Proteomics of the Nucleus Ovoidalis and Field L Brain Regions of Zebra Finch Alexander Benjamin,† Moses Kashem,† Camille Cohen,† Jennifer A. Caldwell Busby,‡ Delanthi Salgado-Commissariat,§ Santosh A. Helekar,§ and Sanjoy K. Bhattacharya*,† Bascom Palmer Eye Institute, University of Miami, Miami, Florida 33136, Translational Research Institute, The Scripps Research Institute, 5353 Parkside Dr-RF1, Jupiter, Florida 33458, and Department of Neurology, Speech and Language Center, The Methodist Neurological Institute, 6560 Fannin Street, Suite 902, Houston, Texas 77030 Received December 20, 2007
The purpose of present study is to analyze the brain proteome of the nucleus ovoidalis (OV) and Field L regions of the zebra finch (Taeniopygia guttata). The OV and Field L are important brain nuclei in song learning in zebra finches; their analyses identified a total of 79 proteins. The zebra finch brain proteome analyses are poised to provide clues about cell and circuit layout as well as possible circuit function. Keywords: Zebra finch • Taeniopygia guttata • Nucleus Ovoidalis • Field L • brain proteome • staining techniques • Kyoto encyclopedia of genes and genome
Introduction A group of model organisms are collectively referred to as songbirds. Nearly half of all avian species are songbirds, which belong to a single monophyletic order: Passeriformes. Songbirds have been prominently utilized in research on topics as varied as stress, reproduction, endocrinology, reproductive strategies, immune function, environmental toxicology, flight physiology, behavioral ecology, and neuroscience.1–3 Songbirds are one of the very few animal groups in which vocal production is learned as in humans.4 Songbirds also have a very welldeveloped visual system with cone rich retinas that allow color perception, and visual cues play a primary role in their life.5 Zebra finch (Taeniopygia guttata) is a member of the songbird group and is a desirable model organism for studies pertaining to speech development,6,7 age-related macular degeneration,5 and for studying cross modality between different sensory systems. The property of learned vocalization has made zebra finches a popular model for studying song learning and other facets of learned vocalization as well as speech development. Male zebra finches learn vocalization from tutor birds, usually their fathers.8 Zebra finches are one of the well-studied models for song development. Interconnected circuitry in the brain that controls song and song learning has been described in songbirds.8 This circuit comprises a set of large, discrete anatomic nuclei, which have evolved uniquely in songbirds. The songbird has well-studied auditory and motor pathways which are adapted for vocal * To whom correspondence should be addressed. McKnight Vision Research Building, Bascom Palmer Eye Institute, University of Miami, 1638 NW 10th Avenue, Room 706A, Miami, Florida 33136. Tel: 305-482-4103. Fax: 305-326-6547. Email:
[email protected]. † University of Miami. ‡ The Scripps Research Institute. § The Methodist Neurological Institute. 10.1021/pr7008687 CCC: $40.75
2008 American Chemical Society
learning. These pathways are analogous to those in mammals, including the brain regions involved in learning human language.4 Unlike the human brain, these pathways are amenable to detailed experimental investigation. The high premotor vocal nucleus or high vocal center9,10 (HVC) and the robust nucleus of the arcopallium (RA) control song production.11,12 An anterior forebrain pathway consisting of lateral magnocellular nucleus of the anterior nidopallium and area X play a role in song learning and form a recursive loop with HVC, RA, and the medial part of the dorsolateral nucleus of the thalamus.11,13,14 HVC projects to both RA (the motor pathway) and area X (the song learning pathway).9,15 The auditory pathway consists of the primary auditory complex called Field L, the nucleus ovoidalis (OV), and secondary auditory areas caudal medial nidopallium (NCM) and caudal mesopallium (CM).16 Nucleus interfacialis (NIf) is involved in the auditory processing of song and is a source of auditory input to HVC.17 Another structure that is involved in the control of song structure and timing is nucleus uvaeformis (UVa).18 UVa is also known to receive input from the visual system.19 Although zebra finches are wellstudied for song development, they are underutilized in more complex studies that involve vision and other behavioral features including song learning. The cross modality of different senses has been recognized due to a number of studies on human subjects.20,21 In early blind human subjects, studies have convincingly showed the utilization of the visual region for auditory tasks.22,23 Because of their features of welldeveloped visual system and learned vocalization, zebra finches are ideal model organisms for molecular details concerning further dissection of the cross modality of sensory systems as well as better understanding of speech development. Convergence of constellation of these features, learned vocalization, for example, is lacking in most laboratory animals such as the mouse. Journal of Proteome Research 2008, 7, 2121–2132 2121 Published on Web 03/25/2008
research articles High-throughput approaches of proteomics and genomics are expected to allow rapid identification of proteins, leading to an understanding of their roles during development, using complementary experimental approaches. Various genomic approaches such as microarray analyses allow the determination of mRNA levels. The information provided by genomics is complementary to that of proteomics, which comprises the identification of proteins and protein modifications indicative of the functional states of the proteins. Proteomic analyses of zebra finch brain regions will also identify proteins that are important for proper axonal guidance of sensory input and circuit establishment during development, as well as during plastic changes in mature organisms. Since brain regions are structured and organized differently, it presents an ideal system to bring the power of proteomic techniques to circuit analysis and network development. Previously, we have performed proteomic analyses on parts of the zebra finch’s primary visual pathway including the retina5 and optic tectum.24 Here we present the proteomic analyses of two different brain structures, nucleus ovoidalis (OV) and the Field L complex, that are involved in auditory processing of sound.16 The proteomic analyses presented here are also expected to help in the design and investigation of studies pertaining to cross modality of sensory systems.
