Protein composition of bronchoalveolar lavage fluid and airway surface liquid from newborn pigs

Abstract

The airway mucosa and the alveolar surface form dynamic interfaces between the lung and the external environment. The epithelial cells lining these barriers elaborate a thin liquid layer containing secreted peptides and proteins that contribute to host defense and other functions. The goal of this study was to develop and apply methods to define the proteome of porcine lung lining liquid, in part, by leveraging the wealth of information in the Sus scrofa database of Ensembl gene, transcript, and protein model predictions. We developed an optimized workflow for detection of secreted proteins in porcine bronchoalveolar lavage (BAL) fluid and in methacholine-induced tracheal secretions (airway surface liquid, ASL). We detected 674 and 3858 unique porcine-specific proteins in BAL and ASL, respectively. This proteome was composed of proteins representing a diverse range of molecular classes and biological processes, including host defense, molecular transport, cell communication, cytoskeletal, and metabolic functions. Specifically, we detected a significant number of secreted proteins with known or predicted roles in innate and adaptive immunity, microbial killing, or other aspects of host defense. In greatly expanding the known proteome of the lung lining fluid in the pig, this study provides a valuable resource for future studies using this important animal model of pulmonary physiology and disease.

Full text

Protein composition of bronchoalveolar lavage fluid and airway surface liquid
from newborn pigs
Jennifer A. Bartlett,
1
Matthew E. Albertolle,
2
Christine Wohlford-Lenane,
1
Alejandro A. Pezzulo,
3
Joseph Zabner,
3
Richard K. Niles,
2
Susan J. Fisher,
2
Paul B. McCray, Jr.,
1
and Katherine E. Williams
2
1
Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa;
2
Department of Obstetrics,
Gynecology and Reproductive Sciences, University of California, San Francisco, California; and
3
Department of Internal
Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Submitted 25 February 2013; accepted in final form 17 May 2013
Bartlett JA, Albertolle ME, Wohlford-Lenane C, Pezzulo AA,
Zabner J, Niles RK, Fisher SJ, McCray PB Jr, Williams KE.
Protein composition of bronchoalveolar lavage fluid and airway sur-
face liquid from newborn pigs. Am J Physiol Lung Cell Mol Physiol
305: L256 –L266, 2013. First published May 24, 2013;
doi:10.1152/ajplung.00056.2013.—The airway mucosa and the alve-
olar surface form dynamic interfaces between the lung and the
external environment. The epithelial cells lining these barriers elabo-
rate a thin liquid layer containing secreted peptides and proteins that
contribute to host defense and other functions. The goal of this study
was to develop and apply methods to define the proteome of porcine
lung lining liquid, in part, by leveraging the wealth of information in
the Sus scrofa database of Ensembl gene, transcript, and protein
model predictions. We developed an optimized workflow for detec-
tion of secreted proteins in porcine bronchoalveolar lavage (BAL)
fluid and in methacholine-induced tracheal secretions [airway surface
liquid (ASL)]. We detected 674 and 3,858 unique porcine-specific
proteins in BAL and ASL, respectively. This proteome was composed
of proteins representing a diverse range of molecular classes and
biological processes, including host defense, molecular transport, cell
communication, cytoskeletal, and metabolic functions. Specifically,
we detected a significant number of secreted proteins with known or
predicted roles in innate and adaptive immunity, microbial killing, or
other aspects of host defense. In greatly expanding the known pro-
teome of the lung lining fluid in the pig, this study provides a valuable
resource for future studies using this important animal model of
pulmonary physiology and disease.
proteomics; airway surface liquid; bronchoalveolar lavage; porcine
lung
THE PASSAGEWAYS OF THE MAMMALIAN respiratory tract are bathed
by secretions that play critically important roles in protecting
the organism from microbial and environmental insults and
promoting normal lung function. In the conducting airways,
this fluid layer is known as airway surface liquid (ASL). ASL,
a complex mixture of secreted proteins, peptides, and mucins
as well as electrolytes and water, represents the combined
contributions of the surface and submucosal gland epithelia of
the conducting airways (5, 85). The varied roles of these gene
products include modulating inflammation, promoting wound
healing, maintaining ASL volume homeostasis, and transport-
ing various nutrients and lipids in the extracellular milieu. In
particular, ASL possesses a redundant and polyfunctional array
of secreted peptides and proteins with host defense and immu-
nomodulatory properties (reviewed in Ref. 7). In the gas-
exchange regions of the lung, epithelia secrete a liquid known
as the alveolar subphase, which contains pulmonary surfactant,
a potent mixture of lipids and proteins necessary for proper
mechanical functioning of the lung. In addition, like the ASL
of the conducting airways, alveolar secretions include a num-
ber of innate immune factors involved in pulmonary host
defenses. The coordinated functions of these secreted products
help maintain lung health.
To better understand the functions of these compartments,
proteomics approaches have been used to help identify the
secreted proteins that comprise the ASL and alveolar subphase
(collectively termed lung lining liquid). In past studies, re-
searchers have used two-dimensional gel electrophoresis or
other fractionation methods coupled with mass spectrometry
(MS) to investigate lung lining liquid composition in a variety
of fluids sampled from the human respiratory tract: nasal
lavage fluid (49, 50), bronchoalveolar lavage (BAL) fluid (14,
28, 44, 47, 48, 50, 53, 58, 83, 84, 87), induced sputum (38, 60),
and apical washings from cultured human bronchial epithelia
(38). A serous cell model of airway submucosal glands using
apical fluid from Calu-3 cells was used to investigate the
regulation of antiprotease and antimicrobial secreted proteins
(37). Notably, these proteomic tools provide a means to assess
global changes in the composition of respiratory secretions in
response to various inflammatory stimuli (12, 47–50) or in
association with certain disease states (44, 53, 58, 83, 84, 87).
Proteomic techniques have also been used to identify the BAL
fluid proteins from wild-type mice (28, 31) and in murine
models of lung injury and disease (13, 17, 28, 89). Here, we
used a shotgun MS approach to define the repertoire of secreted
peptide and protein components of the porcine lung.
