J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 4 3 –1 05 4

Available online at www.sciencedirect.com

www.elsevier.com/locate/jprot

Analytical constraints for the analysis of human cell line

secretomes by shotgun proteomics

Véronique Malard⁎, Laetitia Chardan, Stamatiki Roussi, Carine Darolles, Nicole Sage,
Jean-Charles Gaillard, Jean Armengaud
CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, F-30207, France

AR TIC LE I N FO ABS TR ACT

Article history: Human cell line secretome represents a valuable source of therapeutic targets and
Received 22 August 2011 candidate biomarkers. Secreted proteins found in biological fluids or culture media are by
Accepted 22 October 2011 essence highly diluted. Secretome investigation with proteomic approaches is hardly
Available online 4 November 2011 compatible with the high content of proteins found in complete cell culture media.
Therefore, many studies are currently done with media containing few or no protein.
Keywords: Such conditions may perturb cell metabolism and proliferation. Here, we compared
Secretome seventeen different compositions of culture media for the human bronchial epithelial
Toxicoproteomics BEAS-2B cell line. Cell viability, proliferation rate and initial protein charge were systematically
LC–MS/MS compared. We have shown that an important difficulty for the proteomic analysis is due to the
Lung presence of detergents such as Pluronic F-68 which hinders peptide mass spectrometry. The
high glucose containing DMEM medium which is free of proteins was shown to preserve a
good viability and proliferation of cells. With this conditioning medium, we identified 81
extracellular proteins in the secretome of BEAS-2B cells. Moreover, to illustrate this approach,
we exposed BEAS-2B cells to a low toxic dose of CoCl2, and found 24 extracellular proteins
modulated by cobalt. This study highlights the possible contribution of such proteomic
approach in the field of toxicology.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction spectrometers and shotgun strategies, offer the possibility of

identifying almost all proteins present in a given biological
Secretion of proteins is a very complex and finely regulated sample [8]. The assessment of the full dynamic range of Sac-
process. It reflects the cellular state of cells as the quantity charomyces cerevisiae and human urine proteomes exemplified
of secreted proteins may vary depending on the stimuli they the current approaches for quantifying proteins by selected
received. Secreted proteins play a crucial role in many physio- reaction monitoring [9,10]. Recent shotgun proteomics reports
logical processes [1]. They include numerous growth factors, revealed large catalogues of exoproteomes of several bacteria
cytokines, hormones, and enzymes. In the recent years, they [11,12] and fungi [13] comprising a large list of items
deserved much attention as among them are therapeutic tar- unsuspected from prior 2D-gel based studies. Regarding
gets or candidate diagnostic and prognosis biomarkers [2–7]. human proteomes, a large panel of secretomes have been
Technological advances in the proteomic field, such as the described from myeloid cells [14], dendritic cells [15], stem
widespread use of new generation of high-resolution mass cells [16], endothelial cells [17], astrocytes [18], and airway

⁎ Corresponding author at: Laboratoire de Biochimie des systèmes Perturbés, CEA Marcoule, DSV, iBEB, SBTN, LBSP, F-30207 BAGNOLS-SUR-CEZE,
France. Tel.: +33 4 66 79 68 18; fax: + 33 4 66 79 19 05.
E-mail address: [email protected] (V. Malard).

1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jprot.2011.10.025
1044 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 4 3 –10 5 4

epithelial cells [19]. The description of specific cancer the most adapted media. We have detected specific polymers
biomarkers being the aim of most secretome studies, more that are systematically present in large quantity in different
than 28 reports presented the catalogue of secreted proteins media. We have shown that they should be avoided as they
from cancer cell lines [5]. However, technical difficulties strongly impact the mass spectrometry analysis. With our
should not be miss-evaluated when analysing the secretome of experimental set-up we comprehensively defined the exopro-
cell lines in culture even when using state-of-the-art mass teome of BEAS-2B cells and evaluated the dynamic of this
spectrometry technology. First, proteins secreted in biological specific fraction after cobalt exposure.
fluids or culture media are by essence highly diluted. Concentra-
tion methods such as filtering on low cut-off membranes with
centrifugation devices [4,20,21] and precipitation of proteins 2. Material and methods
[14,22] are frequently used. These experimental steps may lead
in some cases to a modification of the protein content. 2.1. BEAS-2B cell culture
Proteolysis and tube coating should be avoided. Moreover, sam-
ple preparation should be done carefully in order to avoid any The BEAS-2B human bronchial epithelial cell line was obtained
cross-contamination from non-secreted proteins. Cell death from the American Type Culture Collection (CRL#9609). Cells
should be as low as possible both at the cell culture and sample were cultured in LHC-9 medium (Invitrogen) in 5% CO2 atmo-
preparation steps. Removal of biological fluid from cells should sphere in 80 cm2 flasks or plates precoated with a mixture of
be soft to avoid cellular spill. Finally, the mass spectrometry BSA (0.01 mg/mL), human fibronectin (0.01 mg/mL) and colla-
analysis should be highly discriminating to allow identification gen (0.03 mg/mL). Cells were passaged by trypsinization (0.25%
of proteins in low abundance compared to those initially present trypsin/EDTA 2.6 mM) at 70–80% confluency every 3–4 days.
in large quantities in the biological fluid or culture medium, such
as serum proteins. Indeed, cells grown in the laboratory need a 2.2. Cell viability and proliferation tests
conditioned environment that contain all the nutritive elements
and growth factors to stimulate cell proliferation and will main- Cells were seeded at 10,000 cells per cm2 in 6 well-plates and
tain the pH over time. These factors are usually extracted from cultured for 72 h to reach 80% confluency. The LHC-9 medium
tissues and added in large excess in culture media, resulting in was then discarded. The cell layer was rinsed four times with
a relatively high protein charge. Fetal serum is largely used for PBS and incubated for 5 min at 37 °C for the last wash. Cells
this purpose for human cell culture. It is usually employed at were then incubated 24 h in the specified medium for
10% in culture medium, resulting in a 6 mg/mL total protein secretome collection. Cells were collected after trypsinization.
charge. Analysis of secretomes using current proteomic Proteolysis was stopped by adding 10% of Fetal Calf Serum
procedures requires a quantity of proteins from between 8 and (FCS). Cells were pelleted by centrifugation for 5 min at
100 mL culture supernatants [4,23]. The use of fetal serum will 2000 rpm and after discarding the supernatant, they were
then introduce between 50 and 600 mg of protein in such resuspended in 1 mL of fresh medium. Cells from 300 μL of this
analysis, thus masking secreted proteins usually found in the suspension were stained with Trypan blue and counted using
microgram range. To overcome this problem, the use of culture an automated cell counting system Cedex (Roche Innovatis).
medium without serum, i.e. basal medium, has been proposed Measurements of viable and dead cells were done ten times to de-
[5,24]. However, as a consequence these non-optimal culture termine the percent and the number of viable cells. A control per-
conditions may disturb metabolism, stop proliferation and acti- formed on 3 wells consisted of an estimation of the number of
vate apoptosis pathways. Such disturbances may affect different viable cells before incubation with the specified medium. All the
mechanisms of secretion, thus leading to experimental bias in experiments were repeated at least twice (biological replicate).
the secretome catalogue. Moreover, due to the change of culture
medium cells should then face a sudden deprivation of serum 2.3. Cell cycle analysis
and thus, a strong metabolic stress usually translated into a
drastic reduction of their proliferation rate [25,26], activation of BEAS-2B cells were harvested after incubation with the differ-
apoptosis pathways [27], and induction of signal transduction ent media by trypsinization and washed with PBS (Gibco).
and expression of genes involved in stress response mecha- They were fixed with ice-cold 70% ethanol/30% H2O (vol/vol)
nisms [28]. Alternatively, several culture media are presently and kept at least 2 h at −20 °C. They were then washed twice
available with reduced protein charges and therefore, could be with PBS and finally stained for 30 min at room temperature
better adapted to secretome analysis. Their direct comparison in the dark with propidium iodide (PI) at 40 μg/mL in PBS
for secretome analysis should be informative if cell biology containing 0.2 mg/mL RNaseA. Fluorescence of 20,000 cells was
parameters are compared together with shotgun proteomics analysed using a FACSCalibur (BD Biosciences) flow cytometer
constraints. To our knowledge, such extensive comparison has (excitation at 488 nm; FL-2H/FL-2A). The cell-cycle phases were
not yet been reported for any human cell line. analysed using ModFit v3.0 software (Verity software House).
The human bronchial epithelial BEAS-2B cell line is a
model well adapted to study lung secretomes and assess the 2.4. Secretome collection and analysis
effect of possible respiratory toxicants. We compared seven-
teen different compositions of culture media for this cell line For secretome analysis, BEAS-2B cells were seeded at 10,000
in terms of cell viability, proliferation rate and initial protein cells per cm2 in LHC-9 medium in 175 cm2 flasks. They were
charge. We determined the experimental constraints for a cultured for 72 h till 80% confluence was reached. After the
shotgun nanoLC–MS/MS analysis of BEAS-2B secretome with medium was discarded, cell layer was rinsed five times with
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 4 3 –1 05 4 1045

