A Mighty Small Heart: The Cardiac Proteome of Adult Drosophila melanogaster

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DOI: 10.1371/journal.pone.0018497 · Source: PubMed
Abstract
Drosophila melanogaster is emerging as a powerful model system for the study of cardiac disease. Establishing peptide and protein maps of the Drosophila heart is central to implementation of protein network studies that will allow us to assess the hallmarks of Drosophila heart pathogenesis and gauge the degree of conservation with human disease mechanisms on a systems level. Using a gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected adult fly hearts. Contractile, cytostructural and mitochondrial proteins were most abundant consistent with electron micrographs of the Drosophila cardiac tube. Functional/Ontological enrichment analysis further showed that proteins involved in glycolysis, Ca(2+)-binding, redox, and G-protein signaling, among other processes, are also over-represented. Comparison with a mouse heart proteome revealed conservation at the level of molecular function, biological processes and cellular components. The subsisting peptidome encompassed 5169 distinct heart-associated peptides, of which 1293 (25%) had not been identified in a recent Drosophila peptide compendium. PeptideClassifier analysis was further used to map peptides to specific gene-models. 1872 peptides provide valuable information about protein isoform groups whereas a further 3112 uniquely identify specific protein isoforms and may be used as a heart-associated peptide resource for quantitative proteomic approaches based on multiple-reaction monitoring. In summary, identification of excitation-contraction protein landmarks, orthologues of proteins associated with cardiovascular defects, and conservation of protein ontologies, provides testimony to the heart-like character of the Drosophila cardiac tube and to the utility of proteomics as a complement to the power of genetics in this growing model of human heart disease.
A Mighty Small Heart: The Cardiac Proteom e of Adult
Drosophila melanogaster
Anthony Cammarato
1,2
, Christian H. Ahrens
3
, Nakissa N. Alayari
1,2
, Ermir Qeli
3
, Jasma Rucker
4
,MaryC.
Reedy
5
, Christian M. Zmasek
6
, Marjan Gucek
7
, Robert N. Cole
7
, Jennifer E. Van Eyk
8
, Rolf Bodmer
1
, Brian
O’Rourke
4
, Sanford I. Bernstein
2
, D. Brian Foster
4
*
1 Development and Aging Program, NASCR Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America, 2 Department of Biology
and the Heart Institute, San Diego State University, San Diego, California, United States of America, 3 Quantitative Model Organism Proteomics, Institute of Molecular Life
Sciences, University of Zurich, Zuric h, Switzerland, 4 Division of Cardiology, Institute of Molecular Cardiobiology, Johns Hopkins University School of Medicine, Baltimore,
Maryland, United States of America, 5 Department of Cell Biology, Duke University School of Medicine, Durham, North Carolina, United States of America, 6 Bioinformatics
and Systems Biology Program, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America, 7 Johns Hopkins Proteomics Core, Johns Hopkins
University School of Medicine, Baltimore, Maryland, United States of America, 8 Division of Cardiology, Departments of Biological Chemistry and Biomedical Engineering,
Johns Hopkins Bayview Medical Center, Baltimore, Maryland, United States of America
Abstract
Drosophila melanogaster is emerging as a powerful model system for the study of cardiac disease. Establishing peptide and
protein maps of the Drosophila heart is central to implementation of protein network studies that will allow us to assess the
hallmarks of Drosophila heart pathogenesis and gauge the degree of conservation with human disease mechanisms on a
systems level. Using a gel-LC-MS/MS approach, we identified 1228 protein clusters from 145 dissected adult fly hearts.
Contractile, cytostructural and mitochondrial proteins were most abundant consistent with electron micrographs of the
Drosophila cardiac tube. Functional/Ontological enrichment analysis further showed that proteins involved in glycolysis,
Ca
2+
-binding, redox, and G-protein signaling, among other processes, are also over-represented. Comparison with a mouse
heart proteome revealed conservation at the level of molecular function, biological processes and cellular components. The
subsisting peptidome encompassed 5169 distinct heart-associated peptides, of which 1293 (25%) had not been identified in
a recent Drosophila peptide compendium. PeptideClassifier analysis was further used to map peptides to specific gene-
models. 1872 peptides provide valuable information about protein isoform groups whereas a further 3112 uniquely identify
specific protein isoforms and may be used as a heart-associated peptide resource for quantitative proteomic approaches
based on multiple-reaction monitoring. In summary, identification of excitation-contraction protein landmarks, orthologues
of proteins associated with cardiovascular defects, and conservation of protein ontologies, provides testimony to the heart-
like character of the Drosophila cardiac tube and to the utility of proteomics as a complement to the power of genetics in
this growing model of human heart disease.
Citation: Cammarato A, Ahrens CH, Alayari NN, Qeli E, Rucker J, et al. (2011) A Mighty Small Heart: The Cardiac Proteome of Adult Drosophila melanogaster. PLoS
ONE 6(4): e18497. doi:10.1371/journal.pone.0018497
Editor: Andy T. Y. Lau, University of Minnesota, United States of America
Received February 2, 2011; Accepted March 1, 2011; Published April 25, 2011
Copyright: ß 2011 Cammarato et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: AC was funded by an AHA Western States postdoctoral fellowship and by AHA 10SDG4180089. CHA and EQ are funded in part by the University of Zurich’s
Research Priority Project Functional Genomics/Systems Biology. CMZ by National Institutes of Health (NIH) R01-GM087218, SIB by NIH R01-GM32443, RB by NIH R01
HL054732 and by The Ellison Medical Foundation, JVE and RNC by the NHLBI Proteomics Initiative Contract N01-HV28180, BOR by P01 HL081427. DBF was supported
by P01 HL081427 (to BOR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have decl ared that no competing interests exist.