The pig offers several advantages as an animal model for
studies of lung biology and disease states. Porcine lungs share
many similarities with human lungs in terms of size and
structure (71). Also, unlike mice, the relatively long lifespan of
pigs (10 –20 years on average) makes them well suited for
studies of progressive lung diseases (71). Porcine lungs have
previously been used in many areas of lung research, including
studies of surfactant composition and function (76), lung de-
velopment (29), infectious diseases including influenza and
porcine specific infections (27, 51, 61, 65), lung transplantation
(46, 52, 59), responses to various types of lung injury (11, 32),
and testing of gene therapy vectors (20, 57). Additionally, pigs
have been used to model a number of lung diseases and
conditions, including pulmonary hypertension (9), bronchioli-
tis obliterans (2), asthma (82), and cystic fibrosis (CF) (15, 43,
71, 77). Notably, the CF pig model has provided insights into
the development of CF lung disease, an aspect that was
Address for reprint requests and other correspondence: K. Williams, 521
Parnassus Ave., C-18, San Francisco, CA, 94143-0665 (e-mail: katherine.
williams@ucsf.edu).
Am J Physiol Lung Cell Mol Physiol 305: L256 –L266, 2013.
First published May 24, 2013; doi:10.1152/ajplung.00056.2013.
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previously difficult or impossible to study in murine CF models
(15, 62, 66, 77).
In contrast to the human and the mouse, the lung lining
liquid composition of pig airways is poorly characterized. The
components of the ASL and BAL in the normal pig lung are
largely unknown and it is likely that there are hundreds of
proteins and peptides that have never been identified. Pro-
teomic analysis of BAL and ASL has posed formidable chal-
lenges due to variable protein composition and small sample
volumes, which make it difficult to identify relevant biomark-
ers against the background of intracellular proteins from cell
shedding and other processes (28, 31). Additionally, the num-
ber of well-annotated porcine protein sequences lags far behind
the available genomic information. In this study, a global
proteomic analysis to identify the proteins in wild-type porcine
ASL and BAL was performed to establish a foundation for
future research. To create a useful pig-specific protein database
for MS data analysis, we annotated Ensembl Sus scrofa
FASTA protein entries with protein and gene names. These
data provided a comprehensive profile of lining liquid compo-
nents in healthy lung and new insights into the biology of this
important animal model. This repository is an important re-
source for future comparative studies of the alterations in
secreted factors that may occur in association with CF and
other porcine models of pulmonary disease states.
MATERIALS AND METHODS
Animal Protocols and Collection of Bronchoalveolar Lavage
and Airway Surface Liquid
Samples were collected from wild-type pigs as previously de-
scribed (62, 71, 72, 77). All experimental techniques were approved
by the Institutional Animal Care and Use Committee of the University
of Iowa.
For BAL collection, six newborn pigs were euthanized within 12 h
of birth by administering Euthasol (90 mg/kg iv) and lungs were
excised by aseptic technique. To lavage, 1/16-in.-diameter sterile
polyethylene tubing was inserted into the mainstem bronchi and lungs
were washed with 5 ml of normal saline. This procedure was repeated
three times for each excised lung and the collected washes from an
individual animal were immediately pooled and placed on ice. Then
each pooled BAL was centrifuged at a low speed (228 g) and the
supernatant transferred to a fresh tube. Clarified BAL was buffer
exchanged against 100 mM tetraethylammonium bicarbonate (Sigma-
Aldrich, St. Louis, MO) by using Amicon Ultra-15 3-kDa molecular
weight cutoff filters (Millipore, Billerica, MA). Total protein concen-
trations were estimated by the Bradford assay. Samples were stored
frozen at ⫺80°C until use.
Porcine ASL was collected from five wild-type pigs within 12 h of
birth. For this procedure, we used an established bronchoscopic
microprobe method (23) to collect native secretions from the trachea
and first generation bronchi. Methacholine was used to stimulate
secretion after initial studies revealed that it was not possible to collect
secretions from newborn animals without stimulation (66). We rea-
soned that this method would allow us to collect the net contributions
of airway and submucosal gland cells in response to a strong neuro-
chemical stimulus. Pigs were anesthetized with a mixture of ketamine
(20 mg/kg im) and xylazine (2 mg/kg im), followed by propofol (2
mg/kg iv); saline was given intravenously to prevent dehydration.
Once an animal had reached the proper plane of anesthesia, the neck
was dissected to expose the trachea. Tracheal secretion was stimulated
by administering methacholine (2.5 mg/kg iv). After ⬃5 min, tracheal
secretions were collected by making a small incision in the tracheal
wall and inserting a sterile polyester-tipped applicator (Puritan Med-
ical Products, Guilford, ME) to swab the lumen of the trachea. Then
the probe was inserted into a microcentrifuge tube and secretions were
recovered by centrifugation. This procedure generally resulted in
recovery of ⬃10 –20 ␮l of ASL from each animal. Samples were
immediately placed on ice and frozen at ⫺80°C until use. Following
ASL collection, pigs were euthanized with Euthasol.
Protein Preparation and Mass Spectrometry
Bronchoalveolar lavage. The workflow for BAL preparation and
processing is shown in Fig. 1. Upon thawing, 100 ␮g of protein was
denatured with trifluoroethanol (TFE), trypsin digested (81), divided
into two aliquots, and fractionated by isoelectric focusing (16, 25, 34)
and alkaline reverse-phase high-pressure liquid chromatography (re-
verse-phase HPLC) (21, 22, 24). For BAL1 and BAL2, eight HPLC
fractions were collected and for BAL3– 6 a total of 30 fractions were
collected. Peptide fractions were analyzed by LCMS on a QSTAR
Elite mass spectrometer (AB Sciex, Foster City, CA).
