Proteomic Analyses of the Developing Chicken Cardiovascular System Els Bon,‡ Regine Steegers,‡,§ Eric A. P. Steegers,‡ Nicolette Ursem,‡ Halima Charif,† Peter C. Burgers,† Theo M. Luider,*,† and Lennard J. M. Dekker† Laboratories of Neuro-Oncology & Clinical and Cancer Proteomics, Department of Neurology, Erasmus MC, The Netherlands, Department of Obstetrics and Gynecology, Division of Obstetrics and Prenatal Medicine, Erasmus MC, Rotterdam, The Netherlands, and Department of Epidemiology, Department of Pediatrics, Division of Pediatric Cardiology, and Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands Received July 13, 2009
Up until today, no proteomics approaches have been described for heart muscle development. We describe a proteomics method to study the proteome of different heart structures at three stages of chicken embryonic development. For this purpose, a combination of gel separation, nanoLC separation and mass spectrometry was used. With this method, we identified in total 267 proteins in different tissue structures of chicken heart. We observed differences in protein abundance for a number of proteins between the different tissue structures and time points of development using spectral counting as a semiquantitative measure of protein abundance. For myosin-heavy chain 6, myosin-heavy chain 7, titin, connectin, collagen alpha-1, and xin, differences in protein levels for the different stages and structures (great arteries, outflow tract and ventricles) have been observed. A pathway analysis is performed in which the identified proteins are related to theoretical protein networks. Most prominent was the ‘cardiovascular system development and function’ network with the abundantly present proteins myosin 6 and myosin 7. We showed that myosin 6 is highly regulated in a stage and heart tissue specific manner. In conclusion, this method can be used to study changes in protein levels of chicken heart tissue in a spatiotemporal manner. Keywords: proteomics • FTMS • Orbitrap • chicken embryo • heart development • muscle development
Introduction The heart is one of the first functioning organs in the developing embryo. Heart development is a strictly orchestrated process. During folding of the heart tube, various tissues differentiate into atria, ventricles and outflow tract. The macroscopical changes in heart development can be expected to reflect a specific expression of proteins in these diverse heart tissues. To observe the changes in the proteome of the heart during development, different mass spectrometry based techniques can be used. The chicken embryo is a frequently used model to study heart development1 because of the high comparability of avian and human cardiac development. Therefore, it is often chosen as a model to study the pathogenesis of congenital heart defects. Several studies have shown that the chicken embryo model is useful to study the effects of various compounds that interact with cardiac neural crest cell migration and other * To whom correspondence should be addressed. Th. M. Luider Ph.D., Erasmus MC, University Medical Center, Department of Neurology, Lab. Neuro-Oncology, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.Phone:+31107038069.Fax:+31107044365.E-mail:
[email protected]. ‡ Department of Obstetrics and Gynecology, Erasmus MC. § Departments of Epidemiology, Pediatrics, and Clinical Genetics, Erasmus MC. † Department of Neurology, Erasmus MC.
268 Journal of Proteome Research 2010, 9, 268–274 Published on Web 10/29/2009
biological processes implicated in normal heart development (homocysteine, retinoic acid, valproic acid).2-4 However, until now, no proteomics research has been performed on chicken embryonic heart. Some proteomics studies have been performed on cerebrospinal fluid,5 retina,6 liver7 and facial development of the chicken embryo. In one study, a complete embryo at Hamburger-Hamilton (HH)8 stage 29 was analyzed with proteomic techniques.9 Most of these studies are focused on one specific tissue in one or two stages of development. In this study, we investigated changes in the proteome of three different heart tissue structures, that is, great arteries, ventricles and outflow tract, throughout three stages of embryonic development aiming to identify proteins that are specific in a spatiotemporal manner for chicken heart development. To do so, we used a mass spectrometry based proteomics method to be able to study a large number of proteins simultaneously. We performed one-dimensional electrophoresis to separate proteins and subsequent nanoliquid chromatography mass spectrometry for identification. We selected a set of intense stained gel bands for FTMS analysis. By comparison of the spectral counts of the identified proteins, we could determine the relative abundance levels of a large number of proteins. We demonstrate here that information 10.1021/pr900614w
2010 American Chemical Society
Proteomic Analyses of the Chicken Embryonic Heart
Figure 1. Photographs of chicken hearts at stage HH26, HH30, or HH36 of development in ventral and dorsal view. Lines in the figure represent the cutting lines along where the tissues have been cut. Tissue of the (future) great arteries (GA), outflow tract (O) and ventricle (V) were separated and further processed separately (atrial tissue ) A).
about changes during development in protein pathways involved in heart muscle development can be obtained by this approach.