* E-mail: dbrianfoster@jhmi.edu
Introduction
Long valued as a prime model of cardiac development, the
utility of Drosophila melanogaster for the study of cardiac pathogenesis
and pathophysiology is growing rapidly [1,2,3], driven by the
development of new research tools and methods [4,5,6]. Adult
Drosophila possess an open circulatory system consisting, in part, of
a dorsal vessel (Fig. 1) which is differentiated into an abdominally-
located ,1 mm long pulsatile heart tube and an anterior aorta
that extends through the thorax and into the head [7]. The
prospect of combining quantitative proteomics of the cardiac tube
with the power of Drosophila genetics promises to provide novel
insights into the mechanisms of human heart disease.
Two significant roadblocks to widespread adoption of Drosophila
as a model system for the study of heart disease need to be
overcome. The first is technical. The small size of the Drosophila
cardiac tube presents a challenge that is being addressed by the
development of adequate dissection protocols [8] and imaging
methods [8,9]. Application of proteomic techniques presents its
own unique challenges, not the least of which is collecting
sufficient protein for study.
A second impediment is the diminishing, yet persistent, view
that the Drosophila cardiac tube is not a ‘‘true heart’’ and that its
study may yield few insights translatable to human disease
mechanisms. However, recently published work would suggest
otherwise [1,10,11,12,13]. The pathological effects of fly and
human mutant protein isoforms, expressed in the Drosophila
cardiac tube, have successfully predicted causal-genes that
are both involved in, and recapitulate the phenotypes of,
specific human cardiomyopathies [1,10,13,14]. Thus, unbiased,
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high-throughput mutagenesis screens in flies followed by
cardiac phenotyping, can be integrated for rapid gene discovery
and novel network reconstruction to greatly facilitate cardiac
systems biology and to elucidate pathogenic mechanisms of
human cardiac disease [15].
A critical component essential for further exploiting the power
of Drosophila in cardiac systems biology is a comprehensive record
of the protein constituents of the Drosophila heart and associated
cardiac tissues. Here we establish, for the first time, a peptide and
protein compendium of the adult Drosophila heart, and assess the
extent of protein conservation with a mammalian model, further
solidifying the relevance of the Drosophila model as a surrogate for
the study of human heart disease.
Methods
Dissection of the Cardiac Tube
yw wild-type Drosophila melanogaster were raised on a standard
yeast-agar medium at room temperature. The cardiac tubes of 145
male and female adult flies, ranging from 1 to 7 weeks of age, were
dissected and exposed according to Vogler and Ocorr (2009) [8].
Briefly, flies were anesthetized and the heads, ventral thoraces, and
ventral abdominal cuticles were removed, exposing the heart
tubes. All internal organs and abdominal fat were carefully
removed leaving the heart and associated cardiac tissues.
Dissections were performed under oxygenated artificial hemo-
lymph at room temperature and all heart tubes were examined for
Figure 1. The Cardiac Tube
of Drosophila melanogaster
. Panel A. TRITC-Phalloidin labeled wild-type Drosophila heart tube and associated
structures (106 magnification). CC = conical chamber; AM = alary muscle; v = internal valve; Os = ostia in flow tract. Inset: luminal surface of TRITC-
Phalloidin-labeled myosin-GFP-expressing heart (206 magnification). Ostia inflow tracts and the striated alternating myosin and actin myofilament
bands are clearly resolved. Panel B. Electron micrograph of a longitudinal section through the conical chamber reveals the contractile myofibrils and
mitochondria (M)(3,8006). Densely stained Z-bands (Z) demarcate individual sarcomeres and bisect the I-bands. Centrally-located A-bands are also
apparent. Panel C. Cross-section through cardiac myofibrils of the conical chamber (10,5006). Individual thick filaments are surrounded by 9–11 thin
filaments. Regions of sarcoplasmic reticulum (SR) can also be resolved. Panel D. 10% Coomassie-stained polyacrylamide gel from 30 Drosophila heart
tubes. Sarcomeric myosin heavy chain (MHC) and actin are highlighted for reference.
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activity prior to removal. The conical chambers (Figure 1A) were
grasped with forceps and the hearts were gently removed and
quickly transferred to an Eppendorf tube containing 1.5 ml of
artificial hemolymph on ice. The hearts continued to beat
immediately following their removal. The tissue was pelleted
(10,000 rpm) and washed three times quickly in distilled deionized
water at 4uC. The sample was then lyophilized and the cardiac
tubes dehydrated and stored at 280uC.
Fluorescence & Electron Microscopy
Fluorescence microscopy was performed as detailed by Alayari
et al. [9]. Briefly, wild-type (yw) Drosophila hearts or hearts
expressing myosin-GFP (obtained from http://flytrap.med.yale.
edu) were labeled with TRITC-phalloidin and imaged with a Zeiss
Imager Z1 fluorescent microscope equipped with an Apotome
sliding module at 10 and 206 magnification. Electron microscopy
was performed with a Philips CM 420 electron microscope
essentially as described by Wolf et al. (2006) [13], however, prior to
fixation with Karnovsky fixative (3% formaldehyde/3% glutaral-
dehyde in 0.1 M Na-cacodylate buffer, pH 7.35), the cardiac tubes
were exposed and dissected free of extraneous debris as described
by Vogler and Ocorr (2009) [8]. Electron micrographs of semithin
sections through the conical chamber were acquired at 3,8006
and 10,5006 magnification.
