A Chick Retinal Proteome Database and Differential Retinal Protein

A Chick Retinal Proteome Database and Differential Retinal Protein Expressions during Early Ocular Development Thomas C. Lam,† King-Kit Li,† Samuel C. L. Lo,*,‡,| Jeremy A. Guggenheim,§ and Chi Ho To*,†,| Laboratory of Experimental Optometry, Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China, The Proteomic Task Force, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, China, The State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Shenzhen, China, and Department of Optometry & Vision Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, United Kingdom Received August 24, 2005

Proteomics approach as a research tool has gained popularity in a growing number of basic and clinical researches. However, proteomic research has yet to gain significant momentum in eye research. Hence, we decided to build a retinal proteome database using postnatal retinal tissue from chick, a commonly used animal model in eye research. Employing 2-D gels with the coverage of 3-10 pH gradients, we were able to resolve hundreds of proteins from young chick retinae. Among them, 155 high abundant proteins were identified by Peptide Mass Fingerprinting (PMF) after the Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS). These proteins were then classified according to their functions. Making use of the retinal database, we were able to identify several differentially expressed proteins that might be involved in early retinal development by comparing the 2-DE maps of chick retinal tissues (3, 10, and 20 days after hatching). With the current proteomics approach, we not only documented the most abundant soluble proteins in the chick retinal tissue, but also demonstrated the dynamic protein expression changes during early ocular development. This represents one of the first steps in building a complete protein database in chick retinae which is applicable to the study of eye diseases from a few selected protein candidates to the whole proteome. Proteomic technology may provide a high throughput platform for advancing eye research in the feasible future. Keywords: two-dimensional gel electrophoresis • chick • development • proteomics • proteome • retina • mass spectrometry

1. Introduction Proteomics leads to a fundamental paradigm shift in research in the post-genomic era. The advances in the two-dimensional gel electrophoresis (2-DE) coupled with the mass spectrometry (MS) technology have been the driving force which revolutionizes the study of proteins into a high-throughput and accurate endeavor. The ability to profile many proteins has allowed global changes in protein expression to be studied at one setting. 2-DE/MS technology has been widely applied in * Authors for correspondence. Prof. Chi-Ho To, School of Optometry, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China; e-mail, [email protected]; tel, (852) 2766 6102; fax, (852) 2764 6051. Prof. Samuel Chun-Lap Lo, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China; e-mail, [email protected]; tel, (852) 34008669; fax, (852) 2364 9932. † School of Optometry, The Hong Kong Polytechnic University. ‡ Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, and The State Key Laboratory of Chinese Medicine and Molecular Pharmacology. | Both author contributed equally. § Cardiff University. 10.1021/pr050280n CCC: $33.50

 2006 American Chemical Society

identifying novel markers and complex protein pathways to develop new molecular-based therapies, for example, the search for novel targets for cancer therapy,1-4 Alzheimer’s disease study,5,6 Severe Acute Respiratory Syndrome disease (SARS) study,7 and so on. However, its application on eye research is yet to be popularized. The retina is part of the central nervous system (CNS), and it is a complex and important component of the vertebrate’s visual system. The retina in most vertebrates is composed of three layers of nerve cell bodies and two layers of synapses. It is a visible neural extension of the brain which consists of a complex neural network and different cellular microcircuits (information on the basic anatomy of the eye can be obtained from, for example, http://thalamus.wustl.edu/course/ eyeret.html). The retina has long been one of the classic models for embryological and neuronal researches. Topics such as the organizations and the signaling pathways involving precise interconnections through photoreceptors, bipolar to ganglion cells and laterally between amacrine and horizontal cells, have been the prime interest of many vision researchers. In addition Journal of Proteome Research 2006, 5, 771-784