Materials and Methods Tissue Preparation. Fertilized White Leghorn eggs (Gallus gallus (L.)) (Drost Loosdrecht BV, Loosdrecht, The Netherlands) were incubated at 38 °C at a relative humidity of 70-80%. At stage HH 26; (∼day 5 of development), HH30/31 (∼day 7 of development) or HH36 (∼day 10 of development) embryos were taken from the egg and the heart was dissected under a binocular microscope. To remove excess blood, the isolated heart was left pumping in Locke solution (0.94% NaCl, 0.0045% KCl, 0.004% CaCl2 (w/v) in Milli-Q) for a short period of time (approximately 1 min). The heart was chilled on ice, and rinsed with cold Locke solution. Subsequent preparation of tissues was performed at 0 °C. Atrial tissues were removed and the vessels of the outflow tract, the ventricles and intermediate area of the heart were dissected from each other (see Figure 1). The separated tissues were rinsed once with cold Locke solution and transferred directly into 0.5 mL of LoBind Eppendorf tubes (Eppendorf, Hamburg, Germany). Tissues were crushed with a glass rod on ice and immediately frozen on dry ice. Tissues were stored at -80 °C until further use. Protein Quantification. Tissues were thawed on ice, and depending on the amount of tissue collected, either 20 µL (tissues of HH26 and HH30/31) or 50 µL (tissues of HH36) of reagent was added (Reagent 2, ReadyPrep Extraction Kit;
research articles BioRad Laboratories, Hemel Hempstead, U.K.). Proteins from the tissues were dissolved by an Ultrasonic Disruptor Sonifier II (Model W-250/W-450; Bransons Utrasonics, Danbury, CT) for 2 min at 70% amplitude with cooling set at -11 °C. The tissues were immediately heated to 99 °C for 5 min to stop proteolytic activity and subsequently cooled down to room temperature. For protein quantification, we used a Pierce BCA protein assay (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s guidelines. The quantification required a small amount of the tissues; the remainder of the tissues was stored at -80 °C. For subsequent sample preparation, the remaining tissues were thawed on ice, and depending on the measured protein content, 5 tissues with the same characteristics for stage and location were combined to form one sample containing approximately 30 µg of protein. Protein Electrophoresis. To each sample, 10 µL of loading buffer (3.8 mL of Milli-Q, 1 mL of 0.5 M Tris-HCl, pH 6.8, 0.8 mL of glycerol, 1.6 mL of 10% SDS, 0.4 mL of 2-mercaptoethanol, and 0.4 mL of 1% bromophenol blue) was added and samples were heated for 5 min at 95 °C. One-dimensional electrophoresis of the samples was performed on a 10% SDSPAGE gel according to the manufacturer’s recommendations (PROTEAN II xi Cell; BioRad Laboratories). The gel was stained with PageBleu protein staining solution according to the manufacturer’s instructions (Fermentas GmbH, St. Leon-Rot, Germany). Another gel was prepared on which sample of stage HH36 ventricular tissue was loaded three times to determine reproducibilityofsamplepreparationincludingmassspectrometry. In-Gel Digestion and Sample Preparation. PageBleu stained protein bands were manually excised using a glass Pasteur pipet. One hundred microliters of Milli-Q was taken up with this pipet, and subsequently half of a protein band was excised. Plugs were transferred into 1.5 mL Eppendorf tubes by expelling the Milli-Q with the excised plugs from the pipet. Each excised plug was washed with 100 µL of Milli-Q for 5 min with shaking. Gel plugs were twice destained with 0.4% (w/v) ammonium hydrogen carbonate (Sigma-Aldrich Chemie BV), 30% acetonitrile (Biosolve, Valkenswaard, The Netherlands) in Milli-Q for 20 min at room temperature with a shaking platform (Eppendorf heating/shaking block). Gel-plugs were quickly washed with Milli-Q and dried in a rotary evaporator (Savant, Farmingdale, NY) for 45 min. Protein digestion was performed