Sample Preparation and Mass Spectrometry
The washed and lyophilized hearts were homogenized in
reducing SDS-sample buffer (NUPAGE, Invitrogen) containing
6M urea. Thirty (30) heart tubes provide sufficient protein to
detect and resolve the major protein constituents via denaturing
SDS-PAGE and Colloidal Coomassie Blue staining (Simply Blue,
Invitrogen). The reported dataset was obtained from homogeni-
zation of 145 hearts (,20
mg protein) and further processing with
a gel-LC-MS/MS proteomics approach. A single gel lane was cut
into 13 tranches. Each tranche was subjected to in-gel trypsinolysis
and peptide extraction by the method of Shevchenko et al. [16].
Extracted peptides were subjected to 4-replicate runs to LC-MS/
MS on a LTQ ion-trap mass spectrometer (Thermo). Details
regarding chromatography, apparatus and instrumentation set-
tings are found in Methods S1.
Database Searching
Tandem mass spectra were extracted by Bioworks 3.3. All MS/
MS samples were analyzed using Mascot (Matrix Science,
London, UK; version Mascot) and X!Tandem (www.thegpm.
org; version 2007.01.01.1). Mascot was set up to search a database
of D. melanogaster reference protein sequences (Refseq) downloaded
from the National Center for Biotechnology Information (NCBI)
in FASTA format. The database was current as of 09/24/2008
and contained 20735 entries. X!Tandem searches were conducted
using the same database. Searches were conducted using trypsin as
the digesting enzyme. Mascot and X!Tandem were searched with
a fragment-ion mass tolerance of 0.80 Da and a parent-ion
tolerance of 1.5 Da. Carbamidomethylation of cysteine was
specified in Mascot and X!Tandem as a fixed modification.
Oxidation of methionine was allowed as a variable modification.
Criteria for Protein Identification
Scaffold (version 2.02.04; Proteome Software Inc., Portland,
OR) was used to validate MS/MS based peptide and protein
identifications. Peptide identifications were provisionally accepted
if they had a .90.0% probability, as specified by Scaffold’s
implementation of the Peptide Prophet algorithm [17]. Proteins
that contained similar peptides and could not be differentiated
based on MS/MS analysis alone were grouped to satisfy the
principles of parsimony [18]. To maximize the sensitivity of
discovery, given limited starting material (145 hearts, <20 ı`g of
protein), identifications were accepted provisionally if they
contained at least 1 statistically-validated unique peptide from 1
assigned spectrum. Recent studies have demonstrated the value of
single hit protein identifications [19,20], as long as care is taken to
remove potential false-positive identifications. Specifically, proteins
identified on the basis of single spectrum/peptide matches were
inspected manually and accepted only if they: 1) were well
fragmented, displaying contiguous b- and y-ion stretches, 2)
showed complementary b- and y-ions, 3) were scored at 90%
probability by Peptide Prophet, and either 4) matched reference
spectra from the dataset of Brunner et al. archived at the National
Institute of Standards and Technologies (Tables S3, S4, S5, S6,
S7, S8), or 5) conformed with well-established peptide fragmen-
tation biases [21](Table S9).
Bioinformatic Analysis
Ontological protein classification and clustering of the Drosophila
cardiac dataset were conducted using the Database for the
Annotation, Visualization and Integration of Data (DAVID)
(http://david.abcc.ncifcrf.gov/) and ProteinCenter (Proxeon).
Ontological and functional domain comparisons between Drosoph-
ila and mouse proteomic datasets were conducted using Ontolo-
gizer 2.0- a multifunctional software tool for GO term enrichment
analysis and data exploration [22]. A discussion of the limitations
and provisos associated with such comparisons can be found in
Methods S1.
Results
Drosophila Cardiac Tubes Used In This Study
The adult Drosophila melanogaster heart tube extends medially
from the first through the sixth abdominal segment close to the
dorsal body wall [7] (Figure 1A). It consists of a single muscular
layer of circular contractile cardiomyocytes that join together to
create the heart wall, three pairs of opposing ‘‘spongy’’ internal
valve cells that project into the lumen from the wall, and five pairs
of ostial inflow tracts [5,7,23]. The anterior conical chamber, the
most pronounced muscular region of the heart tube, is ,120
mm
wide and tapers gradually through the first two abdominal
segments. The remainder of the heart tube is roughly 50
mmin
diameter along its length. In addition to the cardiomyocytes
highlighted in figure 1, the cardiac tube also closely associates with
a ventral longitudinal muscle layer, pericardial cells, extracellular
matrix and is innervated by neurons (not shown). The dissected
cardiac tubes used in subsequent proteomic studies contained all
aforementioned structures.
Overview of Proteins from Adult Drosophila Cardiac Tube
Data collected from 145 Drosophila hearts resolved by 1D-gel
electrophoresis initially yielded 1520 protein candidates that met
the minimal statistical threshold for provisional acceptance (one
peptide with .90% probability). 766 proteins were identified by at
least 2 unique high-quality peptides (.90% peptide probability)
with a protein identification probability .99.9%, on the basis of
Scaffold’s implementation of the empirical Bayesian algorithms,
Peptide Prophet and Protein Prophet, respectively [17]. To extend
the heart proteome coverage to lower abundance and shorter
proteins [19], we also considered proteins identified by a single
unique peptide as described in the methods section. The merits of
including 1-hit proteins in datasets have been addressed recently
The Drosophila Cardiac Proteome
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[19,20]. To minimize false discovery, single-peptide hits among
the 1520 provisional proteins were filtered using a stringent
multistep cross-validation process as outlined in Methods S1.