Airway surface liquid. The workflow for ASL preparation and
processing is shown in Fig. 1. ASL proteins were solubilized in
sodium dodecyl sulfate (SDS), reduced, alkylated, and trypsin di-
gested either by a filter-assisted solubilization protocol or by in-
solution digestion followed by SDS removal using strong cation
Newborn pig
ASL
Swab collection
LCMS
RP HPLC
pH 10 IEF
Trypsin digest
in solution
-80
C
BAL
ice
Lavage 3X
RP HPLC
pH 10
Trypsin digest
in solution
Trypsin digest
on filter
TFE
denaturation
SDS
denaturation
160 3344 514
ASL BAL
Total
4018
proteins
identified
Centrifuge 228 xg
Fig. 1. Workflow for collection, processing, and mass spectrometric analyses
of pig bronchoalveolar lavage (BAL) and airway surface liquid (ASL). TFE,
trifluoroethanol; IEF, isoelectric focusing.
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exchange (68, 75). Peptides were fractionated offline (n⫽20) by
using alkaline reverse-phase HPLC followed by LCMS on an LTQ
Velos Orbitrap (Thermo Scientific, San Jose, CA).
Pig Protein Sequence Database Development and Protein
Identification
The Ensembl Sus scrofa 10.2.67.pep. all protein FASTA database,
containing 23,118 entries, was annotated with protein and gene names
as follows. First, a program was developed to query all Ensembl
entries for each protein accession code. The gene name, description,
database source (e.g., UniProt, NCBI, HGNC), and entry name, if
present, were parsed out and assembled to replace the original En-
sembl annotation. For those entries for which the description was
“uncharacterized protein” or “novel transcript,” the gene name, if
present, was used to search the human UniProt Knowledgebase
v2012_07 and the human protein description used. The source for
these entries is designated UniProtKB(Hu). The final database con-
tained protein sequences and Ensembl accession codes for all of the
original 23,118 entries with 18,664 entries fully annotated with
descriptive protein names.
Protein identification was accomplished by using ProteinPilot 4.0
software (AB Sciex) and the integrated false discovery rate (FDR)
analysis function (79) with a concatenated reversed database. Search
parameters were trypsin enzyme specificity, carbamidomethyl cys-
teine, and thorough search effort. Proteins with ⱕ5% local FDR and
peptides with ⱕ1% global FDR were reported. For pig Ensembl
entries that did not contain a protein name, the gene name was mapped
to the human protein name. For the novel transcripts and uncharac-
terized proteins lacking a gene name that were detected at an FDR
threshold of ⱕ5%, a sequence similarity search was performed by
using BLAST (4) and the protein with the highest score was reported.
If equivalent top-scoring BLAST matches occurred, the human match
was reported whenever present. A subset of the data was also searched
by using mammalian sequences in the UniProt SwissProt database.
For both BAL and ASL, proteins detected from each individual
sample were aligned to a master search result comprised of all data by
using the Protein Alignment Template V2.000p beta (78). The master
search was a reference protein identification list produced by search-
ing the MS data from all samples to produce a single result. To
perform the analysis of the intersection of protein identifications, the
threshold for the master search was set at 1% global FDR and the
threshold for the individual samples set to 5% local FDR. These
settings were chosen to ensure that high-quality identifications from
each set were matched. The annotation of protein molecular function
and biological processes was performed by using PANTHER Gene
Ontology (GO) (80).
SDS-PAGE and Immunoblotting
To visualize proteins in lung lining fluid, BAL and ASL samples (2
␮g total protein per lane) were electrophoresed through 4 –20%
Tris·HCl gradient gels (Bio-Rad Laboratories, Hercules, CA) and
silver stained by use of the Silver Stain Plus kit (Bio-Rad Laborato-
ries). To immunoblot for PLUNC and SP-D, total protein from pig
BAL and ASL was separated on 4 –20% Tris·HCl gels (20 ␮g/lane for
BAL and 5 ␮g/lane for ASL) and transferred to PVDF membranes,
followed by blocking overnight in TBS-Tween containing 2% BSA.
To detect PLUNC protein, membranes were incubated with a mono-
clonal antibody recognizing human and porcine palate, lung, nasal
epithelium clone (PLUNC; R&D Systems, Minneapolis, MN) diluted
1:250 in TBS-Tween, for 1.5 h at room temperature. Membranes were
washed four times with TBS-Tween, then incubated with secondary
antibody (Immunopure goat anti-mouse conjugated to horseradish
peroxidase; Thermo Fisher Scientific) at a 1:20,000 dilution for 1 h.
After five more washes in TBS-Tween, protein bands were detected
with SuperSignal West Pico Chemiluminescent Substrate (Thermo
Fisher Scientific). For SP-D detection, membranes were incubated
with anti-porcine SP-D diluted 1:500 in TBS-Tween for 1.5 h at room
temperature, followed by four washes in TBS-Tween. Membranes
were incubated with secondary antibody (Immunopure goat anti-
rabbit conjugated to horseradish peroxidase; Thermo Fisher Scien-
tific) at 1:20,000 for 1 h, followed by five washes in TBS-Tween and
chemiluminescent detection as described above. Immunoblotting for
MUC5AC was carried out under nonreducing conditions as described
by Lacunza and colleagues (45). Briefly, BAL and ASL samples were
diluted in SDS-PAGE sample buffer containing 25% SDS (125 mM
Tris·HCl, pH 6.8; 25% SDS; 20% glycerol; 0.004% bromophenol
blue) prior to boiling at 95°C. Samples were loaded onto 7.5%
Tris·HCl gels (5 ␮g/lane) and electrophoresed for ⬃2 h. Then proteins
were transferred to PVDF membranes by electroblotting overnight at
4°C, by using methanol-free transfer buffer containing 0.1% SDS. To
block nonspecific immunoreactivity, the membranes were incubated
for 1–2 h at room temperature in TBS-Tween containing 2% BSA.
Then they were incubated for 1.5 h with a monoclonal antibody
recognizing MUC5AC (Clone 45M1; Thermo Fisher Scientific) di-
luted 1:500 in TBS-Tween. Next they were washed four times in
TBS-Tween and incubated for 1 h with secondary antibody (Immu-
nopure goat anti-mouse conjugated to horseradish peroxidase;
Thermo Fisher Scientific) at a 1:20,000 dilution. After five washes in
TBS-Tween, bands were detected as described above for PLUNC.