by addition of 4 µL of 0.1 µg/µL trypsin mass spectrometry grade (Promega, Madison, WI) to each gel plug and incubation overnight at room temperature. Thereafter, to each gel plug 50 µL of 50% acetonitrile, 0.1% trifluoroacetic acid (Biosolve, Valkenswaard, The Netherlands) in Milli-Q was added, sonified for 2 min and subsequently incubated for 15 min at room temperature. The supernatant was transferred to a new Eppendorf tube, and this process was repeated three times. The collected supernatants (150 µL) were dried in a rotary evaporator. The dried tryptic digested peptides were stored at -80 °C until further use. Mass Spectrometry. For Orbitrap mass spectrometry measurements, lyophilized samples were dissolved in 15 µL of 0.1% TFA in water. Five microliters of peptide sample was injected on to a nanoliquid chromatography system (nanoLC Ultimate 3000; Dionex, Sunnyvale, CA). After preconcentration and washing of the sample on a C18 trap column (1 mm × 300 µm i.d.), peptides were separated on a C18 PepMap column (150 mm × 75 µm internal diameter) (Dionex, Amsterdam) using a linear 90 min gradient (4-40% acetonitrile/H20 0.1% formic acid) at a flow rate of 250 nL/min. The separation of the Journal of Proteome Research • Vol. 9, No. 1, 2010 269
research articles peptides was monitored by a UV detector (absorption at 214 nm). The nanoLC was coupled with a nanospray source of a linear ion trap Orbitrap (LTQ-Orbitrap) mass spectrometer (LTQ Orbitrap XL, Thermo Electron, Bremen, Germany). All samples were measured in a data dependent acquisition mode. Before each run, a blank MS run was performed to monitor system background. The peptide masses are measured in a survey scan with a maximum resolution of 30 000 in the Orbitrap. To obtain a maximum mass accuracy, a prescan is used to keep the ion population in the Orbitrap for each scan approximately the same. During the high-resolution scan in the Orbitrap, the 5 most intense monoisotopic peaks in the spectra were fragmented and measured in the linear ion trap. The fragment ion masses are measured in the linear ion trap to have a maximum sensitivity and the maximum amount of MS/MS data. Protein Identification. From the raw data files of the FT tandem mass spectrometer, MS/MS spectra were extracted by Mascot Deamon version 2.2.2 using the Xcalibur extract msn tool (version 2.07) into mgf files. All mgf files were analyzed using Mascot (Matrix Science, London, U.K.; 2.2). Mascot was set up to search the IPI.CHICK.v3.12 database (version 3.12, 28 229 entries) assuming trypsin digestion. The Mascot search engine was used with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 10 ppm. Oxidation of methionine was specified in Mascot as a variable modification. The Mascot server was set up to display only peptide identifications with Mascot ion scores greater than 25. Scaffold (version Scaffold_2_02_03, Proteome Software, Inc., Portland, OR) was used to summarize and filter the MS/MS based peptide and protein of all the measurements results obtained by Mascot Deamon. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm.10 Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.11 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped. Spectral counts of proteins identified using the above-mentioned criteria were used for semiquantitative analysis of the various tissue types and development stages. This results in a threshold for the spectral count of at least two spectral counts. Venn diagrams were made based on the identified proteins in the different tissue types and development stage in Scaffold software (Proteome Software, Portland, OR). Ingenuity Pathway Analysis. Ingenuity Pathway (Ingenuity Systems, Inc., Redwood City, CA) analysis was performed with version IPA 7.1. The conversion tool used for IPI code to Uniprot code is http://www.ebi.ac.uk/Tools/picr/.
