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Biosci. Rep. (2010) / 30 / 209–215 (Printed in Great Britain) / doi 10.1042/BSR20090045
Proteomics identification and annotation of
proteins of a cell line of Bombyx mori, BmN cells
Hui-peng YAO, Lin CHEN, Xingwei XIANG, Ai-qin GUO, Xing-meng LU and Xiao-feng WU1
College of Animal Science, Zhejiang University, Hangzhou 310029, People’s Republic of China
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Synopsis
A cell line is an important experimental platform for biological sciences as it can basically reflect the biology of its
original organism. In this study, we firstly characterized the proteome of cultured BmN cells, derived from Bombyx
mori. Total 1478 proteins were identified with two or more peptides by using 1D (one-dimensional) SDS/PAGE and
LTQ-Orbitrap. According to the gene ontology annotation, these proteins presented diverse pI values and molecular
masses, involved in various molecular functions, including catalytic activity, binding, molecular transducer activity,
motor activity, transcription regulator activity, enzyme regulator activity and antioxidant activity. Some proteins related
to virus infection were also identified. These results provided us with useful information to understand the molecular
mechanism of B. mori as well as antiviral immunity.
Key words: bioinformatics, BmN cell, gene ontology, LTQ-Orbitrap MS, one-dimensional gel electrophoresis,
proteomics
&
INTRODUCTION
The silkworm, Bombyx mori, is an important economic insect for
production of silk, and recently it is also being developed as a
suitable model insect similar to the fruitfly for biological science
due to its excellent biological characteristics such as ease of
rearing, large body and abundant genomic information available
[1,2]. However, the larva depends on its natural food, mulberry
leaves, and thereby is limited by the seasons. For this reason,
B. mori cell lines were established [3], and they provided the
researchers great convenience to prepare experimental materials
through the year without limitation of seasons. In other aspects,
it is known that positive results could not be observed if the
in vivo experiment is done first, so many experiments have to
be done first in vitro using the cell line, and B. mori cell line is
being applied extensively in various fields. Maeda et al. [4] first
applied BmN cells to produce recombinant viruses and used it
as vector to synthesize human α-IFN (α-interferon) in silkworm
[4]. Previously, the silkworm cell line NISES-BoMo-Cam1 was
used as an in vitro model of the immune system organs of B. mori
to clarify the signalling pathway of antimicrobial peptide gene
activation in lepidopteron insects [5]. Besides, the B. mori cell
line was found to be highly susceptible to BmNPV (B. mori nucleopolyhedrovirus) [6], and thus the cultured cells could also
%
be used as a good tool for studying pathology such as detecting
the proteins involved in virus infection. Although the silkworm
genome has already been completed [1,2], little is known about
the proteome of its cultured cells.
In the present study, we first characterized the proteomics of
Bm cell line in order to make clear the essential background information about this experimental platform. Developments in MS
have dramatically increased the capability of analysing complex
proteomes in depth. It is shown that LTQ-Orbitrap MS, which has
been recently developed, is better than LTQ-FT MS [7] to identify
protein spots. In the present paper, the proteins from BmN cells
were analysed by using 1D SDS/PAGE and LTQ-Orbitrap, and
the MS data were determined by using Bioworks (v3.2) search
algorithm and TurboSequestTM (v3.2; ThermoFinnigan). A total
of 1478 proteins were identified and annotated.
MATERIALS AND METHODS
Sample preparation
The B. mori cells (BmN), originally derived from B. mori ovary
[3], were grown in TC-100 medium (AppliChem) containing 10%
(v/v) FCS (fetal calf serum) and 0.26% bacto-tryptose (Gibco
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Abbreviations used: 1D, one-dimensional; EMCV, encephalomyocarditis virus; IFN, interferon; GBP-1, IFN-induced guanylate binding protein-1; ICE5, interleukin-1β-converting enzyme
5; MAPK8, mitogen-activated protein kinase 8; MS/MS, tandem MS; BmNPV, Bombyx mori nucleopolyhedrovirus; GO, gene ontology; VSV, vesicular-stomatitis virus.
