

Discussion

One of the notable features of anoxia followed by reoxygenation is the protein synthesis seen in the second late response
after 24 h of reoxygenation [5]. Therefore, to allow for differ 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ences initiated during the period of anoxia and reoxygenation to be fully induced we investigated the changes in protein expression seen after anoxia followed by 24 h of reoxygenation.
In chemical anoxia, cells are rapidly depleted of ATP [45],
and it is therefore considered a hard stressor. In a study on
eel hepatocytes, ATP levels drop immediately after anoxia
obtained by flushing with N2 and reach a plateau after 2 h
[46]. Therefore, in order to achieve a realistic comparison
between N2-induced anoxia and chemical anoxia, we completely depleted the medium of oxygen by flushing with N2.
Recently, in the RTHDF cell line we have demonstrated that
www.proteomics-journal.com

Cell Biology

Proteomics 2008, 8, 20352044

2041

4.1 CR vs. NR

Figure 1. Representative 2-DE of proteins extracted from rainbow trout hypodermal fibroblasts cell line: Effects of CR and NR.
A total of 100 mg protein was separated by charge in the first dimension (Immobilline Drystrip pI 47) and then by size in the
second dimension (12% SDS-PAGE). Proteins were visualized by
silver staining. All proteins labeled by their spot number changed
significantly (p ,0.05) in level by stimulation. The proteins indicated by a white arrow have not been identified while those
indicated by a striped arrow have been identified by MS/MS sequencing. (A) Proteins changing their abundance solely in response to CR. The gel is representative of six gels separating
proteins from cells submitted to CR. (B) Proteins changing their
abundance solely in response to NR. The gel is representative of
five gels separating proteins from cells submitted to NR. (C) Proteins changing their abundance in response to both CR and NR.
The gel is representative of four gels separating proteins from
control cells.

the change in the p44ERK (extracellular signal-regulated


kinase) level induced by 2 h of anoxia (obtained by flushing
with N2) followed by reoxygenation (NR) is similar to that
induced by 30 min of chemical anoxia (10 mM azide) followed by reoxygenation (CR) [30]. These durations of anoxia
and reoxygenation were therefore used in the present
attempt to explore and compare the overall protein expression pattern of the two different anoxia models.
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

It was observed that 45 protein spots were regulated in response to CR while 35 protein spots were regulated in response to NR. There was no difference between the two
stimuli in the distribution of proteins that were up/downregulated, as in response to both stimuli more proteins
were down-regulated than up-regulated. The number of
proteins changing their expression was larger in response to
CR than NR, supporting the notion that CR is a more severe
stressor.
Since the approach of using 2-DE to compare different
anoxia models is novel, it is difficult to find comparable
studies in the literature. In an examination of a few proteins,
Petroni et al. [32] likewise observed that anoxia obtained by
flushing with N2 compared to chemical anoxia (10 mM
NaCN) is somehow different even if the difference was less
compelling than in the present study.
Most striking when comparing the expression patterns
of CR and NR was that of the 75 proteins affected by the
two stimuli, only six proteins (Fig. 1C) changed in response to both stimuli. Even when taking into consideration the differences between the ways anoxia was effectuated in CR compared to NR the limited overlap between
the two models was highly surprising. The differences
might originate from the way the two types of anoxia were
effectuated, the obvious difference being the level of oxygen. During chemical anoxia, oxygen was present whereas
it was completely removed during N2-induced anoxia. The
drop in oxygen during N2-induced anoxia, activates
hypoxia-inducible factor (HIF), which is the main regulator
of transcription during anoxia [47]. In a study on rat CB
glomus cells, Baby et al. [48] observed that 5 mM of
sodium azide does not influence the level of HIF-1a. This
marked difference between the two situations can account
for some of the differences seen in the expression pattern
of the two models. However, the proteins regulated following NR in the present study were not typically associated
with HIF.
4.2 CR vs. NR: similarities
The direction of the change in expression of all six protein
spots (Fig. 1C) was the same independent of stimuli, making them highly interesting, as they might represent a
uniform way in which RTHDF cells responds to anoxia
followed by reoxygenation. Two of the three identified proteins were ribosomal protein large P0-like protein and
acidic ribosomal phosphoprotein (see Table 3). Both are
involved in protein synthesis and their down-regulation
might indicate that the RTHDF cells are responding to
both types of cellular stress by down-regulating their protein synthesis as an overall adaptive response to reoxygenation. Interestingly, only acidic ribosomal phosphoprotein has previously been linked to processes connected to
anoxia [49], and it is likewise normally assumed that upwww.proteomics-journal.com