Results Differences in protein expression between the different stages and tissues were observed on the SDS/PAGE gel level (Figure 2). PageBleu staining intensity is different for multiple bands at various molecular weights. Bands A and B at molecular weight 220 kDa (Figure 2) show differences in staining intensities as a function of differentiation stage and the three different embryonic heart tissue structures. Furthermore, the intensity of the staining in bands A and B increases related to development of the three embryonic differentiation stages investigated. Bands A and B show a similar staining intensity and are close 270
Journal of Proteome Research • Vol. 9, No. 1, 2010
Bon et al.
Figure 2. Image of the PageBleu stained SDS-PAGE gel visualizing the proteins for various stages and tissues of the chicken embryonic heart. Gel plugs were taken from each sample from bands at level A, B and C. Abbreviations: GA, great arteries; V, ventricles; O, outflow tract.
to each other on the gel. From both bands (A and B), gel plugs were excised and analyzed separately to determine to which extent there was overlap in proteins. In addition, band C, also an intense stained band that did not show large differences in PageBlue staining for the different stages and tissue structures, was excised. These three bands (A, B, and C) were in a random order measured by mass spectrometry in one consecutive measurement session. The complete series of samples were measured using the same analytical column to minimize technical variations. Between samples, wash runs were included to minimize carryover. These runs are measured by mass spectrometry and were used to determine the background of each individual sample. The sample of stage HH36 ventricular tissue was used to investigate the reproducibility of the sample preparation and mass spectrometry. This sample was selected because enough material at this development stage could be obtained to run it three times on gel. Gel bands at the same height as band C were excised and prepared for nanoLC Orbitrap analysis. A database search resulted in the identification of 148 proteins of which 105 were identified in each of the three samples (on average 127 identified proteins per sample). The CV for the spectral counts of these 105 proteins was on average 14%. The proteins that could not be identified in each of the three samples have typically only 2 or 3 spectral counts. The highest number of spectral counts of a protein that is not identified in all three samples is 4. Orbitrap mass spectrometry measurement of the gel plugs of all the stages and tissue types resulted in the identification of 267 proteins of which one protein was only present in the blank wash runs, resulting in the identification of 266 proteins in bands A, B, and C combined. In Table 2 (Supporting Information), a complete overview of all 267 proteins is given for the three stages and the three heart tissue structures. The Orbitrap measurement of band C of stage HH30/31 outflow tract tissue was excluded from analyses. This measurement was interrupted for technical reasons resulting in a partial measurement of the sample and a significant lower number of identified proteins compared to the other gel bands. Bands A and band
research articles
Proteomic Analyses of the Chicken Embryonic Heart
Figure 3. Venn diagrams indicating the overlap in identified proteins. Venn diagrams A and B show the overlap in identified proteins for the different developmental stages and different tissues. The numbers in parentheses indicate the total number of identified proteins in the samples of a specific day or tissue.
B show, as can be expected, a large overlap in identified proteins (n ) 54). The overlap of identified proteins between band C and band A and B combined is in total only 18 proteins (band A + B + C, 15; band A + C, 2; band B + C, 1). Further examination of these overlapping proteins showed that these proteins are not specific for a gel band and can also be observed in a gel band on which no protein has been loaded (negative control) (n ) 9), or keratins that can be marked as apparent contaminations (n ) 3), or proteins that are also present in the blank wash runs (n ) 6). When the identifications of band A and B are combined and compared to band C for tissue structure and developmental stage, band C shows a higher overlap in the number of identified proteins between especially the different tissues, but also