PBS, with 10 min incubation at 37 °C for the last wash. Cells the MS/MS, static modifications of carbamidomethyl Cys and
were then incubated 24 h in 50 mL of the specified medium dynamic modifications of oxidized Met, acetylation (polypep-
for secretome collection. The conditioned medium from tide N-terminus), deamidation (Asn & Gln residues). The
three identical flasks was collected and centrifuged 10 min maximum number of missed cleavages was set at 2. All peptide
at 800 rpm to discard cell debris. A mixture of protease matches with a peptide score below a P-value of 0.01 were
inhibitors (one tablet of complete Mini EDTA-free protease in- filtered by the IRMa 1.28 parser [29]. A protein was considered
hibitor cocktail (Roche) per sample) was added. The samples validated when at least two different peptides were detected
were then stored at −80 °C until further processing. Samples in the same experiment. The false-positive rate for protein iden-
were thawed and filtered on a 0.22 μm filter. Each sample was tification was estimated using the appropriate decoy database.
dialysed at 4 °C against 10 mM NH4HCO3 for 22 h using 3.5 kDa
cut-off dialysis membranes (Spectra/Por). The dialysis bath 2.7. Protein quantification by spectral count and statistical
was renewed with fresh NH4HCO3 solution three times. analysis
Samples were then concentrated using Amicon ultracentrifugal
filter devices with a 3 kDa cut-off (Millipore) operated at 4 °C by The number of MS/MS spectra per protein was determined in
centrifugation at 4500 g. Samples were further reduced to 20 μL the different nanoLC–MS/MS analysis. Spectral counts were
by means of Vivaspin 500 devices (Sartorius) operated at 4 °C by then compared with the TFold method of the PatternLab
centrifugation at 16,000 g. LDS3X solution (Invitrogen) was software (version 2.1.0.20) [30]. Fold change and P-value cut-
added to the samples, and then proteins were resolved by offs were set at 2.0 and 0.05, respectively. Cellular function
SDS-PAGE at 200 V on 4–12% gradient NuPAGE (Invitrogen) gels and localization of identified proteins were obtained with
operated with MOPS buffer (Invitrogen). After a short migration the Ingenuity Pathway Analysis software (Ingenuity Systems)
of 8 min, polyacrylamide gels were stained with Coomassie Blue and used to classify the secretome components.
Safe stain (Invitrogen). After overnight destaining in water, each
gel lane corresponding to a secretome sample was sliced into 2.8. Cell toxicity assays
8 bands of 1.5 mm height from top to bottom. Polyacrylamide
bands were processed as described earlier for further destaining The effects of CoCl2 on the proliferation of human BEAS-2B
and iodoacetamide treatment (de Groot et al., 2009). Samples cells were evaluated using the CyQUANT assay (Invitrogen)
were then proteolyzed with trypsin using the proteasMAX sur- according to manufacturer's instruction. This assay is based
factant (Promega) as described (Clair et al., 2010). The resulting on measurement of cellular DNA content via fluorescent dye
peptide mixtures were diluted 1/20 in TFA 0.1% prior tandem binding. Because cellular DNA content is highly regulated, it
mass spectrometry. is proportional to cell number. The extent of proliferation
was determined by comparing cell counts in samples treated
2.5. NanoLC-MS/MS with cobalt to untreated controls. Cells were plated at 10,000
cells per cm2 in white 96-well plates, and grown for 72 h. The
Proteolyzed proteomes were analysed with a LTQ-Orbitrap XL medium was then replaced with cobalt solutions (0–300 μg/ml),
hybrid mass spectrometer (ThermoFisher) coupled to an Ulti- and the plates were incubated. DNA content in the wells was
Mate 3000 LC system (Dionex-LC Packings). This system was measured 24 h after exposure; the medium was removed after
operated as described previously (Zivanovic et al., 2009). a centrifugation at 900 g for 5 min. Then, 100 μL fluorescent
Peptide mixtures (5 μl out of 7 μL) were loaded and desalted dyes were added followed by incubation for 80 min at 37 °C.
online on a reverse phase precolumn C18 Pepmap 100 column Fluorescent intensity of each sample was measured at 480/
(LC Packings). Then, they were resolved on a nanoscale C18 520 nm with Fluorescence Microplate Reader (FluoStar (BMG)).
Pepmap 100™ capillary column (LC Packings) at a flow rate Four independent biological experiments, each with quadrupli-
of 0.3 μL per min with a CH3CN gradient prior injection into cate technical measurements, were carried out. The results are
the mass spectrometer. The gradient consisted of a 90 min expressed as the percentage of fluorescence measured for
linear gradient from 5 to 60% solvent B (0.1% HCOOH/80% treated cells compared to untreated cells. Results were analysed
CH3CN) with 0.1% HCOOH/100% H2O as solvent A. The using GraphPad Prism (GraphPad Software).
full-scan mass spectra were measured from m/z 300 to 1800
with the LTQ-orbitrap XL mass spectrometer operated in the
data-dependent mode using a TOP3 strategy. In brief, a scan 3. Results
cycle was initiated with a full scan of high mass accuracy in
the Orbitrap, which was followed by MS/MS scans in the linear 3.1. Comparison of culture media in terms of cell viability
ion trap on the three most abundant precursor ions with dy- and protein charge
namic exclusion of previously selected ions.
3.1.1. Selection of eleven reference culture media
2.6. Database mining Table 1 lists the eleven different culture media that we
selected in the present secretome comparative study with
Using the MASCOT search engine (version 2.3.02), we the human bronchial epithelial cell line named BEAS-2B, and
searched all MS/MS spectra against the SwissProt database their characteristics. The reference medium for BEAS-2B
(SwissProt_2011_07.fasta). Searches for trypsic peptides were cells is LHC-9 (Invitrogen). This medium is largely used for
performed with the following parameters: full-trypsin specifici- culture of primary human lung cells and contains less than
ty, a mass tolerance of 5 ppm on the parent ion and 0.5 Da on 0.2 mg/mL of proteins. It consists of basal medium (LHCB)
1046 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 4 3 –10 5 4

Table 1 – List of commercially available media used in the comparative study.