Materials and Methods Tissue Procurement. Animal use procedures were approved by the Texas A&M University Institute of Biosciences and Technology and the Methodist Hospital Research Institute IACUC committee. The founder birds for this colony were of gray zebra finch strain, acquired from Acadiana Aviaries, Franklin, LA. These birds were housed in large wire cages that hold up to 8 birds per cage. Adult (g100 days old) zebra finches were bred and raised in the Methodist Hospital Research Institute vivarium under standard 12-h light-dark cycle with ad libitum food and water. Animals were euthanized following approved procedures. The dissection of OV, Field-L, and other regions of the brain was carried out on fresh isolated brain under microscope using a scalpel immediately after euthanizing the animal. Four pairs of adult male T. guttata brain (8 brains total) were analyzed for proteomic studies and four adult male brains were used for immunohistochemical studies. Protein Analyses. Proteins were extracted using a Kontes hand-held homogenizer from dissected zebra finch brain regions, which were placed in tubes immediately after dissection and stored in -80 °C until further analysis. Proteins were extracted using a base buffer (containing 125 mM Tris-HCl buffer, pH 7.0, and 100 mM NaCl) and different detergents (1% Triton X100, 1% ASB-14, 1% Tween-20, 0.1% Genapol C-100, 1% SDS, and 1% DM) during optimization.25 Subsequently, all extractions were performed with a combination of detergents 0.1% Genapol C-100 (EMD Biosciences, CA), 1% Triton X 100, and 0.2%SDS in base buffer. Insoluble materials were removed by centrifugation (8000g for 5 min), and soluble proteins were quantified by bicinchoninic acid (BCA) and Bradford assay.26 Proteins were fractionated over 4–20% gradient SDS-PAGE (Invitrogen, Inc., Carlsbad, CA). The gels were subjected to staining with SYPRO Ruby (Molecular Probes, Invitrogen, Inc., Carlsbad, CA), silver (Amresco, Solon, OH), and Coomassie blue (Pierce Biotechnology, Inc., Rockford, IL). Coomassie blue stained protein bands were excised, destained, reduced with dithiothreitol (DTT), alkylated with iodoacetamide, and subsequently processed for mass spectrometric analysis. For 2122
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Benjamin et al. detection of total glycosylated proteins, a GelCode Glycoprotein Staining Kit (Product no. 24562; Pierce Biotechnology, Rockford, IL) was utilized as per the recommendation of the manufacturer. This kit is designed for specific staining of glycoproteins directly on gels. A Pro-Q Emerald 300 glycoprotein gel and blot stain kit (P21857; Molecular Probes, Invitrogen, Inc., Carlsbad, CA) with suitable modification to stain gels was also used to stain identical gels as with the GelCode kit. The two kits differ with respect to their ability to stain proteins. Pro-Q Emerald 300 Glycoprotein stain is 50-fold more sensitive than GelCode staining kit. The GelCode staining kit uses periodic acid-Schiff base modification of carbohydrate groups that are stained with acidic fuchsin dye. Pro-Q Emerald 300 utilizes a proprietary stain that reacts with periodate-oxidized carbohydrate groups to create bright fluorescent green signals on glycoproteins. Mass spectrometric compatibility for proteins stained with the GelCode kit remains to be established. Glycosylated protein bands stained by Pro Q Emerald 300 were excised, destained, reduced and alkylated (iodoacetamide), and subjected to mass spectrometric analysis. Mass Spectrometry. For protein identification, gel slices were excised and digested in situ with sequencing grade trypsin (Promega Biosciences, Inc., CA). Digestion mixtures were loaded onto precolumns (360 mm o.d. × 100 mm i.d. fused silica, Polymicro Technologies, Phoenix, AZ) packed with 3 cm irregular C18 (5–15µm nonspherical, YMC, Inc., Wilmington, NC) and washed with 0.1 M AcOH for 5 min before switching in-line with the resolving column (7 cm spherical C18, 360 × 100). Once the columns were in-line, the peptides were gradient-eluted with a gradient of 0–100% B in 30 min where A was 0.1 M AcOH in nanopure H20 and B was 0.1 M AcOH in 80% MeCN. All samples were analyzed using a Thermo Electron Finnigan LTQ (San Jose, CA). Electrospray was accomplished using an Advion Triversa Nanomate (Advion Biosystems, Ithaca, NY) with a voltage of 1.7kV and a flow rate of approximately 250 nL/min. The mass spectrometer was operated in datadependent mode , and the top 5 most abundant ions in each spectrum were selected for sequential MS/MS experiments. The exclusion list was used (1 repeat, 180 s return time) to increase dynamic range. All MS/MS spectra were searched with Sequest (version 2.7) and Mascot using NCBI nonredundant, Ensemble, and Swiss-Prot databases. The database search entries were restricted to Metazoa (animals) and allowed a maximum of two missed cleavages. The protonated molecule ions “MH+” and “Monoisotopic” were defined for the peak mass data input. For protonated “MH+” peptides SEQUEST Sp and Xcorr, cutoff scores were 500 and 1.8. For Mascot searches, the scores were greater than 78. All spectra were visually inspected to determine the correct database assignment. The potential chemical modifications of a peptide such as the alkylation of a cysteine [Carbamidomethyl (C)] and the oxidation of a methionine residue (M) and acetylation of lysine (K) were also considered in the search. No restriction was applied for either the protein isoelectric point and molecular weight during searching. Subsequently, searches were also performed using the Songbird Brain Transcriptome Database (http://songbirdtranscriptome. net/) and the songbird EST database (http://titan.biotec.uiuc. edu/cgi-bin/ESTWebsite/estima_seqQuery?seqSet)songbird). Database search results were tabulated and visually inspected using Scaffold visualization software (Proteome Software, Portland, OR). These methods are routinely used in our laboratory. A combined list of all identified proteins was prepared. The Swiss-Prot and GenBank accession numbers for