for the developmental stages, as can be expected, since for this band, no difference in the staining pattern for the different tissues and developmental stages was observed on gel. In Venn diagrams (Figure 3), the overlap in protein identifications between the different spatial-temporal conditions is shown. Venn diagram A visualizes the overlap of proteins between the three different developmental stages examined. An increase in the total number of proteins can be observed when development progresses. At stage HH36, there is also an increase in the number of differentiation stage specific proteins, defined as “only present in that tissue or stage” (n ) 35), compared to HH26 and HH30/31 (each 17 specific proteins). In Venn diagram B, 54 specific proteins (only present in that tissue) for the great arteries are listed, 16 were specific for ventricle and 11 for outflow tract. Further analysis of the proteins that were specific for just one developmental stage or tissue showed that most of these proteins were found in only one sample just above the threshold level of two spectral counts (Supplementary Data). Except for the specific proteins for the great arteries, from the 54 proteins, 18 were found in multiple samples, the 5 with the highest spectral counts are shown in Table 1. Seven of the stage and tissue specific proteins show on gel a protein staining that positively correlates to the relative quantitation observed in the mass spectrometer. These were the proteins myosin heavy chain, xin, connectin, connectin/ titin, desmoplakin, supervillin and ankyrin3. Supervillin was identified in band A and ankyrin3 was identified in band B; the others were identified in both bands. These proteins were present in all tissue structures at stage HH26, albeit the number of spectral counts is lower in the great arteries. At stage HH30/ 31 and HH36 of heart development, for these proteins no spectral counts are observed in the samples of the great arteries;
in contrast, the spectral counts for the outflow tract and the ventricle increase considerably in the later embryonic stages (Table 1). Ingenuity Pathway Analysis. There were 266 proteins identified from which, for 151 proteins, Uniprot codes were found. From these proteins, an Excel list was created with 9 observations; 3 different stages and 3 different tissues. This list was uploaded in Ingenuity resulting in 109 proteins which were found in Ingenuity. These 109 proteins are coupled to different stages and tissues. From each observation, networks were created. These networks were overlaid onto theoretical networks which are involved in the following: (a) cardiovascular system development and function, (b) skeletal and muscular development and function, and (c) tissue development, amino acid metabolism, cellular growth and proliferation, drug metabolism. The identified proteins found in this study were mostly abundant in the pathway for cardiovascular system development and function (Figure 4). The following proteins that relate to stage or tissue are found in this pathway: myosin heavy chain cardiac muscle alpha 6, myosin heavy chain cardiac muscle beta 7, titin, actin alpha cardiac muscle, and myosin light chain kinase. In addition, tropomyosin, myosin binding protein C and cardiac alpha actinin were identified as part of the network, but for these proteins, no large changes in spectral counts for stage or tissue are observed (Figure 4). The remaining proteins that are specific for stage or tissue structure have not been included in this network or any of the other theoretical networks, for instance, collagen alpha-1 (XII) which is only observed in the great arteries at the later stages investigated.
Discussion In this study, we investigated the development of the chicken heart in a spatial-temporal way by analyzing three heavily stained bands of a one-dimensional protein SDS polyacrylamide gel loaded with the protein extracts of tissue of different heart structures at different stages of embryonic development. The number of proteins that were identified per individual sample of a specific stage and heart structure were comparable (approximately 100 proteins per sample). The protein concentration of each individual sample was determined before the samples were analyzed on gel to ensure that for every sample the same amount of peptides was loaded. From each gel, band areas with the exactly the same dimensions were excised. From all samples, comparable