Three-step evaluation removed 292 single-hit protein candidates,
yielding a final complement of 1228 proteins clusters, identified by
5169 unique peptide matches from 29862 assigned spectra (Tables
S1, S2).
Specificity of the Drosophila Cardiac Proteome:
1. Comparison with the Extensive Drosophila Proteome
of Brunner et al [24].
To assess the tissue specificity of our
proteome we compared our dataset to the landmark work of
Brunner et al [24] (Figure 2A), an extensive Drosophila peptidome/
proteome compiled from a variety of Drosophila cell lines and body
segments. Of the 5169 unique peptides we observed (Table S1)
1293 were not found among the 72281 detected previously and
are, therefore, novel to the heart tube proteome. Importantly, only
25 peptides of the 5169 peptide matches found from searching the
Refseq database were not present in BDGP3.2 database used
previously [24]. Therefore, the bulk of the novel identified
peptides do not arise simply from the use of different databases
for analysis, but rather, stem from the use of isolated Drosophila
cardiac tubes, which had not been analyzed in the Brunner et al.
study.
The 1293 novel peptides mapped to 237 protein clusters (19%)
that were unique to our cardiac tube proteome dataset (Table S10).
Ontological enrichment analysis of the unique proteins showed that
metabolic, mitochondrial and muscle-related ontologies are more
Figure 2. Specificity of the
Drosophila
Cardiac Proteome. Panel A. The cardiac tube proteome was compared with the extensive Drosophila
proteome of Brunner et al [24]. To minimize complications arising from the use of different databases (Refseq vs. BDGP3.2), comparison at the level of
peptides is preferred. 1293 peptides, or approximately 25% of those identified in this study, were uniquely detected in our heart dataset and
ultimately mapped to 237 protein clusters that were novel to the cardiac tube dataset. Panel B. The cardiac tube proteome was cross-referenced with
the developing heart transcriptome of Zeitouni et al [25]. Protein and transcript datasets were mapped onto CG gene models to facilitate comparison
(see Methods S1).
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prominent than would be expected by chance (Benjamini-corrected
p,0.05; Figure 2B). Enriched biological processes included
carbohydrate metabolism (GO:0005975) muscle contraction
(GO:0006936) and muscle system (GO:0003012). Among enriched
molecular functions, ion pumping ATPases (GO:0042623), and
oxidoreductase activity (GO:0016491), likewise figure prominently
(see Table S10).
2. Comparison with a Drosophila Cardiac Tran-
scriptome.
As an independent assessment of specificity, we
compared our dataset with transcriptomic data from a published
study of early Drosophila heart development [25]. In their time-
course study, covering 8 time points from 21 to 48 hours after
puparium formation, Zeitouni et al. had found about 4800 gene
models to be consistently expressed above background levels (for
details, see Methods S1). 854 of our 1207 gene models (encoding
the 1228 proteins) were observed in both proteomic and
trancriptomic datasets (71%; Figure 2B). Another 285 gene
models encoding proteins found in the adult cardiac tube (24%)
were present on the microarray but not expressed above the
chosen threshold. These may represent gene models that are more
prominently expressed during later stages of heart development.
Taken together, comparison with the broader extensive Drosophila
proteome and the transcriptome of early heart development
demonstrates that dissection has successfully led to a cardiac tube-
enriched proteome.
Abundant Protein Classes in the Drosophila Cardiac
Proteome
To get a qualitative assessment of the relative abundance of
identified proteins, we examined the number of total assigned
spectra for each protein. Spectral assignments followed an 80/20
distribution, i.e. 20% of identified proteins (245) accounted for
nearly 80% (78.8%) of the total assigned spectra. Figure 3A shows
these most abundant proteins categorized manually, guided by
annotation terms available from NCBI and Flybase. Consistent
with the electron micrographs of Drosophila cardiac muscles
(Figures 1B, 1C) depicting alternating arrays of sarcomeres and
mitochondria, the list is dominated by myofilament, cytostructural
and mitochondrial proteins. Myosin heavy chain alone, owing to
its abundance and high molecular weight, accounts for fully 10%
of all assigned spectra. The most abundant myofilament and
cytostructural proteins, together, account for 31% of assigned
spectra among the top 245 proteins (23% and 8% respectively).
Mitochondrial proteins were also among the most abundant.
Spanning diverse functions including fatty-acid oxidation, tricar-
boxylic acid (TCA) cycle and oxidative phosphorylation, they also
accounted for about 31% of the spectra. Proteins of the basal
lamina that provide structural integrity of the cardiac tube,
including several laminins and collagens, accounted for a further
12%. Other noteworthy classes include the proteins involved with
protein synthesis, ion transport, heat-shock response and carbo-
hydrate metabolism. Individual proteins within each group are
shown in Table S11.
Functional Annotation Enrichment Analysis
To determine which biological functions were over-represented
in our cardiac-tube dataset, we undertook functional annotation
and enrichment analysis using tools available from DAVID
[26,27]. By this ontological analysis, each entry was categorized
according to their biological processes, cellular components, and
molecular functions (Table S12). Functional clustering and
enrichment analysis found that approximately 928 of the 1228
proteins could be classified into 47 functional categories. Figure 3B
lists functional annotation clusters, ranked by degree of enrich-
ment within the dataset. Notably, ribosomal and mitochondrial
ribosomal functional annotations were particularly over-represent-
ed. Consistent with our assessments of protein abundance,
functions commonly associated with mitochondria were also
over-represented, commensurate with the high energy demands
of this myofilament rich contractile tissue (Fig. 1B, 1C, 3A). Ca
2+
-
binding proteins and proteins with thioredoxin-folds are highly
enriched, attesting to the importance of Ca
2+
handling and redox
regulation in the Drosophila heart. Likewise, proteins involved in
protein folding (chaperones and cyclophilins), glycolysis, and
varied oxidoreductase enzymes figure prominently by this
measure. Among signaling proteins, those of the low molecular
weight ras-like GTPase superfamily are well represented, including
ras, several rab proteins, rac1, rho1 and cdc42. Kinases identified
include Ca-calmodulin dependent kinase II, casein kinase,
integrin-linked kinase and pyruvate dehydrogenase kinase.
