June 2021

Medical Mass Spectrometry Vol. 5 No. 1

Research Paper

Identiﬁcation of nine proteins in whole lung extract derived
from bleomycin-induced interstitial pneumonia model mouseby two-dimensional electrophoresis/in-gel digestion/
MALDI-TOF MSMS analysis
1
1
1,2
2,3
1
Takaaki Ishida , Takuya Kotani , Yoko Tabushi-Matsumura , Toyofumi Nakanishi *, Tohru Takeuchi
1

Department of Internal Medicine IV, Osaka Medical and Pharmaceutical University,
2‒7 Daigaku-cho, Takatsuki, Osaka 569‒8686, Japan
2
Central Clinical Laboratory, Osaka Medical and Pharmaceutical University Hospital,
2‒7 Daigaku-cho, Takatsuki, Osaka 569‒8686, Japan
3
Research and Development Center, Osaka Medical and Pharmaceutical University,
2‒7 Daigaku-cho, Takatsuki, Osaka 569‒8686, Japan
Abstract The etiology and pathogenesis of interstitial pneumonia have not been fully elucidated. A proteomics-based
approach using two-dimensional electrophoresis/in-gel digestion/matrix-assisted laser desorption time-of ﬂight mass spectrometry (MALDI-TOF MS) was applied to identify new candidate biomarkers in whole lung extract from the bleomycin-induced interstitial pneumonia model mouse. First, the mouse lung extract was subjected to two-dimensional electrophoresis and then the bleomycin-induced interstitial pneumonia (BLM-IP) speciﬁc spots underwent in-gel digestion. The
proteins in the speciﬁc spots were identiﬁed by MALDI-TOF MS and MSMS analyses. Nine proteins including anti-oxidative stress proteins, transgelin-2 (TGLN-2), vimentin, transthyretin, fatty acid-binding protein, and sepiapterin reductase
were identiﬁed by MALDI-TOF MS and MSMS. The expression levels of these 9 proteins were clearly up-regulated whencompared with those of the healthy mouse lung.
In conclusion, the proteomics-based approach enabled us to identify nine up-regulated proteins, especially TGLN-2, inthe BLM-IP model mouse. The up-regulated modiﬁed TGLN-2 might be an early marker of smooth muscle cells diﬀerentiation induced thicken of the alveolar walls and play an important role in pulmonary ﬁbrogenesis.
Key words: bleomycin-induced interstitial pneumonia, whole lung extract, proteomics-based approach, oxidative stress, transgelin-2

Introduction

tium, the walls of alveoli become thick, hard, and show

Idiopathic interstitial pneumonia (IIP) is an intractabledisease in which inﬂammation occurs in the lung intersti-

ﬁbrosis, and ﬁnally it becomes diﬃcult for oxygen /carbon
1)dioxide exchange to take place in lung alveoli . The etiology and pathophysiology are still not well-understood, but* Corresponding author

abnormal wounds are noted due to pathological ﬁndings ofToyofumi Nakanishiﬁbroblast proliferation, extracellular matrix deposition, andResearch and Development Center, Osaka Medical and Pharmaceutical University, 2‒7 Daigaku-cho, Takatsuki, Osaka

1‒3)scarring. It is considered that excessive ﬁbrosis due to569‒8686, Japan
Tel: ＋81‒72‒683‒1221 (ex 2658), Fax: ＋81‒72‒684‒6548

healing and some inﬂammatory reactions are involved. It isalso thought that alveolar epithelial cells acquire the charac-

Received: April 19, 2021.

epithelial cells and cause activation of ﬁbroblasts . IIP is alife-threatening pathological condition that causes respira-teristics of mesenchymal cells due to damage to alveolar

E-mail: nakanisi@osaka-med.ac.jp

4)

Accepted: May 19, 2021.