UV traces were obtained indicating a similar concentration of peptides on column. In this way, a controlled comparison of the different samples could be made. By use of spectral counting,12 a semiquantitative comparison between stages and tissues was obtained that gave fast results about differences in abundance levels in heart development. Just by taking these snapshots of proteins, we could observe significant differences in protein networks of cardiac muscle development. At stage HH26, for myosin heavy chain 6, relative high spectral counts are observed in all heart tissues investigated. Myosin heavy chain 6 is not detected in the great arteries at the later stages investigated. For myosin heavy chain 7, relative moderate spectral counts are observed in the outflow tract and ventricle with its peak at stage HH30/31. In the great arteries, myosin heavy chain 7 is detected at HH26, but is not observed at later stages. At stage HH30/31, a further rigorous protein restructuring of muscles can be observed especially in the ventricles, as fragments of titin, connectin, actin, xin (actinbinding proteins part of the Z-disk) are increasingly observed. Journal of Proteome Research • Vol. 9, No. 1, 2010 271
272
Journal of Proteome Research • Vol. 9, No. 1, 2010
Myosin heavy chain PREDICTED: similar to Desmoplakin Xin Connectin Connectin/titin (Fragment) Chick atrial myosin heavy chain PREDICTED: similar to erythroid spectrin beta, partial PREDICTED: similar to supervillin isoform 2; membrane-associated F-actin binding protein p205; archvillin PREDICTED: similar to ankyrin 3 isoform 1; ankyrin-3, node of Ranvier; ankyrin-G Isoform Long of Collagen alpha-1(XII) chain precursor PREDICTED: similar to latent transforming growth factor beta binding protein 2 Claustrin PREDICTED: similar to A-kinase anchor protein 12 isoform 1; kinase scaffold protein gravin; A-kinase anchor protein, 250 kDa; myasthenia gravis autoantigen gravin Isoform 1 of Myosin light chain kinase, smooth muscle Isoform 1 of Low-density lipoprotein receptor-related protein 1 precursor
1 2 3 4 5 6 7
IPI00582419
IPI00570742 507 kDa
211 kDa
117 kDa 245 kDa
157 kDa
IPI00577169
IPI00593493 IPI00580754
341 kDa
IPI00574203
614 kDa
IPI00582908
kDa kDa kDa kDa kDa kDa kDa
263 kDa
223 323 216 663 465 222 114
mol. weight
IPI00588496
IPI00577818 IPI00602986 IPI00571008 IPI00581990 IPI00596489 IPI00586103 IPI00586498
IPI nr
GA
0
0
0 0
0
0
0
0
102 16 10 12 8 6 0
O
0
0
0 0
0
0
0
7
153 87 15 17 7 23 0
V
0
0
0 0
0
0
0
8
178 83 18 22 9 7 0
14
0
9 12
0
3
0
0
0 0 0 0 0 0 0
GA
band A
0
0
0 0
0
0
0
13
183 70 17 10 5 44 0
O
HH30/31 V
0
0
0 0
0
0
0
15
195 79 30 22 13 8 0
14
8
5 3
6
10
0
0
0 0 0 0 0 0 0
GA
0
0
0 0
0
0
0
4
41 24 8 0 8 15 0
O
HH36 V
0
0
0 0
0
0
0
12
227 58 22 30 15 0 0
0
0
0 0
0
2
0
0
51 16 2 5 2 3 2
GA
0
0
0 0
0
2
4
0
112 31 24 12 6 29 11
O
HH26 V
0
0
0 0
0
0
14
0
145 24 18 15 8 29 13
0
3
2 2
6
45
0
0
0 0 0 0 0 0 0
GA
band B
0
0
0 0
0
14
4
0
189 27 19 5 2 76 4
O
HH30/31 V
0
0
0 0
0
0
14
0
228 21 33 10 7 37 13
6
18
0 0
10
75
0
0
0 0 0 0 0 0 0
GA
0
0
0 0
0
9
8
0
189 16 19 6 6 65 3
O
HH36 V
0
0
0 0
0
0
14
0
220 10 24 16 8 0 15
a For a complete overview of the identified proteins, see Supplementary Material. Proteins nr 1-9 show a staining pattern on gel for band A and B that correlates with the spectral counts observed. Protein nr 10 is the only protein that shows a specific pattern for the great arteries and outflow tract and is only present in band A and B. Proteins nr 11-15 are only identified in the great arteries. Abbreviations: GA, Great Arteries; O, Outflow Tract; V, Ventricle.
15
14
12 13
11
10
9
8
protein name
nr
HH26
Table 1. The Spectral Counts Per Analyzed Sample for a Selection of the Identified Proteinsa
research articles Bon et al.
Proteomic Analyses of the Chicken Embryonic Heart
research articles
Figure 4. Eight of the identified proteins were found in the displayed theoretical network that is related to cardiovascular system development. All other theoretical networks contained less identified proteins. The nodes in the network displayed in red are identified or relate to an identified protein in the proteomics analyses; the other proteins are part of the network but have not been identified. For the proteins in the network which are identified, the spectral counts per tissue structure and development stage are summarized in the table below the figure. In this table, the results for the different gel bands per sample are summed-up.