Proteomics of Brain Regions of Zebra Finch
research articles
Figure 1. Determination of efficacy of detergent for extraction of zebra finch brain proteins. (A) Spectrophotometric determination of protein concentrations in indicated detergent in a base buffer (125 mM Tris-HCl, pH 7.0, 100 mM NaCl). Solid and hollow bars represent estimation of proteins using Bradford’s and bicinchoninic acid (BCA) methods, respectively. Protein amount per milligram of wet tissue is presented and standard deviation from three independent experiments is provided. (B) Representative protein profile of zebra finch brain extracted using indicated detergents in a base buffer. Approximately 10 µg of proteins was loaded onto a 4–20% SDS-PAGE and stained with Gelcode blue.
the proteins were obtained, and the latter was used for a search against the DRAGON database (http://pevsnerlab. kennedykrieger.org/annotate.htm). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/ kegg/pathway.html) entries for biochemical pathways were obtained for each protein accession number using DRAGON interface. A program to interface with databases and to retrieve the NCBI, GenBank accession numbers, and KEGG Pathways has been written (Supplementary document 1 in Supporting Information) and step-by-step instructions for using this program are also available (Supplementary document 2 in Supporting Information). The identified proteins were also subjected to network analyses using Genego portal (portal. genego.com; GeneGo, Inc., 500 Renaissance Drive, Ste 106 St. Joseph, MI 49085). Histochemistry. Histological evaluations were made following published protocols for songbird and other neuronal tissues used in our laboratory.5,24 Brain sections were fixed in 4% paraformaldehyde in phosphate buffer and OCT frozen sections (about 10 µm) were prepared and stained with antibodies. A battery of commercially available antibodies was used to determine the identified proteins in this proteomic analyses or previously reported proteins for their presence in the T. guttata brain region OV and Field L. The endogenous peroxidase was blocked by immersion in 0.6% hydrogen peroxide in methanol for 15 min. After washing with phosphate buffer, sections were blocked with 0.2% BSA in phosphate buffered saline (PBS). The presence of select proteins was also verified using fluorescence analyses (data not shown). For the fluorescence analyses, secondary antibodies coupled with Alexa 488, Alex 595, FITC, or Rhodamine fluorophores were commercially procured (Invitrogen Molecular Probes, Eugene, Oregon) and images were taken on a Leica TSP5 confocal microscope.
Results and Discussions Extraction, Fractionation, and Staining of Proteins from Different Brain Regions. Our prior studies on the zebra finch retina,24 bovine,27 and porcine ocular tissues25 have indicated
profound effects of detergents on protein extraction from tissues. The extraction of brain proteins was performed in a base buffer containing 125 mM Tris-HCl and 100 mM NaCl with six different detergents: Triton X-100, ASB-14, Tween-20, Genapol C-100, Sodium Dodecyl Sulfate (SDS), and Dodecylmaltoside (DM). Protein extractions from these detergents were evaluated using BCA and Bradford’s method (Figure 1A). The Bradford protein assay is limited by its low detergent tolerance; for example, only up to 0.125% SDS is tolerated by Bradford’s. The assay relies on the interaction between the Coomassie blue dye and basic amino acid residues. These interactions are easily perturbed by the addition of detergent, leading to inaccurate estimations. The BCA assay is based on the reduction of Cu2+ to Cu+ upon the addition of proteins. The released Cu+ binds with bicinchoninic acid (BCA) which shows color at 562 nm. The BCA protein assay has a higher detergent tolerance than the Bradford assay (for example, up to 5% SDS). As shown in Figure 1A, the Bradford assay deviated much more than the BCA assay in presence of detergents; for ASB-14, the Bradford measurement significantly deviated from an accurate estimate. Protein profiles (10 µg protein load) for different detergent extractions were also obtained using SDS-PAGE analysis (Figure 1B). Careful examination reveals the presence of two prominent bands in the 22–36 kDa region in SDS extraction but not in Triton X-100. On the basis of careful examinations of protein bands for different extraction detergents, a combination of three detergents, 1% Triton X 100, 0.1% Genapol C-100 (EMD Biosciences, CA), and 0.2% SDS in base buffer, have been used for all further protein extractions from different zebra finch brain regions. Staining of Zebra Finch Brain Proteins. The extracted proteins from different T. guttata brain regions were fractionated on 4–20% gradient 1D SDS-PAGE (Invitrogen, Inc., Carlsbad, CA). A number of the brain regions are very small, and thus, 1D rather than 2D SDS-PAGE was utilized for fractionation primarily to minimize protein loss as was published previously.5,24 We probed the T. guttata brain proteins for the presence of statherin and histatin, the small (e8 kDa proteins) Journal of Proteome Research • Vol. 7, No. 5, 2008 2123
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Figure 2. Efficacy of different staining methods for detection of zebra finch brain proteins. Proteins (10 µg) from indicated brain regions were fractionated on a 4–20% SDS-PAGE and stained for detection. (A) Silver staining using Silver BULLit silver staining kit (Amresco, Solon, OH). (B) Staining using Gelcode blue (Pierce Biotechnology, Inc., Rockford, IL). Arrow indicates proteins not detected using silver stain. (C) Staining using SYPRO Ruby (Molecular Probes, Invitrogen, Inc., Carlsbad, CA). OV ) Nucleus ovoidalis; NCM ) caudomedial nidopallium; RA ) robust nucleus of arcopallium; HVC ) high vocal center; LMAN ) lateral magnocellular nucleus of the anterior nidopallium; Area X and Field L are song nucleus, synonymous zebra finch brain regions.