The Drosophila Cardiac Proteome in Context
1. Identification of Cardiac Proteins Essential for Fly
Survival/Orthologs of Vertebrate Proteins Critical for
Heart Function.
We recently performed a genome-wide
RNAi screen to identify conserved cardiac genes whose products
are essential for Drosophila survival under conditions of stress [14].
Heart-restricted silencing of 498 genes significantly increased
mortality when the flies were exposed to elevated temperatures.
These gene candidates, when knocked down, likely result in severe
cardiac functional abnormalities since the Drosophila heart can be
dramatically altered and not necessarily initiate organismal death.
Seventy-four (74) of these vital 498 genes (15%) had protein
products detectable in our proteome. Furthermore, 73% of the 74
genes corresponded to orthologs found in the cardiovascular
system of vertebrates (humans and/or mice; determined via the
NextBio web-based platform (http://www.nextbio.com/b/
nextbio.nb)) and, 40% have orthologs implicated in diverse
cardiac related disorders including cardiomyopathy, myocardial
infarction, cardiac arrest and heart failure (See Table S13).
2. Comparison with the Mouse Heart Dataset. To assess
the similarity between Drosophila and mammalian hearts, we
compared the functional ontological profile of its proteome with
that of a reference heart dataset from mouse (Figure 4). Analysis of
the Drosophila cardiac proteome revealed 866 protein family (Pfam)
domains. Of these, 706 (82%) were conserved in the mouse heart
(Table S13). Comparison of gene-ontology annotations is
summarized in Figure 4. Note the similarities between the
Figure 3. Annotation and Classification of the
Drosophila
Cardiac Proteome. Panel A By abundance: Using the number of assigned spectra as
a measure of relative protein abundance, the top 245 proteins (20%) were annotated manually with information from NCBI and Flybase. Total
assigned spectra within a group are expressed as a percentage of the total number of spectra assigned to the top 245 proteins. The chart provides a
measure of the relative abundance of proteins that comprise each group. Panel B. By clustering & enrichment of gene-ontology terms: 928 proteins for
which functional annotation was available among the 1228 proteins were subjected to functional clustering and enrichment analysis using the
Functional Classification tool at the DAVID knowledgebase. Approximately 600 proteins were grouped into 47 functional classes based on the
similarity of their gene-annotations. The annotation clusters are ranked by their enrichment score (2log(p-value)). The number of proteins per cluster
is indicated in parentheses. A score of .2() denotes high probability that a class is enriched.
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Drosophila cardiac tube and the mouse heart at the level of cellular
components, biological processes and molecular function (color-
matched ontology terms). GTPase, oxidoreductase and other
mitochondrial activities dominate the molecular function category
in both the Drosophila cardiac tube and the mouse heart. Among
biological processes, the enrichment of terms associated with
protein synthesis (translation, translational elongation) in the
Drosophila cardiac tube is mirrored in the mouse heart. Glycolysis
and ATP synthetic processes are likewise highly-enriched in both
species. Annotations of the cellular component category reaffirm
what could be deduced from microscopy, namely that the cardiac
tube and the mouse heart are dominated by myosin complexes (i.e.
myofilaments) and mitochondria.
Our proteome is ontologically distinct from other smaller
Drosophila proteomic datasets (e.g. Drosophila seminal fluid [28]; not
shown) though it is similar to that of other mitochondria-rich
organ sets, such as mouse liver [29]. The liver dataset, however,
lacks the structural and myofilament protein complement -major
components observed in mouse heart and the Drosophila cardiac
tube. Note p-values in Figure 4 are not directly comparable
between Drosophila and mouse datasets. They are indicative of
enrichment within each dataset only. Finally, at this stage, it would
be premature to attribute ontological differences (grey) to bona fide
biological differences between the two species, since they might
well stem from the differences in methodology and instrumenta-
tion bias (see Methods S1).
Drosophila Cardiac Peptidome: A Resource for Multip le-
Reaction-Monitoring Mass Spectrometry
One of the goals of quantitative proteomics is to robustly assess
the levels and even the posttranslational status of any protein, or
group of proteins, in the cell at a given time. Traditional shotgun
proteomic strategies that identify as many proteins as possible from
an enzymatic protein digest suffer the inevitable shortfall that low-
abundance proteins are systematically under-sampled, making
quantification difficult. One technology that promises to yield
more sensitive protein maps has been used for the quantitative
mass spectrometry of small molecule analytes for years. Known as
single reaction monitoring (SRM) or multiple reaction monitoring
(MRM) [30], it offers up to 100-fold greater sensitivity than
shotgun proteomic approaches [31], and is uniquely suited for the
targeted quantification of specific known peptides, based on
characteristic chromatographic retention time, parent ion masses,
and MS
2
-ion transitions. Yet, before MRM approaches can be
successfully applied, certain criteria must be met. Firstly, of the
tryptic peptides observable theoretically, only a fraction has
physicochemical properties that favor detection by mass spec-
trometry. Secondly, even fewer peptides lend themselves to
unambiguous protein identification. Types of peptides range from
those that uniquely identify a specific protein isoform from a single
gene, to those that arise from multiple unrelated proteins and
therefore provide little protein/gene information.