Name Proteins a(g/L) Pluronic F-68 a(g/L) Additives Glucose a(g/L)

LHC9 < 0.2 0 – 1.1

EX-CELL HeLa 0.0011 1 2% PS b + 6 mM L-Glu 6
EX-CELL 293 0.0011 1 2% PS + 6 mM L-Glu 6
EX-CELL 302 < 0.001 1 2% PS + 6 mM L-Glu 3.4
Opti-MEM 0.015 0 2% PS 1
ITS 0.015 (ITS: 1%) 0 This product is an additive (DMEM) 4.5
DMEM 0 0 2% PS 4.5
PeproGrow-1 0 0 This product is an additive (LHCB) 1.1
CD-CHO 0 – 2% PS + 8 mM L-Glu –
HBSS 0 0 2% PS 1
LHCB 0 0 2% PS 1.1
a
Data from the suppliers except for LHC-9 (micro Bradford).
b
Penicillin Streptomycin.

supplemented with proteins and growth factors (retinoic acid, dead cells were determined by means of the trypan blue meth-
epinephrin, insulin, EGF, bovine pituitary extract, transferrin, od using an automated cell counter. Fig. 1 (Panel A) shows the
hydrocortisone, and triiodothyronin) but does not contain results obtained from at least two biological replicates. In
FCS. For this reason, the protein concentration is much most cases, we found that cell viability is higher than 95%.
lower than in media supplemented with FCS, but remains in- However, LHC-9 dilutions, HBSS and LHCB media show a
appropriate for secretome analysis by shotgun proteomics. marked decrease of cell viability with 82, 73, and 86%,
The recommended basal medium, LHCB, is free of proteins respectively. Fig. 1 (panel B) shows cell proliferation obtained
and was also selected for comparison. We further selected in the seventeen conditions. The higher rate of cell proliferation
three EX-CELL media (Sigma-Aldrich) designed for the was obtained with the LHC-9 medium. This result was expected
production of recombinant proteins, namely EX-CELL HeLa, as it is the recommended medium for this cell line. Compared to
EX-CELL 302 and EX-CELL 293. They have a very low concen- what was found for LHC-9 medium, cell proliferation was re-
tration of proteins originating from plants. Interestingly, duced but in a limited extent (above 80%) for EX-CELL HeLa,
these proteins should be easily differentiated from human EX-CELL 302, Opti-MEM and DMEM media, irrespective of the
proteins by tandem mass spectrometry using the appropriate amount of ITS added. LHC dilutions, HBSS, LHCB, PeproGrow-1
mixed database, but their abundance may still impact the (PG1) and CD-CHO media led to drastic changes in cell
detection of secreted proteins by suppression effects. To our proliferation with decreases up to 70%. We further analysed
knowledge, none of these three media have been yet used in the physiological state of cells by analysing their distribution
a secretome study. Opti-MEM medium (Invitrogen) was also in terms of cell cycle. We analysed systematically by flow
selected as it contains a low concentration of proteins (insulin cytometry the cells after 24 h of incubation. Fig. 2 shows the re-
and transferrin), as well as Dulbecco's Modified Eagle Medium sults obtained in LHC-9 medium where most cells are found in
(DMEM) supplemented with protein (insulin and transferrin) G0/G1 phase. The profiles obtained with the other media are
and selenium. CD-CHO, HBSS and PeproGrow-1 (PG1) are similar, except for the LHCB medium where cells were found
media without proteins like LHCB. The former medium has mostly stopped at the G2/M transition. Thus, incubation with
been already used in secretome analyses [23,31]. PeproGrow- this medium drastically stressed the cells and modified the
1 is a mixture of recommended solutions for supplementing physiological state of the whole population of cells. In conclu-
basal medium. Comprising lipid extract and a mixture of min- sion to this cellular assay, we selected two media that should
erals and metal ions, this alternative culture medium has allow high level of cell viability and a priori could be compatible
been developed for the production of pro-inflammatory with shotgun proteomics due to their very low protein content.
proteins such as cytokines. Besides these ten defined media, EX-CELL HeLa is a medium containing low quantities of plant
we also prepared four different dilutions of LHC-9 into LHCB proteins while DMEM is a protein-free medium. Despite the
as additional culture media, as well as three complementation good results obtained in terms of viability and proliferation,
of DMEM medium with different quantities of Insulin–Transfer- the medium Opti-MEM was excluded because of its high protein
rin–Selenium-A liquid media supplement (ITS) from Sigma content.
(0.125, 0.5, 1%) containing a mixture of bovine insulin, human
transferrin and sodium selenite prepared in Earle's Balanced 3.2. NanoLC–MS/MS experimental constraints for
Salt Solution without phenol red. secretome analysis

3.1.2. Assay of cell viability and cell proliferation in seventeen 3.2.1. Procedure for collecting secretome and shotgun proteomics
different culture conditions We designed a protocol to concentrate the secretome and
BEAS-2B cells were cultured in LHC-9 medium to 80% conflu- render robust its analysis by tandem mass spectrometry.
ence, rinsed extensively with PBS, and then incubated with Dialysis and concentration by ultrafiltration with a low
the selected culture medium for 24 h. Numbers of viable and permeability filter (3.5 kDa cut-off) on a centrifugation device
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 4 3 –1 05 4 1047

Fig. 1 – Cell viability and proliferation. Cells were incubated into the different media during 24 h. Then, cell viability was
assessed using the Trypan Blue exclusion test: % of viable cells (A), number of viable cells expressed in % of control cells grown
in LHC-9 (B). Free-protein media are indicated in red.