salivary calcium bound phosphoproteins that have been detected in neuronal systems in humans28 and found the presence of their homologues (data not shown) among a repertoire of zebra finch brain proteins using Western blot analysis. Staining total proteins with silver stain, however, did not reveal any protein in the lower molecular region, that is, e8 kDa proteins (Figure 2A). The staining of silver-stained SDS-PAGE of T. guttata brain proteins with Coomassie blue revealed the presence of small proteins (Figure 2B; indicated with an arrow); some other proteins were also detected stained selectively with Coomassie blue but not silver (Figure 2B). In contrast, several proteins were stained with silver but not Coomassie blue. We also utilized SYPRO Ruby stain and used fluorescence detection scanner to procure an image (Figure 2C). Although SYPRO Ruby is sensitive in detecting many proteins present in smaller amounts, there was a select set of proteins, which on careful examination were found not stained with SYPRO Ruby as well (Figure 2C). Taken together, while most reagents (silver, Coomassie or SYPRO Ruby) stain the majority of proteins, there is a small set of proteins among different stains which is stained by one reagent but not others (Figure 2). Such lack of staining may have consequences for protein band/spot identification if unstained regions are not subjected to mass spectrometric analyses. Analyses of Zebra Finch Brain Proteome. Liquid chromatography and tandem mass spectrometry of in-gel trypsindigested zebra finch brain region proteins (Figure 2A) identified 79 proteins (Table 1). About 36 proteins were identified from the OV and 72 proteins were identified from Field L. A search for protein identification with MS/MS sequence data was performed using NCBI nonredundant as well as the Ensemble and Swiss-Prot databases. Protein identities with 95% or better confidence were the ones retained. Protein identities were also searched using the Songbird Brain Transcriptome and Songbird EST databases. The OV and Field L regions, while very important for song learning and song control, are small regions in terms of dissected tissue mass. Lack of songbird genome is likely to be another reason that a low number of proteins has been identified from these regions. We carried out the fractionation of the T. guttata OV and Field L proteins on 1D SDSPAGE as previously performed for the retina and optic tectum.5,24 This is in accordance with previous studies where a comparatively higher number of proteins were captured using 1D than 2124
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2D SDS-PAGE from tissues yielding small dissected mass.29,30 The losses incurred in the fractionation steps such as gel fractionation are the foremost factor that limits the capture of proteins and hinders subsequent protein identification by mass spectrometry. Analyses of Glycoproteins from Different Brain Regions. The proteins fractionated on 4–20% gradient SDS-PAGE were subjected to detection for total glycosylated proteins using a GelCode Glycoprotein Staining Kit. The lowest sensitivity of detection is about 50 ng of glycoprotein. After completing the procedure, the glycols are stained, yielding magenta bands with a light pink or colorless background. The total proteins from different zebra finch brain regions showed a number of heavily glycosylated proteins in the regions e6 kDa. Although there were a few other proteins bands which showed glycosylation, these had very weak signals (Figure 3A). The same gel stained with Coomassie blue (Figure 3A′) shows total proteins. The proteins for which only fractions of their total pool are modified or that are not uniformly glycosylated (different molecules of the proteins are glycosylated differently) are likely to fall below the detection limit of this kit. We also utilized Pro-Q Emerald 300 Glycoprotein stain (Figure 3B). This stain, claimed to have superior sensitivity (ability to detect as little as 0.5ng of glycoproteins), is about 50-fold more sensitive than the standard periodic acid-Schiff base method using acidic fuchsin dye. The reagent is proprietary material of the manufacturer. The stain reacts with periodate-oxidaized carbohydrate groups, creating bright fluorescent green signals on glycoproteins. The total protein was detected using Coomassie blue staining (Figure 3B′). As evident from Figure 3A and B, the two staining methods clearly differ in sensitivity. The mass spectrometric compatibility of GelCode glycoprotein staining remains to be determined; therefore, select glycoprotein bands, marked 1–8 in Figure 3B, from the OV and Field-L regions were subjected to in-gel digestion and mass spectrometry, followed by a search in the NCBI nonredundant database (with T. guttata as organism), which led to the identification of 7 proteins (Table 2). This proteomic analysis identified and now confirms the existence of a number of proteins that were previously known only at the transcription level [for example, activity-regulated cytoskeletal-associated protein and melatonin receptor 1b (Table 2)]. This analysis therefore cross-confirms genomic information and confirms the lack of post-translational modi-