To extract peptide-protein-gene model relationships, we
subjected our 5169 unique cardiac tube peptides to a Peptide-
Classifier analysis according to Qeli and Ahrens [32], and ranked
them in order of information content (Table S14). Of the 5169
peptides, 4984 were classified as either ‘‘proteotypic’’ or
‘‘information-rich’’. Proteotypic peptides provide sufficient infor-
mation to distinguish specific protein isoforms (Classes 1a, 1b, and
3a in Table 1). Information-rich peptides are common either to a
subset, or to all protein isoforms encoded by a specific gene model
(Classes 2a and 2b in Table 1). Particularly noteworthy are the
3112 proteotypic peptides among which 774 are newly-identified.
Moreover, the remaining 2338 peptides identified previously in
Drosophila cell lines and body segments [24], can now be assigned a
role in the heart (Table 1).
Discussion
As Drosophila melanogaster is used increasingly as a model of heart
disease, it behooves us to characterize its cardiac tube more fully,
to better gauge the prospects and limitations of the system.
Specifically, extending new insights from Drosophila to mammals
demands a better understanding of the similarities between these
hearts at a molecular level. Since the heart is, in part, the sum of its
protein components, we undertook a proteomic approach.
Here, we have shown that the Drosophila cardiac proteome
conforms, in terms of protein abundance and functional
enrichment, to what one might expect given its ultrastructure by
electron microscopy. But more importantly, the classes of proteins
identified and enriched in our dataset mirror those found in a
recently published comprehensive mouse heart proteome [33],
which we used as a benchmark. Specifically, we note the
similarities at the level of myofilament, structural and mitochon-
drial function. Moreover, Drosophila hearts share the redox
buffering and Ca
2+
-handling proteins found in mammalian hearts.
The comprehensive mouse heart proteome does include proteins
under-represented in our dataset, however, notably kinases,
certain ion channels and transmembrane receptors. We suspect
this could well stem from differences in methodology, as the mouse
hearts were fractionated into their subcellular components prior to
analysis, which would favor identification of lower abundance
proteins from the cytosol and membranes. Efforts are currently
underway to identify under-represented Drosophila protein classes
whose presence is predicted by preliminary cardiac transcriptome
work (AC, NA, RB, SIB, DBF unpublished).
Genetic lesions expressed in the Drosophila cardiac tube are
already revealing remarkable parallels with human heart disease.
We recently demonstrated that knockdown of CCR4-Not
components in Drosophila and in mice resulted in cardiomyopathy
and heart failure and that a common NOT3 SNP (rs36643) in
humans correlates with altered cardiac QT intervals, a frequent
cause of sudden cardiac death [14]. The degree of protein
conservation observed here suggests that Drosophila heart studies
will continue to provide a convenient extension of widely-used
genetic mouse models of heart disease and provide translatable
insights into human cardiac dysfunction. For example, identifica-
tion of rac1 in the Drosophila heart suggests that it may provide a
valuable model to complement mouse studies of rac1-mediated
hypertrophy [34,35].
Figure 4. Comparison of the
Drosophila
and Mouse Heart Proteomes. Functional descriptions of protein domains (as defined by the Pfam
database [42]) of the Drosophila cardiac proteome (left) and the mouse heart proteome (right) were subjected to Gene-ontology term enrichment
analysis using Ontologizer 2.0 software [22] with the Topology-Elim algorithm [43] and Bonferroni correction. The table is laid out according to the
three branches of ontology: Molecular Function, Biological Process or Cellular Component. Annotation terms within each section are listed in
descending order of enrichment (lowest p-values at the top). Within each branch of ontology Drosophila terms are color coded from red (lowest
p-value) to blue (highest p-values). These colors were mapped onto related ontological terms found in the mouse to highlight commonalities (colors)
and differences (grey).
doi:10.1371/journal.pone.0018497.g004
The Drosophila Cardiac Proteome
PLoS ONE | www.plosone.org 8 April 2011 | Volume 6 | Issue 4 | e18497
The conservation between the Drosophila and mouse heart
proteomes also bodes well for the implementation of systems
biology approaches that include, among other techniques,
computational modeling and proteomic network perturbation, to
assess mechanistic commonalities between these model organisms.
For instance, it will be important to test whether Drosophila cardiac
function can be adequately described by the latest models of
excitation-contraction coupling integrated with mitochondrial
energetics (ECME) [36]. Our Drosophila proteome identifies and
provides unique mass spectral signatures for many of the proteins
integral to the model. These include major determinants of
intracellular calcium regulation (voltage-gated Ca
2+
channels,
ryanodine receptors, SERCA, and PMCA), K
+
and Na
+
(Na
+
/K
+
ATPase, Na
+
/Ca
2+
exchanger), NADH production (TCA cycle
proteins) and ATP levels (adenylate kinase, ATP synthase) (see
Table S13).
Finally, compendia of experimentally observable isoform-
specific peptides will be highly valued resources as we strive
toward the goal of complete proteome coverage. Mass spectrom-
etry techniques such as multiple-reaction monitoring are among
those at the forefront of quantitative proteomic approaches [30]
whose experimental design benefit greatly from observed peptide-
spectrum matches. In this study, we have classified and ranked all
5169 identified peptides in order of the information they impart
about a gene-model. Fully 3112 of these peptides were mapped to
specific protein isoforms found in the cardiac tube. This peptide
set will serve as an excellent complement to current proteotypic
peptide prediction algorithms [37,38] as well as existing peptide
repositories such as PeptideAtlas [39], and should thereby expedite
efforts to quantify of particular proteins of interest in the Drosophila
heart by MRM.