Epub June 17, 2021.

tory failure when it progresses. The collective term of inter-

DOI: 10.24508/mms.2021.06.006
38

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Medical Mass Spectrometry Vol. 5 No. 1

stitial lung disease comprises various lung conditions,markers and new therapeutic targets. Previously, our groupwhich are characterized by thickening of the alveolar walls

reported that new biomarkers, 4 citrullinated proteins could

by inﬂammation or ﬁbrosis. Lung inﬂammation is treatedbe used to diﬀerentiate between rheumatoid arthritis and11)
osteoarthritis patients by the proteomics-based technique .

with steroids and immunosuppressants, and pulmonaryﬁbrosis is treated with anti-ﬁbrosis agents such as pirfeni-

In the present study, we applied the novel proteomics-based investigation using two-dimensional electrophore-done and nintedanib. However, many cases are treatment-resistant and the outcome is poor in all disease types,

sis/in-gel digestion/matrix-assisted laser desorption time-of
ﬂight mass spectrometry (MALDI-TOF MS) to investigate

which is problematic. The disease is associated with a very

new biomarkers in whole lung extract from the BLM-IP
model mouse, and identiﬁed nine up-regulated anti-oxida-

poor prognosis, with most patients dying within 5 years ofdiagnosis.

tive and vesicle stress-related proteins.

On developing IP, KL-6, a sialylated carbohydrate antigen, is excessively produced by type II alveolar epithelial

Materials and Methods

5)

cells with inﬂammation . The extremely high levels of
KL-6 in blood showed at the cases of interstitial pneumonia

Culture of mice for drug-induced pulmonary ﬁbrosis

as same as other diseases, such as tuberculosis and malig-

The Institutional Animal Care and Use Committee ofnant tumors, but other lung diseases and bacterial / viral
6,7)pneumonia were limitedly . So, it is not possible to con-

Osaka Medical and Pharmaceutical University approved all

clude that the levels of KL-6 in blood could be used to diag-

including the surgical procedures and animal care, and allnose IP. It is important to identify a speciﬁc biomarker formethods were performed in accordance with the relevant

diﬀerentiations between IP and other diseases.

guidelines and regulations.
Female 13‒14-week-old C57BL/6J mice (SHIMIZU Lab-of the following research protocols (approval ID: 27053),

Bleomycin (BLM) is the most widely used agent to
induce pulmonary ﬁbrosis in experimental animals. Afteroratory Supplies, Kyoto, Japan) were anesthetized with ani.p. injection of 400 mg/kg 2,2,2-tribromoethanol (Avertin,

the systemic delivery of BLM, endothelial cells weredirectly injured, and this was subsequently followed by epi-

Sigma-Aldrich Japan K.K.). The administered BLM was8)thelial cell injury, inﬂammation, and ﬁbrosis . With the

prepared by mixing sterile BLM sulfate powder (Nippon

intratracheal, intranasal, or vascular administration of

Kayaku, Tokyo, Japan) with normal sterile saline. A dose of

BLM, lung epithelial cells were initially injured, but the

3 mg of BLM in a total volume of 100μL of sterile salinedevelopment of endothelial cell damage has not been fully

was injected subcutaneously by osmotic minipump (Alzet

elucidated. It would be a very important to identify new
biomarker for the understanding critical cause of pulmo-

2010, DURECT, Cupertino, CA, USA) from days 0 tonary ﬁbrogenesis and thickening of the alveolar walls in the
BLM-induced interstitial pneumonia (BLM-IP).

opened and the cardiopulmonary was exposed, we wereProteomics-based identiﬁcation facilitates high-throughput studies of protein expression, and enables the identiﬁca-

using 2 mL of saline containing 100 U of heparin. The micetion of molecular mechanisms that are responsible for thedevelopment of speciﬁc diseases. For example, in compari-injection, the lungs were harvested for histological analysis,

7

12,13). After anesthetizing the model mouse, the thorax was

injected from the right ventricle for pulmonary perfusion
were sacriﬁced at 28 days after the initiation of bleomycin
and stored at −80 degrees prior to use.

son with RNA-sequence or microarray-based proﬁling of

Two-dimensional polyacrylamide gel electrophoresis(2D-PAGE)

gene expression, which were previously applied to idiopathic pulmonary ﬁbrosis, proteomics analysis oﬀers various advantages. In particularly, it indicates the eﬀective
existence of functional proteins in clinical samples. In addi-

Complete Mammalian Proteome Extraction Kit (Merck Milli-tion, a high transcription level leading to an abundant quan-pore, Darmstadt, Germany) and cOmplete Mini

tity of messenger RNA does not always mean an abundanceof the corresponding protein or its eﬀectual activity. There-