2042

T. Wulff et al.

regulation of protein synthesis is a part of the response


following long-term reoxygenation, though this topic is still
under investigation [7, 8].
In response to CR (Table 1), we also observed down-regulation of two other proteins involved in protein synthesis,
namely asparaginyl-tRNA synthetase and eukaryotic translation initiation factor 3, subunit 5, and in response to NR
(Table 2), the eukaryotic translation elongation factor 2 was
likewise down-regulated. A general down-regulation of protein synthesis was also supported in the present study by the
observation that of the 75 different proteins regulated the
majority of them were down-regulated (51 proteins). The
combined regulation of protein synthesis experienced in response to both stimuli showed that even when considering
the difference in how anoxia was introduced, there seems to
be a general response to both stimuli. This down-regulation
of protein synthesis might be due to an effort of the cells to
save energy. However, the up-regulation of 24 other proteins
showed that the cells were fully capable of increasing synthesis of some selected proteins, if they were needed, following reoxygenation.
4.3 Anoxia in RTHDF cells
In a parallel study [17] on RTHDF cells investigating the
effects of long-term anoxia, six of the protein spots identified
in the present study (Tables 1 and 2, no. 5, 6, 10, 14, 26 and
34) were also found to change in response to 24 h of anoxia.
The up-regulation of the glycolytic protein phosphoglycerate
mutase 1 (Table 2, no. 34) would be expected during longterm anoxia where an up-regulation of metabolism often is
observed [12]. In the present study, the up-regulation of
phosphoglycerate mutase 1, together with the previouslymentioned down-regulation of protein synthesis, indicated
that the RTHDF cells after 24 h of reoxygenation were still
influenced by the lack of energy experienced during the
initial period of anoxia.
4.4 Protective proteins
What is interesting is that peroxiredoxin 6 (Prdx 6; Table 1)
was up-regulated both in response to CR and 24 h of
anoxia, which showed a potential protective role of this
protein both during the anoxic challenge and in the period
of reoxygenation. Prdx 6 is a member of the relative newly
discovered family of antioxidant enzymes that possesses
peroxidase activity [50] and it could therefore reduce H2O2
and different types of hydroperoxides [51] to protect the cell
from oxidative damage. The observation that Prdx 6 was
up-regulated in response to CR is consistent with the general notion that a rise in H2O2 [52] is observed in response
to IR and the protein level of Prdx 6 therefore most likely
is increased as a protective reaction towards H2O2 [5355].
Since Prdx 6 in the present investigation was up-regulated
in CR and not in NR, it indicates that CR leads to the
production of more H2O2 than NR, confirming its role as a
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2008, 8, 20352044

harder stressor. No up-regulation of proteins involved in


apoptosis was observed although apoptosis is a well-known
consequence of severe hypoxia/anoxia followed by reoxygenation [56]. On the contrary, we observed that caspase 3,
which is known to be involved in apoptosis [57], was downregulated following CR. In addition, we found that Hsp27
was down-regulated in response to CR and chaperone protein GP96 (Hsp90b1) was down-regulated in response to
NR (see Table 2). Both proteins are well known to be
involved in the protective response following different
stressors [58]. Taken together, these results indicate that
neither CR nor NR stressed the cells to an extent that
demanded activation of all the protective mechanisms
present in the cell.

4.5 Keratins
Supporting the observation that CR and NR induced a
down-regulation of the protein synthesis, we found that the
largest group represented in Tables 1 and 2, namely the
keratins, were indeed down-regulated (spots 3, 4, 8, 17 and
26). The finding that down-regulation of different types of
keratin might be a general phenomenon in response to
anoxia followed by reoxygenation is novel and has not previously been described in the literature. The regulation of
different types of keratin is another common response in
the two anoxia models, emphasizing the usefulness of
chemical anoxia as an experimental alternative to N2induced anoxia.
In conclusion, we found that of the 45 detected protein
spots that changed following CR and the 35 protein spots
that changed following NR, only six proteins changed in response to both stimuli. In studies using chemically-induced
anoxia, this should be clearly stated, and preferably a distinction should be made between anoxia induced by depletion of oxygen and chemically-induced anoxia. However,
changes in protein synthesis seem to be uniform in the two
types of anoxia and both insults clearly stress the cells, leading to a general change in protein expression. Even considering such similarities, data should be discussed in context with the applied stimuli, and great care should be taken
before making direct comparison of data obtained using different anoxia models.

The research was supported by grants from the Danish Network in Aquaculture and Fisheries Research (www.fishnet.dk)
financed by the Danish Ministry for Food, Agriculture and Fisheries and the Danish Agricultural and Veterinary Research
Council ass well as Danish Institute for Fisheries Research and
Forskeruddannelsesrdet. We would like to thank Hanne
Jakobsen and Andrea Lorentzen for great technical support, and
Karin Hjern for critical reading of the manuscript.
The authors have declared no conflict of interest.
www.proteomics-journal.com

Proteomics 2008, 8, 20352044

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