allow removing salts and other low molecular components of polymer molecule named Pluronic F-68 [32,33] is added in this
the culture medium while limiting loss of small proteins. A medium to reduce hydrodynamic stresses. It appears that
volume of 25 mL of conditioned medium was collected after such molecule is commonly added to culture media without
24 h incubation, treated by dialysis and then concentrated by serum with the objective to reduce the shear forces applied to
ultrafiltration. The concentrated fraction was mixed with the cell membrane. Given the molecular weight of this mole-
Laemli buffer and subjected to SDS-PAGE. After electrophoret- cule (8.2 kDa), it is probably not eliminated during dialysis,
ic migration and Coomassie blue staining, the polyacrylamide nor during concentration by ultrafiltration. The concentration
bands containing the exoproteins were treated and in-gel of this molecule is relatively high in the culture medium
digested with trypsin. The resulting peptides were analysed (1 mg/mL, i.e. 122 μM). Pluronic F-68 was found to be highly
by nanoLC–MS/MS. concentrated after sample processing. It is therefore very likely
that its final concentration is largely exceeding the critical
3.2.2. Secretome obtained with EX-CELL HeLa medium is micelle concentration. The resulting high viscosity of the
masked by Pluronic F-68 polymer sample renders impossible its analysis by our mass spectrome-
The peptide mixture obtained from the secretome prepared try approach even if the gel-based strategy could have partially
with EX-CELL HeLa medium could not be properly analysed cleaned the sample. We checked and found that the mass
by nanoLC–MS/MS. The reverse phase chromatography spectrometry signal of bovine myoglobin in infusing mode is
resulted poorly resolving with such sample. The pre-column completely suppressed in an ion trap in presence of 5% of
and column were found to be irreversibly damaged after sam- Pluronic F-68 (data not shown). We tried to remove PF68 in
ple injection and could not be cleaned with usual procedures. the sample using two different approaches. First, absorption
We checked the composition of the medium. A hydrophobic of the Pluronic F-68 reagent on a hydrophobic resin developed
1048 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 4 3 –10 5 4

reagent such as Pluronic F-68 should be definitively avoided

700 1400 2100 2800
LHC9

700 1400 2100 2800

LHC9 in order to perform shotgun nanoLC-MS/MS proteomics.
1/10
3.2.3. Secretome obtained with DMEM medium
G2/M : 10% G2/M : 7%
We performed an extensive study of BEAS-2B secretome.
For this, we repeated the protocol for three biological repli-
cates independently generated with cells incubated in DMEM
0

0
0 50 100 150 200 0 50 100 150 200
for 24 h, but with a larger volume (50 mL). The viability of
700 1400 2100 2800
1400 2100 2800

cells was found greater than 98% whatever the replicate.

LHCB Optimem
After sample processing, their protein content was resolved
G2/M : 30% by SDS-PAGE with a short migration of 8 min at 200 V on a
G2/M : 13% 4–12% gradient NuPAGE gel (Invitrogen). After staining, each
gel lane was cut into 8 bands which were proteolyzed with
trypsin. The resulting peptides were analysed by nanoLC–
MS/MS. From the recorded MS/MS spectra data set along
0

0 50 100 150 200 0 50 100 150 200

these 24 nanoLC–MS/MS runs, a total of 57,619 MS/MS spectra
1400 2100 2800

700 1400 2100 2800

DMEM could be assigned (P-value below 0.01). They are listed in

CD-CHO Supplementary Table S1. They correspond to 12,359 unique
ITS 1%
Events

peptides signing the presence of 561 proteins with at least

G2/M : 12% two different peptides (false positive rate below 0.1%).
G2/M : 9%
Supplementary Table S2 shows the list of validated proteins
and their characteristics. Fig. 3 shows a Venn diagram where
0

0 50 100 150 200 0 50 100 150 200

the protein sets of each replicate are compared. A total of
349 common proteins were detected in the three replicates
representing an average reproducibility of 62% between runs.
1400 2100 2800

700 1400 2100 2800

HBSS DMEM
ITS 0.125% 3.3. Extensive analysis of BEAS-2B secretome by shotgun
G2/M : 14% proteomics
G2/M : 10%

3.3.1. Cellular localization of identified proteins

0
0

0 50 100 150 200 0 50 100 150 200

We analysed the cellular localization of the whole set of
proteins detected in the secretome of BEAS-2B predicted
700 1400 2100 2800

700 1400 2100 2800

Excell DMEM with the Ingenuity Pathway Analysis (IPA) software. This pre-
diction is reliable as based on a very well documented
HELA
G2/M : 7% G2/M : 10%
82
Re

:4
pl

1
0

e
ic

0 50 100 150 200 0 50 100 150 200

at
at

il c
e

p 26
Re
2

DNA content
:4

73
26

34
Fig. 2 – Effects of media on cell cycle distribution. Cell cycle
distribution of BEAS-2B cells was analysed by flow cytometry
after 24 h exposure to the different media. For each condition, 349
20,000 events were recorded. The standard deviations
measured for the estimation of cells in the G2/M cell cycle was
within 3%. 34 17

28
to eliminate ionic detergents and non-ionic surfactants in
protein purification procedures (Calbiosorb Absorbent, Calbio- Replicate 3 : 428
chem) resulted in a sample leading to the same detection prob-
lem. Second, we precipitated proteins with 7.5% (final) TCA Fig. 3 – Number of proteins identified by LC–MS/MS in
prior to SDS-PAGE according to Chevallet [14]. However, once BEAS-2B cells secretomes. The Venn diagram shows the
again the resulting peptides could not be detected appropriate- overlap of the three independent replicates together with the
ly (data not shown). We concluded that the presence of a total number of proteins identified per replicate.
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 4 3 –1 05 4 1049

database on human proteins comprising their subcellular these counts with the molecular weight of each protein as a
location on the basis of scientific publication and experimen- large protein may give rise to a higher number of peptides
tal data. A relatively large number of proteins (54%) in our and thus of spectral count. The normalized spectral abundance
secretome catalogue are known to be cytoplasmic, while 16% factor (NSAF) values for each detected protein are indicated in
are nuclear, 12% belong to the plasma membrane, 14% are ex- Supplementary Table S2. In terms of quantities, we found
tracellular, and 4% are not yet characterized or their location that secreted proteins represent 22% of the total spectral
is unknown. That a relatively high number of intra-cellular counts while in terms of percentage of cumulated NSAF values
proteins are found in BEAS-2B secretome was expected as a total of 16% is found. These data show that although the
this has been systematically observed in all previously number of secreted proteins is rather low in the total catalogue,
published secretome studies [17,23,34]. During cell cultivation, they are significantly over-represented in terms of quantities
some cells die resulting in the release of intracellular proteins compared to other proteins.
into the conditioned medium. Despite our efforts to choose
cell culture conditions to minimize cell death (less than 2% 3.3.2. Assignments of Biological Function to extracellular
observed in DMEM medium), these intracellular proteins are proteins
detected with sensitive mass spectrometers. Noteworthy,
similar estimations of intracellular protein release has been The biological functions of the 81 extracellular proteins
reported in secretome analyses performed with in vitro label- detected in the secretome of BEAS-2B cells were predicted
ling [17,35]. Burghoff et al. [17,35] analysed the secretome of with the IPA software. The distribution of these proteins is
endothelial cells under static, laminar, and oscillatory flow. the following: 10% peptidases, 12% enzymes, 7% transporters,
To differentiate between endogenously expressed and added 7% cytokines, 6% growth factors, 1% kinase and 56% others. It
proteins, isolated human umbilical vein endothelial cells were is worth to mention that growth factors represent 1% of the
labelled with L-Lysine-13C6,15N2 and L-Arginine-13C6,15N4. global secretome, and are enriched to 6% in the extracellular
Proteins from the supernatants were isolated, trypsinized, population. In the same way, cytokines represents 1% and
and finally analysed using LC–MS/MS (LTQ). Under static 7%, respectively. These proteins are mainly involved in
control condition 395 proteins could be identified, of which 78 intercellular communication and are enriched in the secre-
proteins were assigned to the secretome according to tome. Fig. 4 shows the ten most representative categories of
Swiss-Prot database. In this study the percentage of secreted cellular functions of these proteins. Cellular growth and pro-
proteins in the conditioned medium is 17%, a value very similar liferation, cellular movement, and cell-to-cell signalling and
to our results (14%). These results confirm the fact that mass interaction are the most numerous categories. These three
spectrometry is so sensitive that a small fraction of intracellu- types of function correspond to those found predominantly
lar proteins can considerably “contaminate” the measurement in the secretome of lung cell lines [23]. Supplementary Table
of secreted proteins. We semi-quantified these proteins with S2 lists the extracellular proteins ranked by their Spectral
a label-free method, namely spectral count. We normalized Count. The most abundant proteins in this secretome list