1 To whom any correspondence should be addressed (email [email protected]).
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BRL, Gaithersburg, MD, U.S.A.) at 27◦ C. The cells were collected by centrifuging at 1000 g for 2 min and washed twice
with PBS (137 mM NaCl, 10 mM phosphate and 2.7 mM KCl,
pH 7.4). Further, the cells were disrupted by suspending in 1 ml
of lysis buffer (8 M urea, 2 M thiourea and 4% CHAPS) for
1 h with vortex-mixing every 10 min. The sample was centrifuged (4◦ C, 40 000 g and 1 h) and the supernatant was then
precipitated by using the following method [8]: approx. 400 ml
of the sample was added to 400 ml of methanol and 200 ml of
chloroform. The sample was vortex-mixed and centrifuged for
2 min at 10 000 g. The upper phase was then removed and
300 ml of methanol was added. The tube was inverted twice
and centrifuged (15 min and 10 000 g) and the supernatant was
discarded. The pellet was allowed to air-dry for 5 min before
the addition of SDS/PAGE loading buffer (0.06 M Tris/HCl,
2% SDS, 5% 2-mercaptoethanol and 0.01% Bromophenol
Blue).
1D (one-dimensional) SDS/PAGE and in-gel
digestion with trypsin
Regular 1D 12% Tricine SDS/PAGE was applied. Briefly, 100 μg
of BmN cell proteins was loaded on to a single lane on a 7 cm
long, 1 mm thick 12% Tricine SDS/PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue
R-250 (GE Healthcare). Protein lanes were excised along the
visible protein bands ranging from 14.4 to 97.0 kDa in molecular
mass after staining; two lanes of the gel were combined and then
sliced into 15 pieces; subsequently they were destained and
in-gel digested with trypsin (Roche Applied Science, Indianapolis, IN, U.S.A.) following standard procedures. In brief, gel
bands were soaked in 75 mM NH4 HCO3 in 40% ethanol and
vortex-mixed every 10 min. Fresh destaining buffer was added
until Coomassie Brilliant Blue was completely removed. Gel
bands were then washed with 25 mM NH4 HCO3 followed by
dehydration with acetonitrile. The process was repeated several
times and the remaining acetonitrile was removed by vacuum
centrifugation for 15 min. Dry gel bands were rehydrated with
50 mM NH4 HCO3 -containing trypsin. Digestion was carried out
at 37◦ C overnight. After digestion, tryptic peptides were extracted with 50 mM NH4 HCO3 , acetonitrile/5% trifluoroacetic acid
(1:1) and acetonitrile. The combined extracts were dried by vacuum centrifugation for 30–60 min and stored at −80◦ C until MS
analysis.
MS
Separation of tryptic peptide mixtures was achieved by nanoscale
reverse-phase HPLC, in combination with online LTQ-Orbitrap.
For the HPLC separation, a nano-MDLC (multidimensional LC)
system (Ettan MDLC; GE Healthcare) was used, employing
a linear gradient of 5–45% buffer B (95% acetonitrile +5%
water+0.1% formic acid) over 60 min. The column system consisted of a trap (0.5 mm×2 mm) and a separation column (Magic
C18 AQ; 3 μm, 200 Å, 0.2×150 mm; 1 Å = 0.1 nm), both purchased from Michrom Company. While column 1, trap 1 was
running, column 2, trap 2 was equilibrated with buffer A (95%
water+5% acetonitrile+0.1% formic acid) to allow continuous
running of the sample through two columns.
LTQ-Orbitrap
The mass spectrometer was operated in the data-dependent mode
to automatically switch between Orbitrap-MS and OrbitrapMS/MS (tandem MS) acquired. Survey full scan MS spectra
(from m/z 200 to 2000) were acquired in the Orbitrap with resolution R = 60 000 at m/z 400. The most intense ions (up to five,
depending on signal intensity) were sequentially isolated for fragmentation; ions were recorded in the Orbitrap with resolution
R = 15 000 at m/z 400.
For accurate mass measurements the lock mass option was
enabled in both MS and MS/MS modes and the polydimethylcyclosiloxane ions generated in the electrospray process from ambient air {protonated [Si(CH3 )2 O]6 ; m/z 445.120025} were used
for internal recalibration. For single SIM (selected ion monitoring) scan injection of the lock mass into the C-trap, the lock mass
‘ion gain’ was set as 10% of the target value of the full mass spectrum. When calibrating in MS/MS mode, the ion at 429.088735
[PCM (pneumatic control module) with neutral methane loss]
was used instead for recalibration.
Target ions already selected for MS/MS were dynamically
excluded for 180 s. General MS conditions were: electrospray
voltage, 1.8 kV, no sheath and auxiliary gas flow; ion transfer tube temperature, 200◦ C; collision gas pressure, 1.3 mtorr
(1 torr = 0.133 kPa); normalized collision energy, 35% for MS,
ion selection threshold was 500 counts for MS/MS. An activation q-value of 0.25 and activation time of 30 ms was applied for
MS/MS acquisitions.