Glyceraldehyde-3-phosphate dehydrogenase Glutamate dehydrogenase 1 L-lactate dehydrogenase B chain
Tubulin beta-2C chain Tubulin alpha 6 ATP synthase subunit beta Heat shock-related 70 kDa protein 2 Dihydropyrimidinaselike 2 Tubulin beta-2B chain ATP synthase Keratin 14 Spectrin, beta, nonerythrocytic 1 isoform 1 Keratin 5 Alpha-enolase, lung specific Gamma-enolase Creatine kinase B-type Keratin, type II cytoskeletal 6A Spectrin alpha chain, brain 14-3-3 protein zeta/delta Tyrosine 3-monooxygenase Pyruvate kinase, muscle
Keratin, type II cytoskeletal 6A Keratin, type I cytoskeletal 10 Tubulin, alpha 1a Keratin, type I cytoskeletal 9 Heat shock protein 90 kDa alpha Sodium/ potassium-transporting ATPase alpha-1 chain Actin, cytoplasmic 1
protein name
NM_002300
NP_002291
M23725
P14618
X07674
M86400 AF142498
P63104 P61981
P00367
J05243
Q13813
EF036498
X13120 M16451 BC139753
P09104 P12277 AAI39754
ABO65084
M21389 X66610
J00314 X59066 NM_000526 M96803
P07437 AAH39135 NP_000517 Q01082
P13647 Q05524
NM_001386
X04098
P63261
NP_001377
D00099
P05023
L26336
X15183
P07900
P54652
NM_006009 Z29074
NP_006000 P35527
X02344 NM_032704 M27132
J04029
P13645
P68371 NP_116093 P06576
M99061
Genbank accession
P35908
accession numbera
3
3
3
3
3 3
3
4 4 4
5 5
6 6 6 5
7
7
7 7 7
8
8
9
11 10
15
16
peptide OV
2
3
3
4
6 5
26
6 3 0
0 0
15 6 0 18
8
9
21 14 11
12
13
14
17 11
15
10
matchesField L
Table 1. Proteins Identified from Indicated Brain Region of Zebra Finches by LC-MS/MS Analysis
37
61
36
58
28 28
285
47 43 60
62 49
0 60 52 275
62
70
50 50 57
42
113
98
50 62
60
65
MW (kDa)
10, 272, 620, 640
251, 330, 471, 910
10, 230, 620, 710, 4910, 4930 10, 1510, 5010, 5040, 5050
4110, 5130, 5131
4530
10 330 1430
1430
190 1430
4360
4540, 5130, 5131
1430, 4510, 4520, 4530, 4670, 4810, 5110, 5130, 5131
4612
4540, 5130, 5131 1430
1430
KEGG pathwayb
8
19
29
15
52 22
10
18 13 10
13 12
67 25 14 15
20
15
29 24 32
28
25
27
51 35
40
44
coverage (%)
3.2
4.62
4.41
4.26
5.52 5.42
4.77
4.83 5.54 4.33
3.38 4.29
6.53 4.45 3.38 4.77
4.15
4.99
3.33 3.98 5.01
5.23
4.81
4.72
5.97 4.66
4.33
4.44
score (Xcorr)
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Journal of Proteome Research • Vol. 7, No. 5, 2008 2125
2126
Elongation factor 1-alpha 2 14-3-3 protein epsilon Malate dehydrogenase Septin 7 Phosphoglycerate kinase 1 Actin, alpha skeletal muscle Destrin Clathrin heavy chain 1 Dynamin-1 Guanine nucleotide-binding protein G(o) subunit alpha 1 Plasma membrane calcium ATPase 1 isoform 1b Vesicle-fusing ATPase Tubulin beta-2B chain ATPase, H+ transporting, lysosomal V0 subunit a isoform 1 Aconitate hydratase, mitochondrial precursor Transitional endoplasmic reticulum ATPase 78 kDa glucose-regulated protein precursor Alpha-enolase Plasma membrane calcium ATPase 1 isoform 1b ADP/ATP translocase 2 GNB1 protein Triosephosphate isomerase Calcium/ calmodulin-dependent protein kinase type II gamma chain Voltage-dependent anion-selective channel protein 1 Histone H4 Hexokinase-1 14-3-3 protein theta
protein name
Table 1. Continued
X70940 U20972 AF047470 S72008 V00572 J00068 S65738 D21260 NM_004408 NM_00107748
X63575
U03985 X79535 NM_005177
U80040 AF100752 M19645 M14328 NM_00100134
M57424 BC114618 M10036 AL713896
L06132
X00038 M75126 X56468
P62258 P40926 Q16181 P00558
P68133
P60981 Q00610 NP_004399 NP_001070957
Q01814
P46459 Q13885 NP_005168
Q99798
P55072
P11021
P06733 NP_001001344
P05141 AAI14619 P60174
Q13555
P21796
P62805 P19367 P27348
Genbank accession
Q05639
accession numbera
Journal of Proteome Research • Vol. 7, No. 5, 2008 0 0 0
0
0
0 0 0
0 0
0
0
0
0 0 0
0
2 0 0 0
2
2 2 2 2
3
peptide OV
3 3 3
3
3
4 4 4
5 5
5
5
5
7 6 5
8
0 26 9 8
0
15 5 3 2
0
matchesField L
11 102 28
31
63
33 36 27
47 135
72
89
85
83 50 96
0
18 192 97 40
42
29 36 49 45
50
MW (kDa)
10, 51, 52, 500, 521, 530 4110, 5130, 5131
4020
4742 4020
10 4020
20, 630, 720
4540, 5130, 5131 190, 5110, 5120
4020
4020, 4540, 4730, 4742, 4912, 4916, 5110
5040
10, 710
4110 20, 620, 630, 710, 720
KEGG pathwayb
42 2 12
11
8
48 41 23
23 4
14
7
9
19 46 11
19
10 22 19 20
7
52 37 17 12
5
coverage (%)
3.26 3.55 4.21
4.19
4.13
3.8 3.68 4.33
5.29 4.96
5.32
4.65
4.51
4.54 7.09 3.91
5.58
2.43 5.09 5.54 4.3
5
5.52 5.17 5.25 5.19
3.15
score (Xcorr)
research articles Benjamin et al.