Fig. 4 – Analysis of biological functions of secreted proteins identified in BEAS-2B secretome. The top 10 functions for
extracellular proteins, as determined by Ingenuity Pathway Analysis, are shown. The bars represent the biological functions
identified, the x-axis identifies it. The y-axis shows the negative log of the P value calculated based on Fisher's exact test. The
dotted line represents the threshold above which there are statistically significantly more genes in a biological function than
expected by chance.
1050 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 4 3 –10 5 4

were Complement component 3 (C3) and Thrombospondin 1 the three controls described above. The viability of cells was
(THBS1), an adhesive glycoprotein that mediates cell-to-cell established to be 99.3%, 97.6% and 98.8% for the three stressed
and cell-to-matrix interactions with 2205 and 824 spectral samples while that of the controls were 99.3%, 99.7% and
counts, respectively. Three other members of the comple- 98.9%. Therefore, the cell viability was not significantly modi-
ment proteins family are also found in BEAS-2B secretome fied by the dose of cobalt chosen. The samples were processed
but in lower amounts: CFB, C1R and C4B with 49, 20 and 11 in parallel. Supplementary Table S3 lists the 63,158 MS/MS
spectral counts, respectively. Among the other extracellular spectra identified in the secretome of BEAS-2B stressed with
proteins, we found IGFBP4, LAMA3, LAMB1, LAMC1 and CoCl2. We found a comparable reproducibility between the
SPARC which are involved in proliferation of epithelial cells, three biological replicates and the control cells, with 4100,
CST6, CYR61, DKK3, GPI, LAMA3, PLAU, SERPINE1, TIMP1 and 3864 and 3911 distinct peptides identified in the three stressed
VCAN which affect adhesion of cell lines, and CSF1, CTGF, samples while 4532, 3797 and 4030 distinct peptides were
CYR61, IGFBP4, LGALS1, PLAU, SAA1, SERPINA1, SERPINE1, found in the controls. We compared with the PatternLab
THBS1, TIMP1, TIMP2 and VCAN which affect migration of software the list of identified proteins in the six samples and
normal cells. their quantities estimated through their corresponding
spectral counts. A total of 684 different proteins were taken
3.4. Comparative secretome analysis to assess the influence into account for normalization and comparison. They are listed
of cobalt on BEAS-2B cells with their spectral counts in Supplementary Table S4. Among
these, 28 proteins were found up-regulated in the stress
3.4.1. Cytotoxic effects of CoCl2 condition (fold change > 2 and P-value < 0.05) while 38 were
Cobalt is a metal widely used in the industry. To further down-regulated (fold change > −2 and P-value< 0.05). Among
document the cellular toxic effect of CoCl2, we compared the these 66 differentially expressed proteins listed in Supplemen-
secretome of BEAS-2B cells exposed to cobalt to the secretome tary Table S4 (red color), 24 are classified as extracellular by IPA
established without stress. First, the cytotoxicity of cobalt was (Table 2). That almost one third of the secretome (24 out of 66
evaluated on BEAS-2B cells. Fig. 5 shows the amount of predicted extracellular proteins) is modulated by CoCl2 indi-
intracellular DNA measured after 24 h exposure which is cates a high impact of this metal on protein secretion. Almost
representative of cell proliferation. A dose-dependent reduc- all are modulated negatively in response to cobalt (Table 2).
tion in cell proliferation was observed in a dose dependant Our data indicate that cells selectively restricted some secreto-
manner after exposure to CoCl2 in comparison to non exposed ry mechanisms in response to cobalt stress. Given the good re-
cells. As reported in Fig. 5, a decrease of 50% of the proliferation sults of viability, these can be considered as non-lethal
(IC50) was observed when cells are exposed to 90 μg/mL CoCl2. metabolic disturbances. The most important changes (fold
We assessed the secretome of BEAS-2B cells stressed with a changes above 10) are observed for fibrillin 2 (−49x), carboxy-
lower dose, i.e. 20 μg/mL CoCl2, corresponding to IC20. peptidase A4 (−32x), biglycan (−20x), complement factor B
(−19x), fibulin 1 (−18x), and cysteine rich transmembrane BMP
3.4.2. Secreted proteins modulated after cobalt exposure regulator 1 (−14x). ERAP1 (endoplasmic reticulum aminopepti-
dase 1) is the only significantly induced protein in the
Three biological replicates (3 × 50 mL secretome) were inde- BEAS-2B secretome with a 2.2 fold change.
pendently generated in DMEM after 24 h incubation with
20 μg/mL CoCl2. These cultures were made in parallel with
4. Discussion
PROLIFERATION TEST
Stress responses can be mediated through the release of solu-
ble factors from cells that have been stressed. These factors
RATIO TEST/CONTROL

1.00 can be transferred through cell culture medium from stressed

to non-stressed cells. This bystander effect has been observed
0.75 in case of irradiation [36]. Inflammation is a frequent response
to stresses. Noteworthy, BEAS-2B cells are able to trigger an
0.50 inflammatory response via the secretion of proteins such as
interleukins, interferons or cytokines when exposed to partic-
0.25 ulate air pollutants [37–39]. Establishing a comprehensive list
of secreted proteins by human cell lines is crucial to further
0.00 document cellular response to stresses. Here, we investigated
0.001 0.01 0.1 1 the analytical constraints for the analysis of human cell line
Co (g/L) secretomes by shotgun proteomics using the BEAS-2B cell
line as model.
Fig. 5 – Cytotoxicity on BEAS-2B cells monitored by CyQUANT Proteins secreted in biological fluids or culture media are
assay. Cells were treated with cobalt for 24 h. The data are by essence highly diluted. We pointed at the difficulties to
expressed as a mean ± standard deviation (S.D.) of two use for proteomic investigation the recommended medium
independent experiments, each point being performed in for most cell lines as they contain large amounts of proteins.
quadruplicate. Values of control cells (unexposed cells) were We also noted that some changes in the physiology of the
considered to be the reference (100%). cells may occur upon a change of medium, which is often
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 4 3 –1 05 4 1051

Table 2 – List of extracellular proteins modulated upon cobalt exposure and their fold changes.
Accession Gene Protein name Fold P value