For the great arteries, this muscle development decreases in abundance and other proteins appear, for instance, collagen alpha-1 (XII). In Table 1 and in Supporting Information Table 2, a listing in a spatiotemporal way of heart related proteins is reported. Supervillin13 and Xin14 are important for organization of actin in cells, which fits nicely in the hypothesis that during stage HH30/31 and later stages massive muscle development takes place. Ankyrin3 is important for normal electrical activity in cardiomyocytes15 and ankyrins are essential for the development of excitable membrane domains in heart tissue.16 This is in agreement with our data, where ankyrin3 is present in the
ventricle where electrical activity takes place and with a lower spectral count in the outflow tract which also contains small amounts of excitable tissue, but not in the great arteries. Desmoplakin is important for cellular organization by stabilizing desmosomes and its presence is fundamental for proper development of the embryonic heart.17 Literature confirms the expression of xin14 and titin/connectin18 in the chicken embryonic heart. In the present study, we show that these proteins differentiate among stages and tissues investigated. Desmoplakin has been shown to be involved in embryonic heart development, but is not yet studied in the chicken embryo. Supervillin and ankyrin3 are Journal of Proteome Research • Vol. 9, No. 1, 2010 273
research articles not previously shown to be involved in chicken embryonic heart development and their cardiac differential expression has, until now, not been described in human or other animals. The proteins that are found only in the great arteries are mostly well-known proteins. Myosin light chain kinase is not described in literature as specific for great arteries, but literature does confirm that it plays a role in vascular smooth muscle cells during chicken embryogenesis.19 Latent transforming growth factor beta binding protein 2 (LTBP-2) has been shown to be expressed in the large arterial vessels in developing rats and mice, but also in the epicardium, pericardium and heart valves.20 It has not been shown before in the chicken embryo. Claustrin is a proteoglycan and is expressed in the nervous system of the developing chicken, but there is no earlier connection to developing vasculature.21 We uploaded the identified proteins into Ingenuity software that relate proteins to known pathways (Figure 4). In a pathway that is related to cardiac vascular system development and function, the largest number of identified proteins were present. In this pathway, myosin 6 and myosin 7 are essential proteins. We showed that the relative abundance of these proteins changed between the different development stages and heart tissue types. Interestingly, a recent paper relates a mutation in myosin 6 to atrial septum defects.22 In this study, we showed that it was possible with the described proteomics method to study the changes in the proteome of different heart structures during embryonic development. Furthermore, this information could be linked to protein pathways that relate to embryonic development. A focus is essential in proteomics approaches, for instance, the toxicology of compounds as diverse as vitamins, new drugs, and compounds that interfere with neural crest cell migration. This method is well-suited for this purpose because a large number of proteins can be studied simultaneously during heart development. With this method, affected and normal tissue structures can be compared in a relatively uncomplicated way.
Supporting Information Available: Complete overview of the identified proteins. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Stern, C. D. The chick; a great model system becomes even greater. Dev. Cell 2005, 8 (1), 9–17. (2) Bouman, H. G.; Broekhuizen, M. L.; Baasten, A. M.; Gittenbergerde Groot, A. C.; Wenink, A. C. Diminished growth of atrioventricular cushion tissue in stage 24 retinoic acid-treated chicken embryos. Dev. Dyn. 1998, 213 (1), 50–8. (3) Whitsel, A. I.; Johnson, C. B.; Forehand, C. J. An in ovo chicken model to study the systemic and localized teratogenic effects of valproic acid. Teratology 2002, 66 (4), 153–63. (4) Boot, M. J.; Steegers-Theunissen, R. P. M.; Poelmann, R. E.; van Iperen, L.; Gittenberger-de Groot, A. C. Cardiac outflow tract malformations in chick embryos exposed to homocysteine. Cardovasc. Res. 2004, 64 (2), 365–73.