Diagnostics, Indianapolis, IN, USA). Homogenates were

9,10）

TM

The lungs were homogenized by ProteoExtract

TM(Rochemixed with lysis buﬀer (5 M urea, 2 M thiourea, 2%
CHAPS, 2% SB3‒10, and 1% dithiothreitol). Protein con-fore, the proteomics-based technique has signiﬁcant strategic beneﬁts for the identiﬁcation of speciﬁc diagnostic bio-

centrations of these samples were measured by Protein
39June 2021

Medical Mass Spectrometry Vol. 5 No. 1(Bio-Rad Laboratories, Hercules, CA,

The gel pieces were then dried in a vacuum centrifuge, and

USA). Aliquots containing 100 micrograms of proteinssubsequently rehydrated in a digestion solution consisting

TM

Assay System

TM(GE
were applied overnight to Immobiline Drystrip
Healthcare Bio-Science, Piscataway, NJ, USA) by in-gelof 50 mM NH4HCO3, 5 mM CaCl2, and 0.1μg/μL modiﬁed
sequence-grade trypsin (Promega). After overnight incuba-

rehydration. The rehydrated gels were then gently driedtion at 37 degrees, the digestion was terminated in 5% tri-with ﬁlter paper to remove excess ﬂuid and isoelectric
focusing (IEF) was performed in a Pharmacia Hoefer Mul-

ﬂuoroacetic acid (TFA) for 20 min. Peptides were extracted
3 times (20 min each) with 5% TFA in 50% acetonitrile, andTMelectrophoresis chamber (GE Healthcare
tiphor II
Bio Science, Piscataway, NJ, USA), according to the manu-

the extracted peptides were pooled and dried in a vacuum

facturer s instructions. Two-dimensional SDS-PAGE wasperformed in 9‒18% acrylamide gradient gels using an Iso-(Millipore) following the manufacturer s protocol and ana-

Dalt electrophoresis chamber. The 2-D gels were stainedwith SYPRO Ruby (Invitrogen, Carlsbad, CA, USA) fol-mass spectrometer and MASCOT database softwareTMcentrifuge. The peptides were puriﬁed with ZipTipC18

lyzed by Ultraﬂex TOF/TOF (Bruker Daltonics) MALDI(Matrix Science).

lowing the manufacturer s protocol. The SYPRO Rubystained protein spots were detected using Molecular Imager
TM(Bio-Rad Laboratories, Hercules, CA, USA) andFX

Results and Discussion

were subjected to in-gel digestion. Image analysis and data-

investigation to identify new candidate biomarkers in the
BLM-IP model for the prediction of etiologies of lung pul-

In the present study, we applied a novel proteomics-based

base management were done using Image Master Platinum
TM(GE Healthcare Bio-Science,
image analysis software

monary ﬁbrosis in comparison with a normal mouse.

Piscataway, NJ, USA).

As shown in Fig. 1, microscopy images of Masson trichrome- stained lung tissues show a deﬁnite change

In-gel digestion and mass spectrometric identiﬁcationof proteins

between the BLM-IP model mouse and normal mouse. A
lung section from the BLM-IP model mouse showed theThis work was performed essentially as described else-

diﬀuse increase collagen deposition compared with normal

4)

where . Protein spots were excised from the dried silver
stained 2-D gels, and rehydrated for 20 min in 100 mM

lung. As expected, we observed lung ﬁbrosis with collagendeposition in lung interstitium 28 days after BLM adminis-

NH4HCO3. The gel spots were then destained for 20 min in

tration.

a solution of 15 mM potassium ferricyanide and 50 mM
thiosulfate, rinsed twice in Milli-Q water, and ﬁnally dehy-

As shown in Fig. 2, the SYPRO Ruby-stained protein
patterns of the BLM-IP model mouse and normal mousedrated in 100% acetonitrile until they turned opaque white.

overlapped. Seventeen spots were diﬀerent between theFig. 1.

Representative microscope images of Masson trichrome-stained lung tissue sections of normal and BLM-IP model
mice 28 days after treatment.
The scale bar indicates 100 μm.