MS data interpretation
The derived MS data sets were converted into generic
format (*.dta) files using the Bioworks Browser (3.2) and
searched against the silkworm proteins database [downloaded
from the website (ftp://ftp.genomics.org.cn/pub/SilkDB/GeneAnnotation/Proteins/SW-ge2k-BGF.pep)], containing all 21302
proteins in the data set using the Bioworks (v3.2) search algorithm, TurboSequestTM (v3.2; ThermoFinnigan). The species
subset was set as B. mori. The number of allowed miscleavages
was set to 2.0 and oxidation of methionine was selected. Intensity threshold was set to 500. The parent ion selection was set to
5 p.p.m., with fragment ion set to 1 p.p.m. The following filters
were set for every peptide: (i) For a peptide charge of 1, the
X corr value was a minimum of 1.5. For a peptide charge of 2,
the X corr value was a minimum of 2.0, and for a peptide charge
of 3, the minimum X corr value was set to 2.5; (ii) the difference
between the first and second ranked assignments by simplified
CorrX (CN) value was greater than 0.1; and (iii) Rsp value was
less than 5. Peak lists were generated using the TurboSequestTM
(v3.2) algorithm. Each product assignment was made based on
two unique spectra from the top hit.
Bioinformatics
The output including the protein IDs from silkworm database were obtained through TurboSequest. Microarray
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Figure 1
Distribution of identified proteins of BmN cells based on the number of peptides used for protein identification
database (http://silkworm.swu.edu.cn/microarray) was used to
find mRNA expression profiles corresponding to the protein
IDs. In order to characterize the complete protein data set, Wego
software (http://wego.genomics.org.cn/cgi-bin/wego/index.pl)
was used to investigate and categorize the GO (gene ontology)
annotations (cellular components, molecular functions and
biological processes). Finally, the newly identified proteins
were annotated by pBLASTp and the pI and molecular mass
of proteins were analysed by using ProtParam software (http://
www.expasy.ch/tool/protparam.html).
RESULTS
Nanospray MS is a very sensitive and versatile method for protein identification. 1D SDS/PAGE was used to prefractionate
the BmN cell protein sample for detecting more low-abundance
proteins. By dissecting the separation gel into slices of approx. 1 mm, the entire spectrum of proteins in the sample could
be recovered from the gel. A total of 15 fractions were individually resolved by reverse-phase chromatography and proteolytically digested before directly spraying into the mass spectrometer. In order to generate a final list of protein identifications
from the information obtained by MS, we run the data
against the silkworm protein database, which was downloaded
from the website (ftp://ftp.genomics.org.cn/pub/SilkDB/GeneAnnotation/Proteins/SW-ge2k-BGF.pep) and predicted by silkworm genome. This is a complete and detailed protein database with minimal redundancy. We set specific filter criteria
to ensure that questionable peptide identification was not considered in our results list. The criteria were largely based on Kapp
et al. [9], who compared several criteria for different search engines and the associated false-positive outcomes. The criteria
have also been applied to analyse the sperm proteomics of human [8] and rat [10]. The peptide information, together with the
associated TurboSequestTM information, is given in Supplementary Table S1 (http://www.bioscirep.org/bsr/030/bsr0300209add.
htm).
In the newly constructed BmN cell proteomic database,
each protein contains at least two peptides. The number of
unique peptides, for each of the reported proteins, can be found
in Supplementary Table S2 (http://www.bioscirep.org/bsr/030/
bsr0300209add.htm). Figure 1 indicates the relation between
the newly identified proteins and the number of peptides used
for protein identification. From Figure 1, we can learn that
as the number of peptides increases, the number of proteins is close to exponential distribution. There are only six
proteins, Bmb004930, Bmb008789, Bmb007857, Bmb012614,
Bmb016644 and Bmb009360, which contain more than 20 peptides.