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Published on Web 02/22/2006

research articles to its importance for vision, the retina was found to be involved in the control of eye growth in many animal species, including chick (Gallus gallus). Chick has long been one of the classical models for the studies of many biological systems,8-10 and its role in the studies of visual functions such as in myopia research11-14 is becoming more prominent. Interestingly, matching between the retina and the image plane (or emmetropization) during postnatal development is found to be fairly independent of signals from the higher order, that is, the brain. Therefore, it is believed that postnatal eye growth is regulated by retinal local processing of visual information. In fact, there is growing evidence to support a major role for the retina in modulating eye growth, although a credible theory on the exact mechanism remains elusive. Retinal development is known as a multiple step process which involves a dramatic increase of complexity and differentiation of many cell types. These events are mostly regulated at the transcriptional level. Studies focusing on the regulatory mechanisms within the retina have been the subject of intense investigation for more than a decade. There was an early report on cultured embryonic chick retina by the 2-DE approach.15 However, only one protein (glutamine synthetase) was studied due to the technological limitations in the gel-based protein separation plus a lack of MS-based protein identification. Today, information on the post-hatching avian retinal growth at the protein level is still very limited. Therefore, we propose to study the retinal growth with a proteomic approach. The present work aims to build the first proteome database for the chick retina. This information may provide a platform for studying or comparing the differential protein expression in the normal ocular growth in chicks and other animal species.

2. Materials and Methods 2.1. Animal and Tissue Preparations. The care and use of the animals in these experiments were in accordance with the ARVO resolution on the Use of Animals in Research and in compliance with university guidelines set forth by the Animal Subjects Research Ethnic Subcommittee (ASEC). Newly hatched white leghorn chicks were kept in temperature-controlled brooders on a 12/12 h light/dark cycle and fed food and water ad libitum. Refractive error changes after hatching and before dissection were measured using a streak retinoscope. The resultant equivalent sphere (S.E. ) spherical power + 1/2 cylindrical power) was calculated based on the two powers along the vertical and horizontal meridians. Chicks were sacrificed by a CO2 overdose, and the eyes were enucleated promptly within 5 min. After removing the extraocular tissues, the eyes were hemisected near to the equator. The anterior segment and vitreous were discarded. The retinal layer was carefully peeled off from the posterior hemisphere, and visible retinal pigment epithelial (RPE) cells attached were either isolated from the retinal tissue or blotted away using Kimwipes (Kimberly Clark Corp.). Tissues were immediately frozen in liquid nitrogen and then stored at -80 °C before further analysis. No sample was stored for more than 12 months. Unless otherwise stated, chemicals of the highest purity available were used. Reagents for 2-DE and silver staining were purchased either from Bio-Rad or Amersham Biosciences (China). Chemicals used for MALDI-TOF MS were either from Sigma-Aldrich or Bruker Daltonics (Germany). A frozen retinal sample was homogenized in a liquid nitrogen-cooled Teflon freezer mill (Mikrodismembrator; Braun Biotech, Germany) with 300-500 µL of lysis buffer containing 772