40June 2021

Fig. 2.

Medical Mass Spectrometry Vol. 5 No. 1

An overlaid image of the two-dimensional gel electrophoresis of proteins in whole lung extract derived from BLM-IP
model mouse (red)and reference
and reference
healthyhealthy
mouse.
mouse (green).
-regulated-regulatedproteins-IP-IPmodelThe redgreynumbers
numbersindicate
indicateupup
proteinsininthe
theBIM
BIM
modelmouse
mousecompared
comparedwith
withthethereference
referencemouse.
mouse.

BLM-IP model mouse and normal mouse. Twelve of these17 spots showed higher expressions in the BLM-IP mouse

molecule by the electrophoresis/western-blot/ ECL detection. As the result, the phosphorylation could not be con-than those of the normal mouse, and ﬁve spots showed
lower expression in the BLM-IP group than in the normal

ﬁrmed, but it might be still caused by a S-sulfonation or
S-nitrosylation in which pI shifts to the acidic side. On themouse. As the results of MALDI-TOF-MSMS analysis coupled with the MASCOT database search, the 12 up-regu-

other hand, spot Nos. 10 and 11, both identiﬁed as vimentin(VIM), had the same pI, but the molecular weight of spot
No. 11 was deﬁnitely higher than that of spot No. 10, possi-

lated proteins in the BLM-IP group could be identiﬁed, but

bly due to modiﬁcation, such as citrullination, glycosyla-

a single spot could not be identiﬁed, as shown in Table 1.
They included two anti-oxidative stress proteins (spot Nos.

tion, or ubiquitination.
Fig. 3a)‒h) show MSMS spectra of the tryptic in-gel

4 and 8), a thyroid hormone-binding protein (No. 2), an
intracellular carrier for active lipids (No. 3), actin-related

digestions of spots (Nos. 2, 3, 4, 7, 8, 9, 10, 11, and 12) inthe whole lung extract of the BIM-IP model mouse. The

proteins (Nos. 5 and 6), a molecule involved in cholesterol
metabolism (No. 7), a molecular chaperon (No. 9), a mole-

MSMS spectra obtained from spot No. 2 matched thecule involved in tetra-hydrobiopterin biosynthesis (No. 10),sequence of two peptides derived from transthyretin (TTR).

and cytoskeleton proteins (Nos. 11 and 12). Interestingly,
spot Nos. 5 and 6 were also identiﬁed as transgelin-2

Figure 3a shows the MSMS spectra from the sequence of atryptic peptide from the 84th to 90th TTR. In the same way,other tryptic peptides were also identiﬁed by MSMS analy-(TGLN-2), but the isoelectric point (pI) of spot No. 6 wasclearly lower than that of spot No. 5. It is of interested
whether this modiﬁed TGLN-2 molecule leads to acceler-

sis coupled with the MASCOT database search. The

ated lung ﬁbrosis compared with the native TGLN-2. So,

MSMS spectra matched the sequences from tryptic peptideof fatty acid-binding protein 5 (FABP5), copper-zinc super-

we speculated that this lower shift of pI may be caused by apost-translational modiﬁcation, such as phosphorylation,oxide dismutase (SOD-1), Apolipoprotein A-1 (Apo A-1),
peroxiredoxin-6 (Perx-6), heat shock protein beta-1 (HSP

but this MSMS analysis unfortunately could not clarify anyb-1), sepiapterin reductase (SPR), and VIM, respectively.
14)

Kulkarni et al.

evidence of the modiﬁcations. And so, we further examined
to identify a post-translational modiﬁcation of the TGLN-2

reported a novel proteomics approach toclarify the proﬁles of expressed proteins in cultured ﬁbro41

June 2021

Table 1.

Medical Mass Spectrometry Vol. 5 No. 1

Up-regulated proteins from 2D gel spots derived from whole lung extract of the bleomycin-induced interstitial
pneumonia model mouse by MALDI-TOF/TOF and MASCOT database

Spot
No.