Electrophoresis and immunoblotting of MUC5AC under these condi-
tions is expected to produce a predominant band of ⬃200 kDa (45).
RESULTS
Diversity of Proteins in BAL and ASL
To investigate the composition of the lung lining fluid in the
newborn pig, we sampled the alveolar subphase and the con-
ducting airways by BAL as well as by direct collection of ASL
from the trachea. The overall abundance and complexity of
proteins in the BAL and ASL samples was assessed by sepa-
rating proteins using SDS-PAGE and visualizing by silver
stain. As shown in Fig. 2, both the BAL and ASL samples
displayed a diverse complement of proteins across a wide
range of molecular weights. Total protein concentrations were
0.050 – 0.42 mg/ml in newborn pig BAL samples and 5–15
mg/ml in ASL.
Shotgun Proteomics Detection of BAL Proteins
To better understand the composition of BAL, six samples
were processed for LCMS analysis (as summarized in Fig. 1)
using TFE for denaturation prior to trypsin digestion. To gain
maximum coverage of the proteome, peptide mixtures were
97.4
66.2
45
31
21.5
14.4
kDa
97.4
66.2
45
31
21.5
14.4
kDa
Fig. 2. SDS-PAGE separation/silver staining of BAL and ASL proteins.
A: BAL fluid from 6 individual newborn wild-type pigs, 2 ␮g protein/lane.
B: ASL collected from 5 individual newborn wild-type pigs, 2 ␮g/lane. MW,
molecular weight standard (SDS-PAGE Molecular Weight Standard, Low
Range; Bio-Rad Laboratories, Hercules, CA).
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fractionated offline by two orthogonal methods, isoelectric
focusing and alkaline pH reversed phase HPLC, prior to LCMS
data acquisition. For the first two samples, a total of eight
offline alkaline pH HPLC fractions were collected. The re-
maining BAL samples were more extensively fractionated for
a total of 30 fractions. To maximize protein identification,
spectra were searched against the pig Ensembl pep. all data-
base, the superset of all translations resulting from Ensembl
known or novel gene predictions. The general format of these
entries was “ID Sequence type:Status:Location:Gene:Tran-
script.” To make this database useful for protein identification,
we developed software to configure the header information for
each protein to include the protein name, the gene name, and
the source database. The annotated database is available for
download at https://wiki.library.ucsf.edu/x/vSzWAw.
The number of proteins detected at ⱕ5% FDR in individual
BAL samples ranged widely, from 46 to 558, with a total of
674 distinct proteins identified from all BAL samples (Table 1).
More extensive fractionation at the peptide level prior to LCMS
resulted in a significant increase in the number of peptides and
proteins detected (BAL 1 and 2 vs. BAL 3– 6). Over 89% of the
proteins identified in the Ensembl database were mapped to porcine
species-specific entries in Swiss-Prot, RefSeq, or TrEMBL (Supple-
mental Table S1; supplemental material for this article is avail-
able online at the Journal website). A sequence similarity
search using BLAST (4) was performed for the 70 confidently
detected sequences listed as “uncharacterized” or “novel tran-
script,” containing neither a protein name nor a gene name. For
all but two proteins, very-high-scoring homologous sequences
were identified (Supplemental Table S2).
As expected, the BAL dataset included many well-recog-
nized lung gene products, such as ␣
1
-antitrypsin, the alveolar
surfactant proteins [surfactant proteins B (SP-B) and C (SP-
C)], and mucins such as MUC1 and MUC16 (Supplemental
Table S1). Of interest, we identified numerous proteins asso-
ciated with mammalian host defense, including lactoferrin,
lipocalin 2, the collectins, SP-A and SP-D, the PLUNC protein
and related family members, lactoperoxidase, S100-A8 (cal-
granulin-A) and S100-A9 (calgranulin-B), histones, and sev-
eral components of the complement system. Additionally, we
identified the pig-specific cathelicidin antimicrobial protein
protegrin-1. Other proteins expected to be found in BAL, e.g.,
PR-39, PMAP-37, and the highly abundant MUC5AC and
MUC5B, were not present in the Ensembl database. To verify
the presence of these abundant BAL proteins in our samples,
we searched the UniProt pig database, which contained full-
length sequences for PR-39 and PMAP-37 and a partial se-
quence of MUC5AC (MUC5B was not in this database).
PMAP-37 and MUC5AC were detected with multiple high-
scoring peptides (Table 2). The relatively small size and high
arginine content (10 of 39 amino acids) of PR-39 limited the
number of observable tryptic peptides to one, which could
explain why this abundant antimicrobial protein was not de-
tected.
To independently verify our results, we selected a subset of
BAL proteins (PLUNC, SP-D, and MUC5AC) to confirm by
immunoblotting (Fig. 3). These proteins were selected for their
known roles in the lung lining liquid milieu and the availability
of cross-reacting antibodies. All BAL samples contained these
proteins as determined by immunoblotting.
ASL Protein Analysis
ASL from the conducting airways was harvested via a
modified collection approach that was designed to avoid sam-
pling the alveolar contribution. On the basis of experience
gained with the BAL analysis, we refined our workflow as
outlined in Fig. 1, using SDS as a denaturant with trypsin
Table 1. Number of unique peptides and proteins detected in
porcine BAL and ASL
Sample Distinct Peptides Proteins
BAL1 826 119
BAL2 149 46
BAL3 4,096 384
BAL4 3,898 161
BAL5 4,270 422
BAL6 4,957 558
Master BAL 9,313 674
ASL1 3,513 1134
ASL2 2,316 803
ASL3 3,207 975
ASL4 18,012 2,959
ASL5 22,888 3,289
Master ASL 30,591 3,858
Data are presented for each individual bronchoalveolar lavage (BAL) and
airway surface liquid (ASL) sample. Distinct peptides at the ⱕ1% global false
discovery rate (FDR) and proteins with a ⱕ5% local FDR are shown. The
“Master” search result of all BAL or ASL sample sets searched together was
used to disambiguate the protein groups and align the proteins across data sets
(Supplemental Tables S1 and S3).