The identified proteins were annotated based on various
aspects. Supplementary Table S3 (http://www.bioscirep.org/bsr/
030/bsr0300209add.htm) shows the results for the values of pI
and molecular mass predicted by ProtParam software. From
Supplementary Table S3, it can be observed that the proteins of
BmN cells are distributed in the pI ranges 5–7 and 8–10 and
in the molecular mass range 8–80 kDa. The tissue distribution
of the proteins was plotted based on a DNA microarray. By
using the microarray data to annotate the identified proteins
(Supplementary Table S4 at http://www.bioscirep.org/bsr/030/
bsr0300209add.htm), it is found that out of 1478 proteins, 509
proteins cannot be found in ten tissues. A total of 790 proteins
can be found in testis and this is the largest number of proteins identified in all tissues. The number of identified proteins
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Figure 2
Tissue distribution of the protein hits reported
Figure 3
GO categories of the identified protein of the BmN cells by using Wego software
in haemolymph is 391, which is the least number for all
tissues (Figure 2). Using Wego software, we were able to
annotate 778 proteins out of 1478 proteins. An analysis of the
subcellular distribution of BmN proteins based on GO categories
(Supplementary Table S5 at http://www.bioscirep.org/bsr/030/
bsr0300209add.htm); Figure 3) revealed 294 proteins belonging
to cell parts in the cellular component, with 127 proteins belonging to organelles. A total of 500 proteins could be annotated
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Table 1 List of proteins identified related to actin filaments and microtubules
actin-RPV, vertebrate actin-related protein; ARP1, actin-related protein 1; RalBP1, RalA-binding protein 1; TSNAXIP1, translin-associated factor
X-interacting protein 1.
Protein ID
Bmb001619
Protein description
Similar to exosome complex exonuclease RRP43 (ribosomal RNA processing protein 43)
(exosome component 8) (p9) (Opa-interacting protein 2) [Apis mellifera]
Bmb001856
Similar to huntingtin interacting protein E; huntingtin interactor protein E [Pan troglodytes]
Bmb002021
Similar to MAPK8 (mitogen-activated protein kinase 8) interacting protein; MAPK8 interacting protein 1 [Gallus gallus]
Bmb002802
Similar to α-centractin (centractin) (centrosome-associated actin homologue) (actin-RPV) (ARP1) isoform 9 [Bos taurus]
Bmb004087
α-Actinin – fruitfly (Drosophila melanogaster)
Bmb004470
Translin-associated factor X interacting protein 1 [Homo sapiens] unknown [H. sapiens] TSNAXIP1 protein [H. sapiens]
Bmb004752
Similar to phosphatase and actin regulator 1 isoform 1 [Canis familiaris]
Bmb006176
RalBP1-associated Eps domain-containing protein 1 (RalBP1-interacting protein 1)
RalBP1-associated Eps domain-containing protein [Mus musculus] RalBP1-associated EH domain protein Reps1 [M. musculus]
Bmb006203
Hypothetical protein Aple02000468 [Actinobacillus pleuropneumoniae serovar 1 str. 4074]
Bmb006486
Similar to F-actin capping protein α-subunit [Ap. mellifera]
Bmb006636
Glutamate receptor interacting protein 1 isoform 2 [M. musculus] glutamate receptor interacting protein 1a-s [M. musculus]
Bmb007009
Similar to regulating synaptic membrane exocytosis protein 2 [RIM2 (Rab3-interacting molecule 2)] [Ap. mellifera]
Bmb007404
Membrane interacting protein of RGS16 [H. sapiens] membrane interacting protein of RGS16 [H. sapiens]
Bmb007769
Similar to Numb-interacting protein 2 isoform 1 [B. taurus]
Bmb008129
PREDICTED: similar to SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5
(sucrose non-fermenting protein (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin A5) (sucrose
non-fermenting protein
Bmb008813
Similar to TBP (TATA-box-binding protein)-interacting protein 120A, partial [Strongylocentrotus purpuratus]
Bmb009416
Similar to TNF (tumour necrosis factor) receptor-associated factor 3 interacting protein 1 (predicted), partial [S. purpuratus]
Bmb010478
Similar to myosin phosphatase-Rho interacting protein isoform 2 [C. familiaris]
Bmb011097
Similar to actin related protein 2/3 complex subunit 2 [C. familiaris]
Bmb011236
Smad- and Olf-interacting zinc finger protein [H. sapiens] KIAA0760 protein [H. sapiens]
Bmb012212
Actin 6 [Aedes aegypti]
Bmb014240
Similar to MAPK kinase 7 interacting protein 1 [Rattus norvegicus]
Bmb014548
Sec23-interacting protein p125 [H. sapiens] Sec23-interacting protein p125 [H. sapiens]
SEC23-interacting protein (p125) phospholipase [H. sapiens]
Bmb015457
Similar to schwannomin interacting protein 1 [Ap. mellifera]
Bmb017622
RAB5-interacting protein [R. norvegicus]
based on their molecular function (Figure 3). From this analysis,
76% of this population possessed a catalytic domain. Proteins
containing a binding domain represented 70% of the annotatable
population, corresponding to the second largest category. This is
important for two reasons: first, by identifying which catalytic
reactions are occurring in the cell, it should be possible to
determine the mechanisms responsible for the relationship
between BmNPV and its host BmN cells and, secondly, the
ability to identify novel proteins related to antiviral activity
or other immune activity relies heavily on whether proteins
possess cell-specific catalytic domains and binding domains. An
analysis of the biological process (636 proteins) demonstrated
that, behind the broader GO definitions of the ‘cellular process’,
384 ranked the highest (Figure 3). Supplementary Table S5
reveals the identity of more than 360 proteins involved in
the metabolism of BmN cells. In addition, 1274 proteins
of the BmN cells were annotated by protein BLAST searching
against non-redundant protein sequences (nr) from all organisms
(Supplementary Table S6 at http://www.bioscirep.org/bsr/030/
bsr0300209add.htm).