Name

MW in
database(KDa)

Observed
MW(KDa)

Description

pI

Localization

Mowse
Score

̶

̶

̶

̶

1

ND

̶

̶

2

Transthyretin

16

15

TTR, thyroid hormone-binding protein

5.77

cytoplasm,
secreted

43

3

Fatty Acid-binding Protein

15

15

FABP5, epidermal type

6.14

cytoplasm

51

4Superoxide Dismutase Cu-Zn

16

16

SOD1, Antioxidant

6.02

cytoplasm,

103

5Transgelin-2

23

20

6

Transgelin-2

23

20

TAGLN2, protein binding, epithelial cell
diﬀerentiation

8.39

cytoskeletal
protein

77
143

7

Apolipoprotein A-I

30

25

ApoA1, cholesterol transporter

5.64

secreted

61

8

Peroxiredoxin-6

2525

PRDX6, antioxidant

5.71

cytoplasm

198

9

Heat shock protein beta-1

23

25

HSPB1, estrogen-regulated

6.12

cytoplasm

104

10

Sepiapterin reductase

28

26SPR, oxidoreductase

5.58

cytoplasm

79

11

Vimentin

53

45

12

Vimentin

53

46

intermediate ﬁlaments

5.06

cytoplasm

63
46

ND: not identiﬁcation.

blasts of mouse lung tissue after the intranasal administrationof BLM. The expression levels of HSPs, TGLN-2, and VIM

anti-oxidative proteins in the BIM-IP model mouse (SOD-1
and Perx-6). Oxidative stress caused by reactive oxygen

were up-regulated and those of MMP-9 were down-regulated

species is involved in the pathophysiology of IIP, and oxi-compared with the normal mouse. They suggested that the
BLM-induced up-regulated proteins were closely associateddative stress is associated with damage to lung epithelial

with phosphoinositide-3-kinase/protein kinase B (PI3K/Akt),repair of the lung, and thus induces ﬁbrosis . Schamberger
21）
AC et al. reported that anti-oxidative molecules, such as

cells and vascular endothelial cells and abnormal tissue
20)

and that nitric oxide signaling pathways are important for the
15,16). On the other hand,

SOD, glutathione-3-peroxidase, and HSP b-1, are highly

reported the expression levels in a model mouse

expressed in interstitial lung diseases of humans and model
mice, as in the present study. HSP b-1, also known as

regulation of pulmonary ﬁbrosis
17)

Della et al.

of lung ﬁbrosis induced by the intratracheal instillation of asingle dose of BLM. The levels of Perx-6, TGLN-2, VIM, andHSP27, is a small heat shock protein involved in many cel-

HSP all showed down-regulated patterns, with all results

lular processes and protects cells against oxidative stressbeing the opposite of our MS analysis. We considered thatand controls cell apoptosis.

this discrepancy was likely due to the route of administrationof BLM, and so the expression pattern of cytokines, prote-

As shown in Figs. 4 and 5, the MSMS spectra obtained
from spot Nos. 5 and 6 matched the sequences of two andsix tryptic peptides of the same protein, TGLN-2, in theases/anti-proteases, growth factors, and signaling pathways

lung tissue extract of the BIM-IP model mouse. TGLN-2,
which belongs to the actin-binding protein family, is an19)

involved in pulmonary ﬁbrosis were diﬀerent. Chua F et al.

also reported that a single intratracheal instillation of BLM
rapidly leads to severe bronchiocentric ﬁbrosis, while intrave-

early marker of smooth muscle cell diﬀerentiation. The
basic function of TGLN-2 is to regulate the actin cytoskele-

nous or intraperitoneal routes of administration characteristically induce subpleural scarring, and mild intermittent ﬁbro-

ton through actin binding, and eventually participate in pro-sis can result from repeated BLM dosing similar to thecesses involving cytoskeleton remodeling. As the present

induction of human pulmonary ﬁbrosis.

results, we considered that the diﬀerentiation of smooth

In the present study, we identiﬁed 2 kinds of up-regulated

muscle cells and reconstruction of the cytoskeleton would
42

June 2021

Fig. 3.