Table 2. MUC5AC and PMAP-37 peptides that were used for protein identification
Accession Name Coverage, % Conf., % Sequence Cleavages
O97866 MUC5AC (Fragment, 357 AA) 56.9
99 AESFPDTPLQALGQDVIC(CAM)DK
99 WFDVDFPSPGPHGGDFETYSNILR
99 LGQVVEC(CAM)RPEVGLVC(CAM)R
P49932 Antibacterial peptide PMAP-37 76.7
99 AVDRLNEQSSEANLYR Missed R-L@4
99 LLELDQPPKADEDPGTPKPVSFTVK Missed K-A@9
99 LNEQSSEANLYR
99 LLELDQPPK
96 ADEDPGTPKPVSFTVK
99 RPPELC(CAM)DFKEN(Dea)GR W-R@N-term
Mass spectrometry (MS) data from BAL samples were searched by using the pig Uniprot SwissProt and TrEMBL databases. Peptides detected with confidence
levels ⱖ95% are shown. Coverage is the percent of matching amino acids (AAs) from identified peptides with any confidence (Conf.) divided by the total number
of amino acids in the sequence. CAM, carbamidomethyl; Dea, deamidation; Cleavages, nontryptic cleavage sites.
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digestion in solution or on filters (55, 86). The number of
distinct peptides identified was 6.5-fold higher with the in-
solution trypsin digestion protocol, averaging 18,872, com-
pared with 2,888 with the filter-assisted digestion protocol.
This translated to an average of 2,407 protein groups compared
with 969, respectively (Table 1). The presence of highly
abundant glycosylated, difficult-to-proteolyze proteins in ASL
could potentially clog the filter and reduce peptide recovery.
Overall, the ASL analyses resulted in a significantly larger
number of unique protein identifications compared with BAL,
with 3,858 distinct proteins detected in toto (Supplemental
Table S3). Confidently detected Ensembl sequences lacking a
protein or gene name were searched by using BLAST as
described for BAL proteins (Supplemental Table S4). Addi-
tional lung and host defense proteins detected in ASL included
MUC-4, -13, and -19, LPLUNC2, lipocalin 1, antileukoprotei-
nase (SLPI), PMAP-23, PLAT, protegrin 3, SPPI, and ser-
pinB5. A subset of proteins (PLUNC, SP-D, and MUC5AC)
was confirmed by immunoblotting and all ASL samples con-
tained these proteins (Fig. 3B).
Functional Classification of BAL and ASL Proteins
We observed significant overlap between the BAL and ASL
proteomes; 88% of BAL-associated proteins were also detected
in ASL. Using PANTHER GO classifications, we categorized
proteins in both datasets by biological process, protein class,
molecular function, and cellular compartment (Fig. 4). The
distribution of proteins was similar between BAL and ASL and
included a broad range of protein classes associated with
diverse processes such as responses to stimuli, immune system
functions, cell communication, and transport. In addition to
extracellular and secreted proteins, a number of intracellular
proteins such as metabolic enzymes were identified.
We previously profiled mRNA expression in trachea, bron-
chus tissue, and well-differentiated primary cultures of tracheal
and bronchial epithelia isolated from wild-type and CF trans-
membrane conductance regulator (CFTR)-null newborn piglets
(66). A set of 973 genes (secreted host defense proteins as well
as those involved in inflammation, bacterial responses, and
wounding) was compared with the proteins detected in BAL
and ASL. A total of 314 proteins were found, 87 of which were
secreted host defense proteins (Table 3).
DISCUSSION
Here we developed techniques to identify proteins from the
porcine lung and conducting airways by using BAL fluid and
ASL as starting materials. To our knowledge, this study pro-
vides the most comprehensive database to date of proteins in
porcine lung lining fluid. Because BAL has traditionally been
the method of choice for sampling lung and airway secretions
for proteomic studies, we began by characterizing BAL from
newborn pigs. Although earlier studies used proteomics-based
approaches to monitor pig BAL for changes in response to
bacterial (33) and viral (88) pathogens, ours is the first attempt
to compile the proteome of normal pig BAL, which resulted in
the identification of 674 proteins. This level of detection was in
line with previous reports that used mass spectrometry-based
methods to identify BAL proteins in human (28, 44, 53, 58,
87), mouse (13, 17, 28, 31, 67), rat (74), cow (8), and horse
(10). For example, Guo and colleagues (31) identified 297
proteins in BAL from a single mouse, and analysis of BAL
from six healthy horses yielded 582 identifications (10). No-
tably, protein discovery has often been greatest in studies
designed to investigate differential protein expression in the
context of various pulmonary diseases or challenges. To date,
the greatest number of BAL proteins reported for any species
was 959, observed in a study investigating the effects of
cigarette smoke inhalation in mice (67). In a study of biomark-
ers in lung transplant patients with chronic graft dysfunction,
Kosanam et al. (44) reported a total of 531 proteins in human
BAL. In another study, as many as 889 proteins were identified
in BAL from antigen-challenged asthmatic patients and control
individuals (87).
One drawback of using BAL fluid as the analyte for pro-
teomics is that the collection technique necessarily involves
significant and variable dilution, potentially complicating ef-
forts to make quantitative comparisons among study subjects.
As an alternative, several groups attempted to characterize lung
lining fluid in humans using induced sputum, which can be
collected without dilution of the sample (38, 60). We addressed
this issue by collecting methacholine-induced tracheobronchial
B
123456 7
37
25
20
15
kDa
72
55
PLUNC
SP-D
MUC5AC
A
123 45 7
37
25
20
15
kDa
72
55
PLUNC
SP-D
MUC5AC
Fig. 3. Verification of selected proteins identified in LC-MS/MS analysis.