In summary, the present study provides the first demonstration
that proteomic studies are possible in Drosophila hearts. Though the
extent of proteome coverage lags that of the well-studied mouse
heart (4906 proteins) [33], through a combination of extensive
dissection (100 Drosophila hearts can be harvested in a day) and
careful data validation, we have compiled 1228 protein clusters
from an organ whose mass is about 1/10
6
that of mouse heart.
Ongoing efforts using new strategies and instrumentation
platforms will seek to extend peptidome/proteome coverage while
reducing the number of hearts required as we lay the framework
for quantitative protein-network approaches [40,41] to study
cardiomyopathy in Drosophila.
Supporting Information
Table S1 Proteins & Peptides Panel 1: Read Me covers
caveats associated with using these table(s) Panel 2: List of 1228
protein clusters, with identification probability and other buttress-
ing peptide information. Panel 3: List of all peptides identified and
associated with given protein isoform or protein cluster. Panel 4:
List of all unique (non-redundant) peptides identified in this study.
Panel 5: List of human homologues of identified Drosophila protein
clusters. Homologues were found using batch searches of
Homologene and Ensemble Compara databases. Panel 6:
Distribution of spectra among the 4 technical replicates associated
with the identified proteins.
(XLSX)
Table S2 Assigned Spectra Panel 1. Read Me covers caveats
associated with using these table(s). Panel 2: List of each spectrum
file assigned to a given peptide.
(XLSX)
Table S3 Spectra ST Matches All proteins identified on the
basis of a single peptide, regardless of the number of assigned
spectra, were cross-referenced against a reference Drosophila
peptide dataset as described in the Methods section and Methods
S1. Panel 1: The specific criteria for inclusion in these tables are
presented. All other panels: Output from Spectra ST searches
showing matches between our query spectra and the reference
spectra.
(XLSX)
Table S4 Spectra ST Matches All proteins identified on the
basis of a single peptide, regardless of the number of assigned
spectra, were cross-referenced against a reference Drosophila
peptide dataset as described in the Methods section and Methods
S1. Panel 1: The specific criteria for inclusion in these tables are
presented. All other panels: Output from Spectra ST searches
Table 1. Drosophila Cardiac Peptidome.
Peptide Class
1
Type of Peptide Evidence
# Identified
Peptides (%) New Peptides
Class 1a identifies one protein - one gene-model 2316 (44.8) 627
Class 1b identifies one protein - encoded by isoforms differing in 59 or
39 UTR of one gene model
783 (15.1) 146
Class 2a identifies a subset of protein isoforms 249 (4.8) 95
Class 2b common to all protein isoforms encoded by a gene-model 1623 (31.4%) 377
Class 3a identifies one protein from multiple gene-models 13 (0.3%) 1
Class 3b peptides common to unrelated proteins 185 (3.6) 47
Proteotypic Peptides
1
Information-Rich Peptides
1
(Class 1a+Class 1b+Class 3a) (Class 2a+Class 2b)
Total: 3112 Total: 1872
New: 774 New: 472
1
See Table S14.
Peptides identified in a shotgun proteomics experiment may be classified into 6 types on the basis of the information they impart about a gene model [32]. Proteotypic
peptides are those that uniquely identify a specific protein isoform and may be encoded by multiple transcripts or multiple genes. Information-rich peptides are shared
among protein isoforms arising from multiple transcripts or genes. Proteotypic peptides are particularly useful for the design of new high-sensitivity quantitative mass
spectrometry methods based on multiple-reaction monitoring. New peptides were not previously in the Drosophila peptide compendium of Brunner et al. [24].
doi:10.1371/journal.pone.0018497.t001
The Drosophila Cardiac Proteome
PLoS ONE | www.plosone.org 9 April 2011 | Volume 6 | Issue 4 | e18497
showing matches between our query spectra and the reference
spectra.
(XLSX)
Table S5 Spectra ST Matches All proteins identified on the
basis of a single peptide, regardless of the number of assigned
spectra, were cross-referenced against a reference Drosophila
peptide dataset as described in the Methods section and Methods
S1. Panel 1: The specific criteria for inclusion in these tables are
presented. All other panels: Output from Spectra ST searches
showing matches between our query spectra and the reference
spectra.
(XLSX)
Table S6 Spectra ST Matches All proteins identified on the
basis of a single peptide, regardless of the number of assigned
spectra, were cross-referenced against a reference Drosophila
peptide dataset as described in the Methods section and Methods
S1. Panel 1: The specific criteria for inclusion in these tables are
presented. All other panels: Output from Spectra ST searches
showing matches between our query spectra and the reference
spectra.
(XLSX)
Table S7 Spectra ST Matches All proteins identified on the
basis of a single peptide, regardless of the number of assigned
spectra, were cross-referenced against a reference Drosophila
peptide dataset as described in the Methods section and Methods
S1. Panel 1: The specific criteria for inclusion in these tables are
presented. All other panels: Output from Spectra ST searches
showing matches between our query spectra and the reference
spectra.
(XLSX)
Table S8 Spectra ST Matches All proteins identified on the
basis of a single peptide, regardless of the number of assigned
spectra, were cross-referenced against a reference Drosophila
peptide dataset as described in the Methods section and Methods
S1. Panel 1: The specific criteria for inclusion in these tables are
presented. All other panels: Output from Spectra ST searches
showing matches between our query spectra and the reference
spectra.