NM_006793 NM_213611 X71490
J04173 M60750 AF092437 NM_002634 X79781
NP_006784
NP_998776 P61421
P18669
P62807 Q9H0U4
NP_937818
Q15286
Journal of Proteome Research • Vol. 7, No. 5, 2008 2127 D16593
X05608 U03271
P84074
P07196
P47756
0
0
0
0 0
0
0
0
0
0
0 0
0
0 0
0
0
0 0
0 0 0
0
0
peptide OV
2
2
2
2 2
2
2
2
2
2
2 2
2
2 2
2
3
3 3
3 3 3
3
3
matchesField L
31
61
22
47 92
44
17
47
23
17
14 22
29
40 40
28
59
30 187
28 22 46
43
24
MW (kDa)
10
251, 252, 272, 330, 350, 360, 400, 401, 710, 950 4020, 4070, 4720, 4740, 4910, 4912, 4916, 5040, 5214 190, 5110, 5120
10
4010, 4020, 4210, 4310, 4360, 4370, 4650, 4660, 4662, 4720, 5030
5040
4110
KEGG pathwayb
7
3
8
16 3
4
26
2
11
20
20 13
10
7 7
18
4
10 5
17 12 10
10
13
coverage (%)
3
3.24
2.5
6.5 3.89
2.75
4.3
3.15
3.35
3
3.99 3.55
3.64
3.32 2.96
3.3
3.3
4.98 3.9
4.09 4.41 3.61
3.05
3.55
score (Xcorr)
The KEGG pathway analysis was performed using Web-based Dragon database (http://pevsnerlab.kennedykrieger.org/dragon.htm) annotate
X16504 AF191298
P13929 Q96QK1
b
X69151
P21283
a Swiss-Prot database and NCBI entries (italicized) are presented. tools. Some accession numbers lacked KEGG pathway entries.
Vacuolar ATP synthase subunit C Beta-enolase Vacuolar protein sorting 35 homologue Neuron-specific calcium-binding protein hippocalcin Neurofilament light polypeptide F-Actin capping protein subunit beta
NM_000944
NP_000935
J04046
NM_002634 NM_007098
NP_002625 NP_009029
P62158
X57346 U14747 D13748
P31946 P62760 P60842
M22632
D90084
P08559
P00505
NM_016131
NP_057215
Ras-related protein Rab-6A Pyruvate dehydrogenase E1 component alpha subunit 14-3-3 protein beta/alpha Visinin-like protein 1 Eukaryotic initiation factor 4A-II Prohibitin clathrin, heavy polypeptide-like 1 Serine/threonine-protein phosphatase 2B alpha isoform Thioredoxin-dependent peroxide reductase Solute carrier family 25 ATPase, H+ transporting, lysosomal, V0 subunit d1 Phosphoglycerate mutase 1 Histone cluster 3, H2bb Ras-related protein Rab-1B Nucleoside diphosphate kinase B Ras-related protein Rab-35 Aspartate aminotransferase Calmodulin
Genbank accession
accession numbera
protein name
Table 1. Continued
Proteomics of Brain Regions of Zebra Finch
research articles
research articles
Figure 3. Staining of zebra finch brain proteins for glycoproteins. Indicated regions of zebra finch brain proteins (10 µg) were fractionated on a 4–20% SDS-PAGE and stained for detection. All staining reagents/kit were from Pierce Biotechnology, Inc., Rockford, IL, unless stated otherwise. (A) Staining for glycoproteins using a kit; (A′) the same gel stained with Gelcode blue for detection of total proteins after staining for glycoproteins. (B) Staining for glycoproteins by ProQ diamond kit; (B′) the same gel stained with Gelcode blue after staining for glycoproteins.