A: BAL fluid from wild-type newborn pigs was electrophoretically separated
by SDS-PAGE under reducing conditions (top and middle) and nonreducing
conditions (bottom). Proteins were transferred to PVDF membranes prior to
immunoblotting with antisera specific for the PLUNC protein (top), surfactant
protein-D (SP-D; middle), and MUC5AC (bottom). Lanes 1-6contained lavage
samples from 6 individual pigs. Secretions from the apical surface of pig
primary airway epithelial cultures (2 ␮l) served as a positive control for the
PLUNC antibody (lane 7). Recombinant porcine SP-D (50 ng) was a positive
control for the SP-D antibody (lane 7; noncontiguous lane from the same blot).
Total protein from pig stomach scraping (10 ng) served as a positive control for
the MUC5AC antibody (lane 7). B: the strategy described for Awas used for
the immunoanalyses of 5 individual porcine ASL samples from wild-type
newborn pigs.
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secretions directly from pig airways (ASL). We originally
collected tracheal ASL using the capillary technique; however,
it was not possible to collect sufficient material for MS analysis
from individual animals. Although the composition of secre-
tions with methacholine induction might be different than
steady state, the proteins detected are those that the epithelium
might release under resting conditions, or in response to
neurohumoral or environmental stimuli (18, 19, 36, 40).
The number of protein identifications increased markedly
when we shifted our focus from BAL samples to ASL. How-
ever, it is not clear whether the ASL samples are intrinsically
more protein rich and diverse, or whether this is a consequence
of refinements in our methods of sample collection and prep-
aration, peptide fractionation, and/or LCMS instrumentation,
making a direct comparison of the data difficult. For example,
increasing the number of BAL peptide fractions analyzed from
8 to 30 resulted in an average 4.5-fold increase in protein
identifications on the QSTAR Elite. Although fewer ASL
fractions (n⫽20) were analyzed, more proteins were detected
by using the LTQ Orbitrap Velos. It is not possible to deter-
mine how much of this effect was due to the MS platform or
inherent to the sample type. We note that both instruments
undergo quality control measures assuring 1 fmol protein digest
sensitivity. A confounding factor in the analysis of lung lining
fluids is the presence of highly abundant proteins (e.g., albu-
min, serotransferrin, ␣
2
-macroglobulin, ␣
1
-acid glycoprotein)
that contribute to the dynamic range of these complex mixtures
and confound detection of medium- and low-abundance spe-
cies. Affinity-depletion strategies have been used to ameliorate
this issue in human BAL, although no pig-specific antibodies
are currently available for this purpose. We found that 88% of
the proteins detected in BAL were also found in ASL. How-
ever, since other studies have reported ⬃900 proteins in BAL
(67, 87), the overlap of common proteins in these fluids could
be greater. We speculate that avoidance of the sample dilution
associated with BAL collection may have enhanced the num-
ber of proteins detected. In addition to minimizing sample
dilution, this approach also afforded the opportunity to inves-
tigate the protein repertoire of the tracheobronchial epithelium,
representative of the conducting airways, separately from the
alveolar compartment. Although this sampling technique ap-
pears to be unique to our study, other investigators have
attempted to examine the composition of ASL by using mass
spectrometry to identify proteins in apical secretions from
0% 5% 10% 15% 20%
Transporter
Transmembrane receptor
Transferase
Transfer/c arrier protein
Surfactant
Signaling molecule
Receptor
Protease
Oxidoreductase
Other
Nucleic acid binding
Membrane traffic protein
Extracellular matrix protein
Enzyme modulator
Defense/immunity protein
Cytoskeletal protein
Chaperone
Protein Class
BAL (870) ASL (3723)
0% 5% 10% 15% 20% 25%
Transport
System process
Response to stimulus
Other
Metabolic process
Immune system process
Developmental process
Cellular process
Cellular component org.
Cell cycle
Cell communication
Cell adhesion
Apoptosis
Biological Process
BAL (1356) ASL (5928)
0% 10% 20% 30% 40%
Transporter activity
Translation regulator activity
Transcription regulator activity
Structural molecule activity
Receptor activity
Ion channel activity
Enzyme regulator activity
Catalytic activity
Binding
Antioxidant activity
Molecular Function
BAL (659) ASL (3248)
0% 10% 20% 30% 40% 50% 60%
Ribonucleoprotein complex
Protein complex
Plasma membrane
Intracellular
Extracellular region
Cellular Compartment
BAL (110) ASL (481)
Fig. 4. Classifications for proteins identified in porcine BAL and ASL. Using PANTHER (80), we classified proteins by Gene Ontology terms describing
biological process (A), protein class (B), molecular function (C), and cellular compartment (D). Results are displayed as percent of genes classified to a category
over the total number of class hits. Class hit means independent ontology terms; if a gene was classified to more than 1 independent ontology terms that are not
parent or child to each other, it counts as multiple class hits. The total number of class hits for each category is shown in parentheses after the sample type.
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air-liquid interface cultures of human primary airway epithelia
(3, 12, 38) or cell lines (37).
In the course of these experiments, we optimized database
searching methods for identification of proteins in pig-derived
samples. A critical factor was selection of the protein database.
The porcine protein databases still have large gaps, although
the number of entries is increasing monthly due to continued
progress on the pig genome (30). The UniProt Knowledgebase
(KB) version used in this study contained only 1,406 manually
curated, nonredundant (SwissProt) entries for Sus scrofa pro-
teins, compared with 20,248 entries in the human and 65,102 in
the mammalian databases (54). To overcome the paucity of
fully annotated entries, MS data from porcine samples are
often searched against either human plus pig or mammalian
protein databases (6, 35, 56, 70). An additional UniProtKB
database, TrEMBL, contains protein sequences associated with
computationally generated annotation but is not reviewed nor
curated for redundancy. Protein fragments, isoforms and vari-
ants, encoded by the same gene, are found in separate entries.