ERAP1_HUMAN ERAP1 Endoplasmic reticulum aminopeptidase 1 2.19 0.0033

IBP7_HUMAN IGFBP7 Insulin-like growth factor binding protein 7 −2.02 0.0080
BGH3_HUMAN TGFBI Transforming growth factor, beta-induced, 68 kDa −2.32 0.0133
FBLN3_HUMAN EFEMP1 EGF containing fibulin-like extracellular matrix protein 1 −2.40 0.0032
GRN_HUMAN GRN Granulin −2.74 0.0022
CO3_HUMAN C3 Complement component 3 −2.75 0.0015
CYTC_HUMAN CST3 Cystatin C −2.92 0.0187
CSF1_HUMAN CSF1 Colony stimulating factor 1 −2.97 0.0134
CSPG2_HUMAN VCAN Versican −3.14 0.0386
FSTL1_HUMAN FSTL1 Follistatin-like 1 −3.69 0.0170
ATRN_HUMAN ATRN Attractin −4.02 0.0243
PXDN_HUMAN PXDN Peroxidasin homolog −4.25 0.0356
A1AT_HUMAN SERPINA1 Serpin peptidase inhibitor −4.27 0.0018
PLBL2_HUMAN PLBD2 Phospholipase B domain containing 2 −4.36 0.0208
LAMA4_HUMAN LAMA4 Laminin, alpha 4 −4.56 0.0241
PCSK9_HUMAN PCSK9 Proprotein convertase subtilisin/kexin type 9 −4.60 0.0012
CH3L1_HUMAN CHI3L1 Chitinase 3-like 1 −4.79 0.0007
LAMA5_HUMAN LAMA5 Laminin, alpha 5 −6.23 0.0001
CRIM1_HUMAN CRIM1 Cysteine rich transmembrane BMP regulator 1 −14.11 0.0291
FBLN1_HUMAN FBLN1 Fibulin 1 −17.83 0.0078
CFAB_HUMAN CFB Complement factor B −18.86 0.0276
PGS1_HUMAN BGN Biglycan −19.56 0.0189
CBPA4_HUMAN CPA4 Carboxypeptidase A4 −31.75 0.0003
FBN2_HUMAN FBN2 Fibrillin 2 −49.45 0.0005

the case, as most of secretome studies are performed with be monitored during secretome studies, one should be sure
the basal medium without addition of fetal calf serum. More- that the physiological state of the cell is not modified by the
over, the extent of the wash step to get rid of the protein conditioning medium.
contaminants from the culture medium may also impact the Among the media tested, some contain additives that
cells [4]. resulted inappropriate with mass spectrometry. As an exam-
LHC-9 is the recommended medium for the culture of ple, the EX-CELL media are protein-free but contain the Pluro-
primary human lung cells and the BEAS-2B cell line. Its nic F-68 polymer that can result concentrated along the
protein content is much lower than in media supplemented procedure for secretome recovery. Finally, DMEM that was
with FCS but remains inconsistent with the analysis of the used for several secretome studies [2,14,21] appeared the best
secretome by shotgun proteomics. We tested a variety of option as serum-free medium. Because its concentration of
alternative media that contained fewer proteins. We observed glucose (an essential carbon source for cellular catabolism) is
that almost all media allow maintaining good cell viability high, a good viability was observed for BEAS-2B cells. Our
after 24 h of incubation. However, clear differences between results compare relatively well with other recent secretome
the media tested were observed in terms of proliferation. To reports as we used a sensitive and high resolution mass spec-
illustrate this point, we can cite the CD-CHO medium which trometer for the characterization of peptides generated in the
does not contain proteins and that has been already used in study and performed the study with several replicates. We
several studies [23,31]. CD-CHO medium did not allow a good identified a total of 561 proteins using stringent parameters
proliferation of BEAS-2B cells. In previous studies, cytotoxicity for their validation (false positive rate 0.1% as estimated with
tests were performed measuring LDH release from dying cells, a decoy database search). This total number of identified
an indication on the level of membrane integrity loss. In these proteins is in the same order as those reported in other studies
cases, good cellular viability was reported. The different [4,31]. From the catalogue of proteins detected in the secre-
behaviour of BEAS-2B cells in this medium may be explained tome, 472 proteins were detected in the three replicates yield-
by the fact that the BEAS-2B cell line is immortalized, non- ing a reproducibility of 62%, a value similar to that reported
invasive and non-tumorigenic [40] while cancer cell lines used by others [23].
in previous studies are invasive. We observed a decrease of We compared the list of proteins identified in the present
the cell viability for the HBSS and LHCB media. While the latter study with the proteomes of lung-related diseases and lung-
has been used in a high-throughput analysis of 48 secretomes related biological fluids. On the first hand, Xiao et al. (18) de-
from lung cancer and bronchial tissue organ culture [34], we ob- tailed the secretome from primary cultures of lung cancer cells
served a cell cycle arrest of BEAS-2B cells in this medium. Such and adjacent normal bronchial epithelial cells of six lung cancer
flow cytometry analyses were not reported previously. With patients. Using one-dimensional SDS-PAGE to fractionate the
these results in hands, we recommend performing such con- sample and nano-ESI–MS/MS for peptide detection, they
trol. Indeed, given that fine cellular metabolism variations can identified 42 secreted proteins. On the other hand, Planque et
1052 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 4 3 –10 5 4