fication of the identified peptides from the regions of proteins, enabling trypsin digestion and sequence determination. The synthetic peptides bearing the sequence of identified peptides will allow generation of antibodies against identified proteins. The knowledge of existence of protein (since now they have been identified at the protein level) will also allow the generation of siRNA against the structural part of the gene. The overall identification of these proteins is based on transcriptome sequence database- and therefore- potential modification at protein level remains to be determined. We refer the analyses of pan-glycosylated proteins (glycoproteome analysis) as subset proteomics. Such subset proteomics have a few advantages: they enable better fractionation, reduce complexity, and allow better capture of molecules and, importantly, within the sphere of the subset of the proteome, provide a broad spectrum investigation of the hypothesis while maintaining the semblance of unbiased specific reagent-independent approach. Histochemical Localization and Comparative Analyses of Identified Proteins. Select identified proteins were localized in the OV and Field L brain regions of zebra finches. The anatomical positions of the OV and Field L regions of the zebra finch brain have been shown (Figure 4A). The dissections of these regions were performed very carefully under a microscope to avoid contamination of tissue from surrounding regions. The positive staining of γ-synuclein and neurofilament protein in the OV and Field-L regions has been shown (Figure 4B). The proteomic identification and subsequent demonstration of the presence of these proteins in specific brain regions suggests proper tissue isolation without contamination from surrounding regions. Immunohistochemical staining against specific proteins can help to identify neuronal populations and facilitate circuit analysis. This analysis corroborates our pro2128
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Benjamin et al. teomic findings and confirms that identified proteins are from the different brain regions and not due to contamination from different tissue regions. Additional analyses were done using fluorescent secondary antibodies (data not shown). Database Searches and Pathway Analyses of Identified Proteins. Proteomic analyses of T. guttata resulted in the identification of 36 and 72 proteins in the OV and Field L regions, respectively (Table 1). Identified proteins for which at least two peptides were captured with good spectra (manually inspected) are presented in the list (Table 1). The current brain transcription database for the T. guttata genome is incomplete, which is reflected by the low number of proteins identified from the OV and Field L regions as well as from glycosylated bands; the latter were subjected to search in the T. guttata database alone (Table 2). The identified proteins from the Swiss-Prot database (Table 1) were also searched in the Songbird Brain Transcriptome database (results not shown). The EST database, which is also available for zebra finches, presents a problem for protein identification using either peptide masses or MS/ MS derived peptide sequence information because they only represent a portion of a gene’s coding sequence. Such segments may not be long enough to cover a sufficient number of peptides observed in the mass spectrometric experiments to allow an unambiguous identification.31 To analyze the proteins in metabolic/biochemical pathways, we performed an analysis with the Dragon annotate database interface and utilized the Kyoto Encyclopedia for Genes and Genomes (KEGG) Pathway database.32,33 The KEGG Pathway database contains metabolic and regulatory processes in the form of wiring diagrams and provides a base for modeling and simulation. We have also used Genego portal (portal.genego.com) for analyses of metabolic and regulatory network (Supplementary Figure 2 in Suporting Information; network symbol detail are provided in Supplementary document 3 in Supporting Information) for the identified proteins. Many other databases are also available for pathway analyses for proteins identified using high-throughput methods of proteomics, for example, Biocarta (http://www. biocarta.com/genes/allPathways.asp) and Gene Ontology information (http://www.geneontology.org/). A number of Webbased and stand alone tools for analyses and the visualization of information derived from databases are also available. For example, KGML-ED, a program (http://kgml-ed.ipk-gatersleben. de) assisting researchers to retrieve and visualize the KEGG database, is freely available to the scientific community.34 The KEGG analyses performed on the identified proteins from the OV and Field L regions showed important differences between these two regions (Figure 5). Calcium signaling, Axon guidance, Long-term potentiation, Taste transduction, GnRH signaling, or Neuroactive ligand–receptor interaction, and Melanogenesis are some of the cellular pathways whose members were more frequently captured in Field L compared to those in the OV region. On the other hand, more frequent members of the pyruvate metabolism and cell junction related pathways were captured from the OV region (Figure 5). Further analyses of identified members in birds of different ages and different conditions will enable determination of which pathway is more activated in which region of the brain. A number of identified proteins belong to glycolysis and amino acid biosynthesis and degradation as well as insulin signaling pathways, suggesting that flux of metabolites in these pathway and key proteins are important regulators of function in these regions. These data also highlight that carbon flux, particularly at key branch points of glycolysis, entry level points
2 2
Glyceraldehyde-3-phosphate dehydrogenase Melatonin receptor 1B
a
Band number as shown in Figure 3B.
2
NP_001041723, ABA19225
3
Cytochrome P450
Activity-regulated cytoskeletal-associated protein
AAF78051
7
Tubulin, beta 2C
ABK56827
AAT46359
NP_001093160
ABM54454
6
Heat shock protein 70
NP_001071647, AAO61783
accession numbers
3
peptide matches
Chromo-helicase DNA-binding protein
protein name
Table 2. Proteins Identified from Glycosyl Stained Bands by LC-MS/MS Analysis
25
41
17
57
50
51
208
MW (kDa)
banda number
APQMNAVVYLGDITSRNMIR, 1 MVLDHLVIQRMDTTGK, NMIRTHEWMHPQTK ARFEELNADLFR, 2, 5 IINEPTAAAIAYGLDK, IINEPTAAAIAYGLDKK, LLQDFFNGKELNK, FEELNADLFR, TTPSYVAFTDTER ALTVPELTQQMFDAK, 6 FPGQLNADLR, IMNTFSVVPSPK, ISEQFTAMFR, KLAVNMVPFPRLHFFMPGFAPLTSR, LAVNMVPFPR, MSATFIGNSTAIQELFK FWREGGLSALHLSMAQKFRR, 2, 5 MLKSTRLLKATIK, NKPYGVLLKTGEAWR LTGMAFRVPTPNVSVVDLTCR, 4 VPTPNVSVVDLTCR MLENGSLRNCCDPGGRGR, 7, 8 RISMSLWMPR NWVEFKKEFLQYSEGTLTRDAIK, 3, 8 WWEYKQDSVKNWVEFK
peptide sequences
15
8
13
9
23
12
3
coverage (%)
1.75
1.12
2.95
0.92
3.57
3.51
1.88
score (Xcorr)
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Figure 4. (A) Coronal section of a male zebra finch brain stained using the cytochrome C oxidase staining method.35,36 (B) Immunohistochemical detection of select proteins (Neurofilament and γ-synuclein) in different zebra finch brain regions as indicated. Primary antibody was omitted in control (as indicated) sections; all sections were subjected to staining with DAPI.