We annotated the 23,118 Ensembl Sus scrofa 10.2.67.pep. all
database entries with protein name, gene name, and source
database. Then we compiled databases that contained various
combinations of the curated and noncurated pig and mamma-
lian entries and tested various search parameters to determine
the optimal strategy for identifying the greatest number of
proteins with low FDRs for peptide and protein detection. In
summary, searching the Ensembl Sus scrofa protein FASTA
database explained the greatest number of spectra at a given
FDR and reduced the number of redundant proteins found
compared with searching the larger mammalian databases. We
detected many pig peptides and proteins whose existence was
previously only predicted from available genomic (DNA) and
transcriptomic (mRNA, EST) data. The Ensembl protein data-
base is not complete, and we noted that some known lung
proteins, such as the mucins MUC5AC and MUC5B as well as
the pig-specific antimicrobial proteins PR-39 and PMAP-37,
are not present. However, MUC5AC and PMAP-37 were
identified in BAL and ASL by using the pig UniProt SwissProt
Table 3. Secreted host defense proteins detected in BAL and ASL
Protein Identified in BAL/ASL Gene from MicroArray Protein Identified in BAL/ASL Gene from MicroArray
Alpha-2-macroglobulin A2M High mobility group protein B1 HMGB1
ATP-binding cassette F1 ABCF1 Hermansky-Pudlak syndrome 5 protein HPS5
RAGE Protein AGER 60-kDa heat shock protein, mitochondrial HSPD1
Angiotensinogen AGT Interleukin-6 receptor subunit beta IL6ST
Serum albumin ALB Integrin, beta 4 ITGB4
Protein AMBP AMBP Kininogen-1 KNG1
Annexin A1 ANXA1 Alpha-lactalbumin LALBA
Annexin A5 ANXA5 Lipopolysaccharide-binding protein LBP
Apolipoprotein A-I APOA1 Protein-lysine 6-oxidase LOX
Apolipoprotein A-IV APOA4 Lactoperoxidase LPO
Apolipoprotein E APOE Lactotransferrin LTF
Beta-2-glycoprotein 1 APOH Lysozyme C-2 LYSC2
Beta-2-microglobulin B2M Lysozyme-like protein 6 LYZL6
Bactericidal permeability-increasing protein BPI Macrophage migration inhibitory factor MIF
Complement C1r subcomponent C1R Growth/differentiation factor 8 MSTN
C4b-binding protein alpha chain C4BPA Mucin 5AC MUC5AC
Complement C5a anaphylatoxin C5 Tissue-type plasminogen activator PLAT
Complement component C6 C6 Plasminogen PLG
Complement component C7 C7 BPI fold-containing family A member 1 PLUNC
Complement component C9 C9 Peroxiredoxin-5, mitochondrial PRDX5
Carbonic anhydrase 2 CA2 Vitamin K-dependent protein C PROC
monocyte differentiation antigen CD14 CD14 Saposin-B-Val Saposin-B PSAP
CD59 glycoprotein CD59 Protein S100-A8 S100A8
CD97 antigen CD97 Protein S100-A9 S100A9
Complement factor D CFD Serum amyloid A2 SAA4
Complement factor I CFI Alpha-1-antitrypsin SERPINA1
Properdin CFP Plasma serine protease inhibitor SERPINA5
Clusterin CLU Thyroxine-binding globulin SERPINA7
Cystatin-C CST3 Leukocyte elastase inhibitor SERPINB1
Cystatin-M CST6 Serpin B5 SERPINB5
Cystatin-B CSTB Heparin cofactor 2 SERPIND1
Lysosomal protective protein CTSA Plasminogen activator inhibitor 1 SERPINE1
Cathepsin B CTSB alpha-2 antiplasmin member 1 SERPINF1
Cathepsin C CTSC alpha-2 antiplasmin member 2 SERPINF2
Procathepsin H CTSH Plasma protease C1 inhibitor SERPING1
Cathepsin L1 CTSL1 Pulmonary surfactant-associated protein D SFTPD
Coxsackievirus and adenovirus receptor CXADR Superoxide dismutase SOD1
Acyl-CoA-binding protein DBI(32-86) DBI SPARC SPARC
Epidermal growth factor EGF Osteopontin SPP1
Coagulation factor XII F12 Serotransferrin TF
Coagulation factor V F5 Thyroglobulin TG
Fibrinogen beta chain Fibrinopeptide B FGB Thrombospondin 1 THBS1
Fibronectin FN1 Tenascin TNC
Fibronectin FN1 Vitronectin VTN
Proteins detected in BAL and ASL were cross referenced to a curated list of mRNA transcripts with recognized host defense and antimicrobial functions (66).
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and TrEMBL. The abundant MUC5B, which has been detected
on the luminal surface of the bronchiolar epithelium of piglets
airways by using immunohistochemistry (41), is not present in
any of the porcine protein databases thus precluding MS-based
identification in these data sets.
The most abundant proteins in both the BAL and ASL
datasets included plasma proteins: serotransferrin, serum albu-
min, complement factor C3, ␣
2
-HS-glycoprotein (fetuin-A),
␣-fetoprotein, ␣
1
-acid glycoprotein 1, ␣
2
-macroglobulin, im-
munoglobulins, fetuin-B, and haptoglobin. In this respect, our
study is in very good agreement with earlier proteomic analy-
ses, which consistently reported plasma proteins in BAL and
other airway fluids (14, 28, 44, 47, 48, 50, 53, 58, 83, 84, 87).
This finding may reflect physiological transudates of plasma
proteins from the circulation into the airways or alveoli and/or
may be due to low level blood contamination introduced during
sample collection procedures. However, there is also evidence
that at least some of these proteins are normal secretory
products of airway epithelia. Proteomic studies of apical se-
cretions from cultured human airway epithelia have docu-
mented release of complement factor C3, ␣
2
-macroglobulin,
␣
2
-HS-glycoprotein, and serum albumin from these cells (3,
12, 38).