al. [23] reported the secreted proteins from four lung cancer cell raise of its shedding observed in presence of cobalt could be
lines in conditioned media. Using a two-dimensional LC–MS/MS linked to angiogenesis (which is known to be indirectly induced
approach, they identified 291 extra-cellular proteins. Among by cobalt [53]). This protein has never been associated to cobalt
the 81 secreted proteins identified in the BEAS-2B secretome, toxicity. It is worth to further investigated its role in response
72% have been identified in the latest study while only 20% in to cobalt stress.
the former. Interestingly, 23 proteins (28%) resulted uncovered In conclusion, our comparative analysis of different media
time in our study, underlining the interest to cumulate such for the investigation of BEAS-2B cells secretome shows that
studies with various cell lines. BEAS-2B cell line is an immortal- the high glucose containing DMEM medium is an attractive
ized cell line which has been obtained by transformation with conditioning medium because it does not modify the
adenovirus SV40. It is non-tumorigenic and non-invasive, thus behaviour of BEAS-2B cells compared to the recommended me-
being quite different from cancer cell lines. The behaviour of dium and is fully compatible to shotgun proteomic strategy. In
these cells is considered closed to that of normal lung cells addition, we pointed at the requirement of a thorough analysis
[40,41]. Whether the differences observed are due to the of cell mortality and cell cycle behaviour when choosing an al-
methodology used or the cell line should be further documen- ternative conditioning medium for secretome analysis and the
ted. Among these proteins the most representatives in term of difficulties arising from the presence of detergents such as
spectral count are (i) Osteonectin (SPARC) which is a matrix- Pluronic F-68. From these results, we recommend to investigate
associated protein that elicits changes in cell shape, inhibits the effect of serum starvation onto cells prior any secretome
cell-cycle progression, and influences the synthesis of extracel- proteomic analysis. Here, we identified 81 extracellular pro-
lular matrix [42], (ii) the chitinase 3-like protein1 which is teins in the secretome of BEAS-2B cells. Moreover, we
thought to play a role in the process of inflammation and tissue highlighted 24 extracellular proteins modulated by exposure
remodelling as already observed in bronchoalveolar fluids, and to a low toxic dose of CoCl2. This study illustrates the future
(iii) the insulin-like growth factor binding protein 4 (IGFBP4) contribution of such proteomic approach in the field of
which is a protein secreted in bronchoalveolar fluids and is toxicology.
known to bind both insulin-like growth factors extending the Supplementary materials related to this article can be
half-life of IGFs and altering their interaction with cell surface found online at doi:10.1016/j.jprot.2011.10.025.
receptors.
Although an essential element for humans as exemplified
by its occurrence in vitamin B12, cobalt is toxic at high concen- Acknowledgements
trations [43,44]. In vitro studies have demonstrated its geno-
toxicity [45,46]. It may also induce oxidative stress [47], We thank the Commissariat à l'Energie Atomique et aux Energies
apoptosis [48] and has the same effect as a deprivation of oxy- Alternatives and the EDF company for financial support. We also
gen [49]. Soluble forms of cobalt have been classified as poten- thank C. Bruley (CEA-Grenoble) for providing the IRMa parser and
tially carcinogenic by IARC [50]. Various respiratory and skin O. Pible for bioinformatic support.
diseases are known to be induced by chronic occupational
exposures to cobalt. The most common damages involve the
respiratory system as cobalt particles can be inhaled by REFERENCES
workers of metallurgy industry. How secretome produced by
the human bronchial epithelial BEAS-2B cell line is modulated
upon cobalt stress is of interest to further document cellular [1] Dowling P, Clynes M. Conditioned media from cell lines: a
cobalt effects. The fact that mostly a restrained protein secre- complementary model to clinical specimens for the discovery
tion is observed indicates that upon cobalt exposure, protein of disease-specific biomarkers. Proteomics 2011;11:794–804.
secretion is reduced. Recent evidence suggests that comple- [2] Caccia D, Zanetti Domingues L, Micciche F, De Bortoli M,
Carniti C, Mondellini P, et al. Secretome compartment is a
ment is activated in human nasal airways in inflammatory
valuable source of biomarkers for cancer-relevant pathways.
states. C3-related fragments are present on cell membranes of J Proteome Res 2011;10:4196–207.
fresh nasal epithelium and can be adsorbed through the [3] Chenau J, Michelland S, de Fraipont F, Josserand V, Coll JL,
epithelial cell membrane in nasal mucosa tissue segments and Favrot MC, et al. The cell line secretome, a suitable tool for
in cell cultures. Complement activation may occur upon the investigating proteins released in vivo by tumors: application
nasal epithelial cell membrane during inflammation in vivo to the study of p53-modulated proteins secreted in lung
and nasal epithelium might regulate this complement activation cancer cells. J Proteome Res 2009;8:4579–91.
[4] Lawlor K, Nazarian A, Lacomis L, Tempst P, Villanueva J.
[51]. We notice upon cobalt exposure a decrease of C3 protein as
Pathway-based biomarker search by high-throughput
well as complement factor B which would indicate an inhibition proteomics profiling of secretomes. J Proteome Res 2009;8:
of the inflammatory response through complement activation. 1489–503.
Moreover, CSF1 protein is a cytokine that controls the produc- [5] Makridakis M, Vlahou A. Secretome proteomics for discovery
tion, differentiation, and function of macrophages that is also of cancer biomarkers. J Proteomics 2010;73:2291–305.
found in lower amount in secretome from cobalt-exposed cells. [6] Paulitschke V, Kunstfeld R, Mohr T, Slany A, Micksche M, Drach
J, et al. Entering a new era of rational biomarker discovery for
On the other hand, the ERAP1 protein is a multifunctional zinc-
early detection of melanoma metastases: secretome analysis of
binding enzyme belonging to the family of aminopeptidases
associated stroma cells. J Proteome Res 2009;8:2501–10.
with roles in regulation of blood pressure, angiogenesis, ectodo- [7] Pavlou MP, Diamandis EP. The cancer cell secretome: a good
main shedding of several cytokine receptors, and processing of source for discovering biomarkers? J Proteomics 2010;73:
antigenic peptides presented to MHC class I molecules [52]. The 1896–906.
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 10 4 3 –1 05 4 1053