274
Journal of Proteome Research • Vol. 9, No. 1, 2010
Bon et al. (5) Parada, C.; Gato, A.; Aparicio, M.; Bueno, D. Proteome analysis of chick embryonic cerebrospinal fluid. Proteomics 2006, 6 (1), 312– 20. (6) Mizukami, M.; Kanamoto, T.; Souchelnytskyi, N.; Kiuchi, Y. Proteome profiling of embryo chick retina. Proteome Sci. 2008, 6, 3. (7) Jianzhen, H.; Haitian, M.; Liming, Y.; Sixiang, Z. Developmental changes of protein profiles in the embryonic Sanhuang chicken liver. J. Vet. Med. A Physiol. Pathol. Clin. Med. 2007, 54 (9), 464–9. (8) Hamburger, V.; Hamilton, H. L. A series of normal stages in the development of the chick embryo. J. Morphol. 1951, 88, 49–92. (9) Agudo, D.; Gomez-Esquer, F.; Diaz-Gil, G.; Martinez-Arribas, F.; Delcan, J.; Schneider, J.; Palomar, M. A.; Linares, R. Proteomic analysis of the Gallus gallus embryo at stage-29 of development. Proteomics 2005, 5 (18), 4946–57. (10) Keller, A.; Nesvizhskii, A. I.; Kolker, E.; Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 2002, 74 (20), 5383–92. (11) Nesvizhskii, A. I.; Keller, A.; Kolker, E.; Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 2003, 75 (17), 4646–58. (12) Liu, H.; Sadygov, R. G.; Yates, J. R., III. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 2004, 76 (14), 4193–201. (13) Khurana, S.; George, S. P. Regulation of cell structure and function by actin-binding proteins: villin’s perspective. FEBS Lett. 2008, 582 (14), 2128–39. (14) Wang, D. Z.; Reiter, R. S.; Lin, J. L.; Wang, Q.; Williams, H. S.; Krob, S. L.; Schultheiss, T. M.; Evans, S.; Lin, J. J. Requirement of a novel gene, Xin, in cardiac morphogenesis. Development 1999, 126 (6), 1281–94. (15) Lowe, J. S.; Palygin, O.; Bhasin, N.; Hund, T. J.; Boyden, P. A.; Shibata, E.; Anderson, M. E.; Mohler, P. J. Voltage-gated Nav channel targeting in the heart requires an ankyrin-G dependent cellular pathway. J. Cell Biol. 2008, 180 (1), 173–86. (16) Cunha, S. R.; Mohler, P. J. Cardiac ankyrins: Essential components for development and maintenance of excitable membrane domains in heart. Cardiovasc. Res. 2006, 71 (1), 22–9. (17) Gallicano, G. I.; Bauer, C.; Fuchs, E. Rescuing desmoplakin function in extra-embryonic ectoderm reveals the importance of this protein in embryonic heart, neuroepithelium, skin and vasculature. Development 2001, 128 (6), 929–41. (18) Lim, S. S.; Woodroofe, M. N.; Lemanski, L. F. An analysis of contractile proteins in developing chick heart by SDS polyacrylamide gel electrophoresis and electron microscopy. J. Embryol. Exp. Morphol. 1983, 77, 1–14. (19) Fisher, S. A.; Ikebe, M. Developmental and tissue distribution of expression of nonmuscle and smooth muscle isoforms of myosin light chain kinase. Biochem. Biophys. Res. Commun. 1995, 217 (2), 696–703. (20) Shipley, J. M.; Mecham, R. P.; Maus, E.; Bonadio, J.; Rosenbloom, J.; McCarthy, R. T.; Baumann, M. L.; Frankfater, C.; Segade, F.; Shapiro, S. D. Developmental expression of latent transforming growth factor beta binding protein 2 and its requirement early in mouse development. Mol. Cell. Biol. 2000, 20 (13), 4879–87. (21) Burg, M. A.; Lee, J. A.; Cole, G. J. An alternatively spliced, 5′-truncated MAP1B isoform is expressed in the developing chick nervous system. J. Mol. Neurosci. 1997, 9 (3), 177–86. (22) Nath, A. K.; Krauthammer, M.; Li, P.; Davidov, E.; Butler, L. C.; Copel, J.; Katajamaa, M.; Oresic, M.; Buhimschi, I.; Buhimschi, C.; Snyder, M.; Madri, J. A. Proteomic-based detection of a protein cluster dysregulated during cardiovascular development identifies biomarkers of congenital heart defects. PLoS One 2009, 4 (1), e4221.
PR900614W