As shown in Fig. 4, we found that the proteins detected in
BAL and ASL were similarly distributed across protein
classes, biological processes, and molecular functions. Inter-
estingly, the largest proportion (⬃25%) of proteins in both
sample types was categorized as having a role in metabolic
processes, likely reflecting the fact that a significant proportion
of the detected proteins was predicted to be cytoplasmic or
intracellular. The detection of intracellular proteins has been
noted in earlier studies of airway secretions (3, 12, 38, 87). It
is possible that some of these proteins may be derived from
apoptotic cells, the result of normal turnover of airway epithe-
lium, or possibly from cell debris due to disruption of the
epithelium during sample collection. However, a review of the
literature suggests that a certain number of intracellular pro-
teins might actually be expected in the lung lining fluid of
mammals, due to the secretory machinery of the airway epi-
thelium and submucosal glands. Studies in humans, mice, and
rabbits have provided evidence for both merocrine and apo-
crine secretion by goblet cells of the airway epithelium (and
Clara cells in mice) (26, 42, 63, 64, 73). In merocrine secretion,
secretory products are packaged into vesicles and exocytosed
directly to the extracellular milieu; apocrine secretion involves
release of cargo as part of membrane-bound vesicles that bud
off the apical surface of the cell. Thus it is anticipated that
apocrine secretion could result in disruption of cell membranes
and introduction of intracellular and membrane proteins into
the ASL. In support of this possibility, a recent proteomic study
demonstrated that membrane-bound granules secreted from
airway epithelial goblet cells contain cytoskeletal and regula-
tory proteins as well as mucins, suggesting that such granules
may be a source for some of the intracellular proteins detected
in ASL (69). Additionally, several groups have reported the
presence of exosomes, microvesicles implicated in host de-
fense and cell communication, in human BAL and cell culture
secretions (1, 39). Exosomes have been shown to be associated
with mucins, cytoskeletal proteins, and cytosolic enzymes,
suggesting that these structures may also be a source of some
of the intracellular proteins we identified.
Among the lung lining liquid (BAL and ASL) proteins
identified were many associated with host defense (Supple-
mental Tables S1 and S3), including histone fragments, lacto-
ferrin, lactoperoxidase, lysozyme, lipocalin 1, lipocalin 2,
PLUNC, LPLUNC1, LPLUNC2, SLPI, surfactant proteins A
and D, S100A8 (calgranulin-A), S100A9 (calgranulin-B), and
the pig-specific antimicrobial cathelicidin proteins (PMAP-23,
PMAP-37) and protegrins-1 and -3. It is also interesting to note
that several proteins of the complement family were present in
lung lining liquid. These included complement components
(C1r, C4, CB, C5, C6, C7, C9) and complement factors (B, D,
H, and I). The complement system is recognized for its roles in
innate immunity, with activities that include opsonization,
chemotaxis, and membrane destruction. However, the func-
tions of complement protein components in lung lining liquid
are not well studied. Several protease inhibitors (e.g., the serine
protease inhibitors, serpins) and proteases (e.g., the cathepsins)
were also resident in lung lining liquid. This speaks to the
importance of a balance of these forces in modulating protein
function. Mucociliary clearance is an important component of
airway host defenses, and both tethered and secreted compo-
nents of the mucin layer were identified, including Muc1, -2,
-3A, -4, -5AC, -13, -16, and -19. Our data focus on an
important early time point after birth. Thus the BAL and ASL
proteome may change over time as the animal develops.
In summary, this study is the first to define the proteome of
the lung lining fluid in the newborn pig. For this task, we used
both BAL fluid as well as methacholine-stimulated tracheal
secretions in an effort to ensure that our results encompass both
the conducting airways and the gas-exchange regions of the
lung. In doing so, we greatly expanded the known proteome of
the porcine lung, while also contributing to the greater body of
literature documenting the composition of airway secretions
across all mammalian species. Our database of porcine airway
proteins should provide a framework for future studies utilizing
porcine models of airway infection, disease, or injury. In partic-
ular, it can serve as a reference for proteomic studies of patho-
genesis and/or progressive lung changes in porcine models of CF
and other inflammatory lung conditions. Additionally, these find-
ings may also be useful for studies investigating responses to
economically relevant pig pathogens such as porcine reproductive
and respiratory syndrome and influenza. Thus this “pig airway
proteome” is a resource that will enhance the utility of the pig as
an animal model for studies of lung biology, disease, and thera-
peutics.
ACKNOWLEDGMENTS
We thank Paula Ludwig for technical support. Polyclonal antiserum against
porcine SP-D was generously provided by Dr. Henk Haagsman (Utrecht
University, The Netherlands).
GRANTS
We acknowledge support from the Cystic Fibrosis Foundation RDP (P. B.
McCray and S. J. Fisher), National Heart, Lung, and Blood Institute Grants
P50 HL-61234 (P. B. McCray) and P01 HL-091842 (P. B. McCray), as well
as the Roy J. Carver Charitable Trust (P. B. McCray). The UCSF Sandler-
Moore Mass Spectrometry Core Facility is partially funded by a National
Cancer Institute Cancer Center Support Grant, the Sandler Family Foundation,
and the Gordon and Betty Moore Foundation.
At the request of the author(s), readers are herein alerted to the fact that
additional materials related to this manuscript may be found at the institutional
website of one of the authors, which at the time of publication is https://
wiki.library.ucsf.edu/x/vSzWAw. These materials are not a part of this man-
L263PROTEOMICS OF PORCINE LUNG FLUID
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uscript and have not undergone peer review by the American Physiological
Society (APS). APS and the journal editors take no responsibility for these
materials, for the website address, or for any links to or from it.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
J.A.B., J.Z., S.J.F., P.B.M., and K.E.W. conception and design of research;
J.A.B., M.E.A., C.W.-L., A.A.P., and K.E.W. performed experiments; J.A.B.,
M.E.A., R.K.N., and K.E.W. analyzed data; J.A.B., P.B.M., and K.E.W.
interpreted results of experiments; J.A.B., M.E.A., and K.E.W. prepared
figures; J.A.B., P.B.M., and K.E.W. drafted manuscript; J.A.B., S.J.F., P.B.M.,
and K.E.W. edited and revised manuscript; J.A.B., M.E.A., C.W.-L., A.A.P.,
J.Z., R.K.N., S.J.F., P.B.M., and K.E.W. approved final version of manuscript.
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