DISCUSSION
GO functional analysis and protein BLAST search of the annotation results of the identified proteins revealed that many of the
proteins related to BmNPV infection are found in our BmN cell
proteome.
Selected examples of these proteins are discussed in detail
below.
Clathrin is a protein that is the major constituent of the ‘coat’
of the clathrin-coated pits and coated vesicles formed during endocytosis of materials at the surface of cells [11,12]. Different
inhibitors proved that functional entry of the budded virus form
of baculovirus into insect and mammalian cells is dependent on
clathrin-mediated endocytosis, which is generally thought to occur through a low-pH-dependent endocytosis pathway, clathrincoated pits [13]. Clathrin is made up of ‘three legs’, each leg
comprising a heavy chain and a light chain [14]. A total of 21
peptides were identified (Supplementary Table S2). Its number
in the protein database is Bmb007857, which was found to be
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similar to CLH-17 (clathrin heavy-17) isoform 6 of dog by using protein BLAST software. The score of the results of protein
BLAST software is 0 (Supplementary Table S6).
Microfilament, microtubule and intermediate filament are the
main components of the cytoskeleton and nuclear matrix of
animal cells. They are also the factors required for baculovirus entering the insect cell nucleus. The ultra-microstructure of midgut,
fat and haemolymph cells indicates that nucleocapsids of PDV
(polyhedra-derived virus) or CRV (cell-released virus) are related
to the tracks of the microtubule [15,16]. Formation of actin filaments facilitates the nuclear import of viral nucleocapsid [17]. In
the present study, we identified a large number of proteins related
to actin filaments and microtubules shown in Table 1.
ICE5 (interleukin-1β-converting enzyme 5), a member of
the caspase protein family, is also named Caspase-1 (cysteinylaspartate-specific proteinase-1) [18] and plays an important role
in the process of cell apoptosis [19]. In the process of viral infection, caspase in the form of zymogen can self-cut or can be
cut by other members of the caspase family [20] and results
in the production of long chains and short chains and forms a
heterodimer containing the amino-acid sequence QACRG [21],
which brings apoptosis of cells and prevents the production of
new virus. Bmb001418 is the ICE5 protein identified in BmN
cells. DNA microarray data indicated that it can be expressed
not only in the midgut of the silkworm, but also in the head and
haemolymph (Supplementary Table S4). In addition, GO annotation of all proteins indicated that Bmb004188 [Apaf-1 (apoptotic protease-activating factor 1)], Bmb014676 and Bmb007835
newly identified also exert much influence on BmN cell apoptosis.
From Supplementary Table S6, it can be learned that
Bmb002206 is a kind of GBP-1 (IFN-induced guanylate-binding
protein-1), which is the factor required for the replication of some
virus [VSV (vesicular-stomatitis virus) and EMCV (encephalomyocarditis virus)] [22]. GBP-1 mediates an antiviral effect
against VSV and EMCV and plays a role in the IFN-mediated
antiviral response against these viruses [23]. This is the first time
that GBP-1 of silkworm is found in BmN cells. We predicted that
it may have antiviral function.
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FUNDING
This work was supported by National 863 [grant number 2007AA10Z159]; the Basic Research Program [grant number
2005CB121003]; and the New-Century Training Programme Foundation for the Talents, Ministry of Education, China [grant number
NCET-06-0524]. The authors declare no conflict of interest.
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23 Anderson, S. L., Carton, J. M., Zhang, X. and Rubin, B. Y. (1999)
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member of the murine interferon-induced guanylate-binding protein
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Received 3 April 2009/29 May 2009; accepted 4 June 2009
Published as Immediate Publication 4 June 2009, doi 10.1042/BSR20090045
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