Figure 5. Analyses of proteins for biochemical pathways using Kyoto Encyclopedia of genes and genome (KEGG) database. Select pathways which occurred twice or greater in proteomic analyses of OV and Field L brain regions (Table 1) have been presented. The pathways are 10, glycolysis; 20, TCA cycle; 190, oxidative phosphorylation; 272, cysteine metabolism; 330, Arginine and praline metabolism; 591, Linoleic acid metabolism; 620, pyruvate metabolism; 630, Glyoxylate and dicarboxylate metabolism; 710, carbon/ sugar metabolism; 720, Reductive carboxylate cycle; 1430, cell junction; 4020, calcium signaling pathway; 4110, cell cycle; 4360, Axon guidance; 4530, Tight junction; 4540, gap junction; 4720, Long-term potentiation; 4742, Taste transduction; 4912, GnRH signaling pathway (Neuroactive ligand–receptor interaction); 4916, Melanogenesis; 5040, Huntington’s disease; 5110, cell adhesion/pore; 5120, Epithelial cell signaling; 5130 and 5131, Pathogenic microbial infection.
in cells, and at branch points of amino acid and sugar metabolism pathway points, are likely to be very important for the OV and Field L regions and opens up the scope of future investigations utilizing key regulating enzymes to determine their effect on the integration of other sensory systems that converge on these regions. Notably, a number of pathway 2130
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members of calcium signaling, long-term potentiation, and axon guidance have been identified in our analysis (Table 1, Figure 5). These findings also suggest that in the adult brain a sufficient number of cells synthesize axon guidance molecules and therefore retain plasticity potential. Localization of these molecules in the cells will provide further clues about the subset
research articles
Proteomics of Brain Regions of Zebra Finch of cells that synthesize these proteins and their target cells involved in rewiring during different instances of plasticity. An understanding of molecular players in neurological disorder or diseases will help expand our understanding of these diseases. At the molecular level, proteins which are critical for normal and aberrant functions of neurological systems remain poorly understood. Studies with model organisms may help to identify homologues and allow the determination of their role in system function, integration, and system aberration, which results in pathologic state. It is important to recognize that the identical capture method reproducibly shows a different pattern of proteins in different regions of brain. The proteome of the primary visual pathway5,24 shows members of KEGG pathways with frequencies different from those of other regions, for example, the OV and Field L regions (Supplementary Figure 1 in Supporting Information) indicating their inherent differences. The alignment of proteins within specific neuronal cell types and neuronal circuits will eventually allow the integration of the molecular level of analysis with the functional level in different pathways. Further rigorous investigation along these lines is expected to enable the formulation of a crude map of pathways in select discrete regions. Further studies using these identified molecules will also lead to a better understanding of the integration of these sensory systems. The molecular identification of proteins is the first step toward utilizing other methods in the determination of their cellular localization and probing their role.
Conclusions In summary, we have identified 79 proteins in the T. guttata OV (36 proteins) and Field L (72 proteins) regions with a high degree of confidence. The proteome analysis of the different brain regions will provide insight into their normal function as well as further our understanding of song learning, song control, and speech development. Knowledge of the proteome may also provide a basis for further analyses and therapeutic interventions in disease states. The T. guttata genome sequencing is currently underway and our mass spectrometric data will assist in the detection of bona fide proteins when the T. guttata genome sequence becomes available. The analyses confirmed the presence of a number of proteins at the protein level and complemented the genomic database. Identified peptides provide definitive information about the lack of post-translational modification on them, which has consequences for developing antibodies. The T. guttata brain proteome will also allow the comparison of other proteomes from this organism and others, including the comparison of different brain regions or from control and treated groups in different neurobiological studies and studies pertaining to integration of sensory systems.
Acknowledgment. This work was partly supported by grants from American Health Assistance Foundation (Thomas Lee Award to SKB) and NIH grants R21MH073900 (S.A.H.), S10 RR019382, and P30 EY014801, an unrestricted grant to the University of Miami and Career Award (S.K.B.) from Research to Prevent Blindness (RPB). During summer, A.B. and M.K. were supported by an HHMI internship. We thank Valerie Cavett and Mabel Algeciras for their superb technical assistance. Authors thank Drs. D. F. Clayton, Sarah London, Michael Gaines, William Searcy and John Metz. Supporting Information Available: The Supplementary Table 1, Supplementary Figures 1 and 2, and Supplemen-
tary documents 1–3. This material is available free of charge via the Internet at http://pubs.acs.org.
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PR7008687