[8] Ahrens CH, Brunner E, Qeli E, Basler K, Aebersold R. [28] Levin VA, Panchabhai SC, Shen L, Kornblau SM, Qiu Y,
Generating and navigating proteome maps using mass Baggerly KA. Different changes in protein and
spectrometry. Nat Rev Mol Cell Biol 2010;11:789–801. phosphoprotein levels result from serum starvation of
[9] Picotti P, Bodenmiller B, Mueller LN, Domon B, Aebersold R. high-grade glioma and adenocarcinoma cell lines. J Proteome
Full dynamic range proteome analysis of S. cerevisiae by Res 2010;9:179–91.
targeted proteomics. Cell 2009;138:795–806. [29] Dupierris V, Masselon C, Court M, Kieffer-Jaquinod S, Bruley
[10] Selevsek N, Matondo M, Sanchez Carbayo M, Aebersold R, C. A toolbox for validation of mass spectrometry peptides
Domon B. Systematic quantification of peptides/proteins in identification and generation of database: IRMa.
urine using selected reaction monitoring. Proteomics 2011;11: Bioinformatics 2009;25:1980–1.
1135–47. [30] Carvalho PC, Fischer JS, Chen EI, Yates III JR, Barbosa VC.
[11] Christie-Oleza JA, Armengaud J. In-depth analysis of PatternLab for proteomics: a tool for differential shotgun
exoproteomes from marine bacteria by shotgun liquid proteomics. BMC Bioinformatics 2008;9:316.
chromatography-tandem mass spectrometry: the Ruegeria [31] Kulasingam V, Diamandis EP. Strategies for discovering novel
pomeroyi DSS-3 case-study. Mar Drugs 2010;8:2223–39. cancer biomarkers through utilization of emerging
[12] Clair G, Roussi S, Armengaud J, Duport C. Expanding the technologies. Nat Clin Pract Oncol 2008;5:588–99.
known repertoire of virulence factors produced by Bacillus [32] Kall L, Krogh A, Sonnhammer EL. A combined
cereus through early secretome profiling in three redox transmembrane topology and signal peptide prediction
conditions. Mol Cell Proteomics 2010;9:1486–98. method. J Mol Biol 2004;338:1027–36.
[13] Peterson R, Grinyer J, Nevalainen H. Secretome of the [33] Palomares LA, Gonzalez M, Ramirez OT. Evidence of Pluronic
coprophilous fungus Doratomyces stemonitis C8, isolated from F-68 direct interaction with insect cells: impact on shear
Koala feces. Appl Environ Microbiol 2011;77:3793–801. protection, recombinant protein, and baculovirus production*.
[14] Chevallet M, Diemer H, Van Dorssealer A, Villiers C, Rabilloud Enzyme Microb Technol 2000;26:324–31.
T. Toward a better analysis of secreted proteins: the example [34] Xiao T, Ying W, Li L, Hu Z, Ma Y, Jiao L, et al. An approach to
of the myeloid cells secretome. Proteomics 2007;7:1757–70. studying lung cancer-related proteins in human blood. Mol
[15] Gundacker NC, Haudek VJ, Wimmer H, Slany A, Griss J, Cell Proteomics 2005;4:1480–6.
Bochkov V, et al. Cytoplasmic proteome and secretome [35] Zwickl H, Traxler E, Staettner S, Parzefall W, Grasl-Kraupp B,
profiles of differently stimulated human dendritic cells. Karner J, et al. A novel technique to specifically analyze the
J Proteome Res 2009;8:2799–811. secretome of cells and tissues. Electrophoresis 2005;26:
[16] Skalnikova H, Motlik J, Gadher SJ, Kovarova H. Mapping of the 2779–85.
secretome of primary isolates of mammalian cells, stem cells [36] Prise KM, O'Sullivan JM. Radiation-induced bystander
and derived cell lines. Proteomics 2011;11:691–708. signalling in cancer therapy. Nat Rev Cancer 2009;9:351–60.
[17] Burghoff S, Schrader J. Secretome of human endothelial cells [37] Fuentes-Mattei E, Rivera E, Gioda A, Sanchez-Rivera D,
under shear stress. J Proteome Res 2011;10:1160–9. Roman-Velazquez FR, Jimenez-Velez BD. Use of human
[18] Dowell JA, Johnson JA, Li L. Identification of astrocyte bronchial epithelial cells (BEAS-2B) to study
secreted proteins with a combination of shotgun proteomics immunological markers resulting from exposure to PM(2.5)
and bioinformatics. J Proteome Res 2009;8:4135–43. organic extract from Puerto Rico. Toxicol Appl Pharmacol
[19] Ali M, Lillehoj EP, Park Y, Kyo Y, Kim KC. Analysis of the 2010;243:381–9.
proteome of human airway epithelial secretions. Proteome [38] Park EJ, Park K. Induction of pro-inflammatory signals by
Sci 2011;9:4. 1-nitropyrene in cultured BEAS-2B cells. Toxicol Lett
[20] Srirajaskanthan R, Caplin ME, Waugh MG, Watkins J, Meyer T, 2009;184:126–33.
Hsuan JJ, et al. Identification of Mac-2-binding protein as a [39] Veranth JM, Reilly CA, Veranth MM, Moss TA, Langelier CR,
putative marker of neuroendocrine tumors from the analysis Lanza DL, et al. Inflammatory cytokines and cell death in
of cell line secretomes. Mol Cell Proteomics 2010;9:656–66. BEAS-2B lung cells treated with soil dust, lipopolysaccharide,
[21] Wu CC, Hsu CW, Chen CD, Yu CJ, Chang KP, Tai DI, et al. and surface-modified particles. Toxicol Sci 2004;82:88–96.
Candidate serological biomarkers for cancer identified from [40] Shen J, Behrens C, Wistuba II, Feng L, Lee JJ, Hong WK, et al.
the secretomes of 23 cancer cell lines and the human protein Identification and validation of differences in protein levels
atlas. Mol Cell Proteomics 2010;9:1100–17. in normal, premalignant, and malignant lung cells
[22] Villiers C, Chevallet M, Diemer H, Couderc R, Freitas H, Van and tissues using high-throughput Western Array and
Dorsselaer A, et al. From secretome analysis to immunology: immunohistochemistry. Cancer Res 2006;66:11194–206.
chitosan induces major alterations in the activation of [41] Reddel RR, Ke Y, Gerwin BI, McMenamin MG, Lechner JF, Su
dendritic cells via a TLR4-dependent mechanism. Mol Cell RT, et al. Transformation of human bronchial epithelial cells
Proteomics 2009;8:1252–64. by infection with SV40 or adenovirus-12 SV40 hybrid virus, or
[23] Planque C, Kulasingam V, Smith CR, Reckamp K, Goodglick L, transfection via strontium phosphate coprecipitation with a
Diamandis EP. Identification of five candidate lung cancer plasmid containing SV40 early region genes. Cancer Res
biomarkers by proteomics analysis of conditioned media of 1988;48:1904–9.
four lung cancer cell lines. Mol Cell Proteomics 2009;8: [42] Bradshaw AD, Puolakkainen P, Dasgupta J, Davidson JM,
2746–58. Wight TN. Helene Sage E. SPARC-null mice display
[24] Chenau J, Michelland S, Seve M. Secretome: definitions and abnormalities in the dermis characterized by decreased
biomedical interest. Rev Med Interne 2008;29:606–8. collagen fibril diameter and reduced tensile strength. J Invest
[25] Cooper S. Reappraisal of serum starvation, the restriction Dermatol 2003;120:949–55.
point, G0, and G1 phase arrest points. FASEB J 2003;17:333–40. [43] ATSDR AfTSaDR-. toxicological profile for cobalt. http://www.
[26] Shin JS, Hong SW, Lee SL, Kim TH, Park IC, An SK, et al. Serum atsdr.cdc.gov/toxprofiles/tp33.pdf Agency for Toxic Substances
starvation induces G1 arrest through suppression of and Disease Registry — U.S. Department of Health and Human
Skp2-CDK2 and CDK4 in SK-OV-3 cells. Int J Oncol 2008;32: Services; 2004.
435–9. [44] Ménager GL, Goyffon, Bresson, Ansoborlo, Malard. Cobalt
[27] Hasan NM, Adams GE, Joiner MC. Effect of serum starvation Chap29. Toxicologie nucléaire environnementale et
on expression and phosphorylation of PKC-alpha and p53 in humaine. Lavoisier; 2009.
V79 cells: implications for cell death. Int J Cancer 1999;80: [45] De Boeck M, Kirsch-Volders M, Lison D. Cobalt and antimony:
400–5. genotoxicity and carcinogenicity. Mutat Res 2003;533:135–52.
1054 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 10 4 3 –10 5 4

[46] Lison D, De Boeck M, Verougstraete V, Kirsch-Volders M. compounds. IARC Monogr Eval Carcinog Risks HumLyon:
Update on the genotoxicity and carcinogenicity of cobalt International Agency for Research on Cancer (IARC) Working
compounds. Occup Environ Med 2001;58:619–25. Group; 1991. p. 1–544.
[47] Salnikow K, Su W, Blagosklonny MV, Costa M. Carcinogenic [51] Varsano S, Frolkis I, Shapiro H, Ophir D. Human nasal
metals induce hypoxia-inducible factor-stimulated epithelium adsorbs complement C3-related fragments and
transcription by reactive oxygen species-independent expresses cell membrane complement regulatory proteins.
mechanism. Cancer Res 2000;60:3375–8. Laryngoscope 1996;106:599–604.
[48] Pulido MD, Parrish AR. Metal-induced apoptosis: [52] Goto Y, Ogawa K, Hattori A, Tsujimoto M. Secretion of
mechanisms. Mutat Res 2003;533:227–41. endoplasmic reticulum aminopeptidase 1 is involved in the
[49] Bruick RK. Oxygen sensing in the hypoxic response pathway: activation of macrophages induced by lipopolysaccharide
regulation of the hypoxia-inducible transcription factor. and interferon-{gamma}. J Biol Chem 2011;286:21906–14.
Genes Dev 2003;17:2614–23. [53] Maxwell P, Salnikow K. HIF-1: an oxygen and metal
[50] Chlorinated drinking-water; chlorination by-products; responsive transcription factor. Cancer Biol Ther 2004;3:
some other halogenated compounds; cobalt and cobalt 29–35.