Medical Mass Spectrometry Vol. 5 No. 1

The MSMS spectra of the tryptic in-gel digestions of spots (Nos. 2, 3, 4, 7, 8, 9, 10, 11, and 12) in whole lung extract of
the BIM-IP model mouse. All Mowse scores of MSMS ion spectra indicate identity or extensive homology (p＜0.05),
which strongly supported these identiﬁcations.

a) MALDI-TOF/TOF mass spectra of peptides
peptides fromfrom thethe inin--gelgel digest
digest of
of2D
2Dgel
gelspot
spotNo.
No.2.2.The
Thegrey
red letters indicate the amino acidsof the tryptic peptide of TTR. The numbers show the residues of identiﬁed sequences of the TTR.
b) FABP-5, c) SOD-1, d) Apo A-I, e) Perx-6, f) HSP b-1, g) SPR, and h) Vim also present the MSMS spectra of tryptic peptides; the-mentioned.
-mentioned.
grey
red letterslettersand
andnumbers
numbersare
arethethesamesameasasabove
above

43

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Medical Mass Spectrometry Vol. 5 No. 1

Fig. 4.

MALDI-TOF/TOF mass spectra of peptides from the in-gel digest of 2D gel spot No. 5.

Fig. 5.

MALDI-TOF/TOF mass spectra of peptides from the in-gel digest of 2D gel spot No. 6.

-2.-2.
The redgreyletterslettersindicate
indicatethe
theaminoaminoacids
acidsofofthethetryptic
tryptic
peptidepeptide
ofof
TGLN
TGLN
The
The
numbers
numbersshow
show
thetheresidues
residuesof ofidentiﬁed
identiﬁed
sequences
sequencesof-2. -2.
TGLN
of TGLN-2.-2.
The redgreyletterslettersindicate
indicatethe
theaminoaminoacids
acidsofofthethetryptic
tryptic
peptidepeptide
ofof
TGLN
TGLN
The
The
numbers
numbersshow
show
thetheresidues
residuesof ofidentiﬁed
identiﬁed
sequences
sequencesof-2. -2.
TGLN
of TGLN

also occur in the BLM-IP mouse. Yu H et al. reported that
TGLN-2 mRNA expression in isolated alveolar epithelial

expression coincides with cellular plasticity. They demonstrated that the up-regulated TGLN-2 plays an important

type II cells was signiﬁcantly up-regulated after BLM

role in pulmonary ﬁbrogenesis.

22)

administration compared with the control mouse. This
up-regulation was also noted in lung species of IP ﬁbrosis

Nonaka et al.

23)

reported that a case of tracheal amyloido-

sis discovered during observation of interstitial pneumonia.
Although the authors considered to be a coincidence of tra-patients. This high expression of TGLN-2 was recognizedin ﬁbroblast-to-myoﬁbroblast transdiﬀerentiation, which ischeal amyloidosis and IP, there are also some reports of thea process leading to endothelial-to-smooth muscle celltransdiﬀerentiation, strongly suggesting that TGLN-2

diﬀuse alveolar septal amyloidosis. They suggested thatfurther study is needed to resolve the relationship between
44June 2021

Medical Mass Spectrometry Vol. 5 No. 1

Nanotoxicology 7(2): 157‒168, 2013..

IP and tracheal localized amyloidosis. Previously, ourgroup had ﬁrst identiﬁed the S-sulfonation of transthyretin

4) Parimon T, Yao C, Stripp BR, Noble PW, Chen P: Alveo-(TTR) in patient with TTR-related amyloidosis by the novel
proteomics-based analysis. The S-sulfonation of TTR is an

lar Epithelial Type II Cells as Drivers of Lung Fibrosis in
Idiopathic Pulmonary Fibrosis. Int J Mol Sci 21(7): 2269‒

important ﬁrst step in the formation of TTR-related amyloid

2290, 2020.

ﬁbril. The shift of the pI value in this modiﬁcation will be5) Ishikawa N, Hattori N, Yokoyama A, Kohno N: Utility ofsimilar with that of the phosphorylation, and the m/z shift is

KL-6/MUC1 in the clinical management of interstitial

the same, ＋80 Da. At the present study, the levels of total
TGLN-2 in the lung extract of BIP model mouse clearlylung diseases. Respir Invest 50: 3‒13, 2012.
6) Kohno N, Kyoizumi S, Awaya Y, Fukuhara H, Yamakido

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M, et al.: New serum indicator of interstitial pneumonitis

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68‒73,1989.

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