(XLSX)
Table S9 High-Quality Spectra Proteins identified on the
basis of high quality spectra, as defined in the Methods section
and Methods S1, but for which no match could be found using
Spectra ST. Panel 1. The specific criteria for inclusion in these
tables are presented. All other panels: High-quality spectra are
presented, along with the explicit attributes of the spectra that
conform to established CID-induced fragmentation biases.
(XLSX)
Table S10 Proteins Absent from the Dataset of Brunner
et al . Panel 1: Protein isoforms and clusters were mapped to their
CG identifiers and screened against the dataset of Brunner et al.
[24]. Overlap is designated with ‘‘1’’ in column C; proteins unique
to our study are designated ‘‘0’’. Panels 2–4: Analysis of the
proteins unique to our dataset with respect to the three branches of
gene-ontology. Panel 2: Biological Processes. Panel 3. Cellular
Components. Panel 4. Molecular Functions.
(XLSX)
Table S11 Relative Protein Abundance List of proteins that
comprise the functional classes depicted in Figure 3A. These 245
proteins represent the most abundant proteins, comprising 20% of
the identified protein isoforms or clusters and nearly 80% of all
assigned spectra.
(XLSX)
Table S12 Functional Annotation and Enrichment Panel
1: Read Me covers caveats associated with using these table(s)
Panel 2: Functional classification of identified Drosophila heart
proteins using DAVID as described in the Methods section and
Methods S1, in a sortable format. Panel 3: Sorted by enrichment
score. Panel 4: Complete GO annotation for identified proteins.
(XLSX)
Table S13 Cardiac Proteins Essential for Fly Survival,
Orthologs of Vertebrate Proteins Critical for Heart
Function, Pfam Analysis of Drosophila and Mouse
Heart Proteomes and Proteins of Interest The Drosophila
cardiac proteome was compared with the work of Neely et al.
[14] 74 identified proteins overlap with the 498 cardiac genes
deemed essential for fly survival. Panel 1: Mapping the human
and mouse orthologs of these proteins. Information includes the
tissue distribution, disease-association and functional classifica-
tion of these orthologues. Panel 2: GO annotation of the 74
overlapping proteins. Panel 3: Graphical representation of the
preponderance of functional classes represented by the 74
overlapping proteins. Panel 4: Pfam domains represented in the
Drosophila cardiac dataset. Panel 5: Pfam domains represented in
the Mouse heart dataset of Bousette et al. [33]. Panel 6: Proteins
in the Drosophila cardiac dataset with multiple isoforms. Panel 7.
Listing of the major myofilament proteins identified. Panel 8:
Proteins of interest with respect to mathematical models of
cardiac function.
(XLSX)
Table S14 Drosophila Cardiac Peptidome Panel 1:
Proteotypic peptides (as defined in the text) that unambiguously
identify a specific protein isoform. Panel 2. Information-rich
peptides (defined in text) that can be used to identify multiple
protein isoforms. Panel 3. PeptideClassifier analysis of all 5169
unique (non-redundant) peptides.
(XLSX)
Methods S1 This supplement contains detailed description of
experimental methods and apparatus.
(DOCX)
Acknowledgments
We thank D. Kent Arrell Ph.D (Mayo Clinic) for helpful discussions and
critical reading of early versions of the manuscript. Special thanks are
extended to Dr. Erich Brunner (Institute of Molecular Life Sciences,
University Zurich) and Dr. Ruedi Aebersold (Institute of Molecular
Systems Biology, Zurich), for sharing proteomic data, and to Dr. Laurent
Perrin (Developmental Biology Institute, Marseille, Luminy) for sharing
transcriptomic data. This paper is dedicated to the memory of Mary C.
Reedy.
Author Contributions
Conceived and designed the experiments: AC DBF. Performed the
experiments: AC NNA JR MCR MG DBF. Analyzed the data: AC CHA
EQ CMZ DBF. Contributed reagents/materials/analysis tools: CHA EQ
RNC JVE RB BOR SIB. Wrote the paper: AC CHA DBF. Edited the
paper: EQ CMZ MG RNC JVE RB BOR SIB.
The Drosophila Cardiac Proteome
PLoS ONE | www.plosone.org 10 April 2011 | Volume 6 | Issue 4 | e18497
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The Drosophila Cardiac Proteome
PLoS ONE | www.plosone.org 11 April 2011 | Volume 6 | Issue 4 | e18497
    • "The orthologs of PDCD10 are found in Caenorhabditis elegans and Dr. melanogaster as ccm-3 and Ccm3, respectively. Ccm3 is a protein-coding gene with unknown function but is expressed in the caudal region of the adult dorsal vessel of fruit fly (Cammarato et al. 2011). In short, PDCD10 could be involved in the development of mammal heart, and its ortholog in fruit fly was reported to be expressed in the organ that acts as insect's heart. "
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    • "Previous reports about similarities of CaMKII in Drosophila and mammals have referred to its presence and functionality in brain [37], [38]. More recently, a CaMKII isoform has been detected in Drosophila heart through mass spectrometry [39]. However, a possible role of CaMKII in Drosophila heart was not identified. "
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    • "Our results identified 502 protein clusters from purified cardiomyocyte nuclei. We note that this number of proteins is less than reported for cardiac protein profiling in the adult fly or mouse heart (Bousette et al., 2009; Cammarato et al., 2011). However, there are several possible reasons for this discrepancy. "
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