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7 M urea, 2 M thiourea, 40 mM Tris, 0.2% (w/v) Biolyte, 1% (w/v) DTT, 2% (w/v) CHAPS, 1% (v/v) ASB14 (Calbiochem) and 1% (v/v) Protease Inhibitor Cocktail (cat. no. P8340, SigmaAldrich) for a total of 5 min at the highest speed. The sample was collected, vortexed briefly, and then centrifuged at 16.1 × 1000 G force for 20 min at 4 °C. The supernatant was collected, and the pellet was discarded. To perform reagent compatible protein quantification, the amount of soluble protein recovered was measured by a 2-D Quant Kit (Amersham Biosciences, China) 2.2. 2-D Gel Electrophoresis. 2.2.1. Development of Chick Retinal Proteome Database. For a quicker process of proteome database development, a robotic platform was first employed to investigate the most abundant protein spots on a colloidal coomassie blue-stained 2-D gel. As the protein recovery for MALDI-TOF MS using the robotic system is not as good as the manual processing due to intrinsic limitations of the system, a number of silver-stained 2-D gels were later prepared for manual in-gel tryptic digestion on those spots which failed to be identified in the robotic PMF run. As refractive errors in chicks were relatively stabilized and the hyperopic variability was largely reduced 1 week after hatching, 7-day-old chicks were selected for building the 2-D retinal proteome database. Soluble retinal proteins extracted from the young chicks (50 or 300 µg) were added to 300 µL of rehydration buffer containing 7 M urea, 2 M thiourea, 0.2% (w/v) Biolyte, 1% (w/v) DTT, 2% (w/v) CHAPS, and 1% (v/v) ASB14 with a trace amount of bromophenol blue. Isoelectric focusing (IEF) was performed using linear IPG strips (17 cm long) with pH gradients of 3-6, 5-8, and 3-10. They were actively rehydrated at 50 V in PROTEAN IEF Cell (Bio-Rad) under constant temperature (20 °C) for 16 h to enhance protein uptake. Subsequently, the protein samples were focused for a total of 42 000 V/h using a linear voltage ramp: 100 V for 2 h, 500 V for 1 h, 100 V for 1 h, 4000 V for 2 h, and finally 8000 V for 6 h. Following IEF, the IPG strips were incubated in the equilibration buffer (6 M urea, 30% glycerol, 50 mM Tris, and 2% SDS) containing 2% DTT for 15 min and then 2.5% iodoacetamide for an additional 15 min. The focused IPG strips were rinsed briefly with Milli-Q H2O and sealed with 0.5% agarose on the top of a 1 mm thick, 25 cm × 20 cm SDS-PAGE gel. Separation of the proteins in the second dimension was achieved by using 12% continuous gels in the Dodeca tank (BioRad) and running at a constant 200 V until completion. 2.2.2. Investigation on Retinal Protein Expressions during Ocular Development. For comparative age-matched animals being used in our myopia study, normal chicks with similar body weight in each well-separated time point of 3-day old (PN3), 10-day old (PN10), and 20-day old (PN20) after hatching were used to study the differential protein expression involving eye development. It was also reported that the eye growth during this experimental period was almost linear.16 The retinal proteins were extracted as described before. Equal portions of protein from each chick (20 µg each) were pooled (total ) 20 µg × 3 ) 60 µg) for each of the 3 time points. 2-DE separations were carried out as described previously using 17 cm IPG strips of pI ranges from pH 3-10. Furthermore, to better visualize protein spots, we repeated the differential protein expression study on the PN3 and PN20 samples using narrow 11 cm IPG strips of pH 5-8. A total of 36 000 V/h was used in the shorter IPG strips. To minimize experimental variations, all samples to be compared were processed and run in the 2-DE systems

A Chick Retinal Proteome Database

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at the same time (17 cm IPGs were run together in parallel, while paired 11 cm IPGs were run side by side on a single gel). 2.3. Protein Visualization and Image Analysis. Proteins profiles in differential expression study were routinely visualized with silver stain performed according to the MS compatible silver staining method,17 except that overnight methanol and acetic acid fixation were adopted and sodium acetate was omitted in our protocol according to our previous optimization result. When colloidal coomassie blue stain was used for the robotic protein identification test, 2-D gels were soaked in 0.0025% (w/v) hot (∼90 °C) R350 (cat. no. 17-0518-01, Amersham Biosciences, China) solution in 10% acetic acid for 10 min and then destained with 10% acetic acid overnight. Stained gels were scanned as uncompressed “TIFF” images at 300 dpi (pixels per inch) resolutions with a flatbed scanner “ScanMaker 4600” from Microtek (Taiwan). The gel images were then exported directly to the ImageMaster 2D Platinum software ver.5 (Swiss Institute of Bioinformatics, Sweden) for protein spots detection, quantification, and annotation. 2.4. Protein Identification. 2.4.1. Manual Processing. Protein spots were excised manually from the 2-D gels before they were washed with milli-Q water and 50 mM NH4HCO3/ACN (1:1, v/v) for 15 min respectively, and then washed with ACN. In the reduction-alkylation steps that followed, the gel plugs were first incubated at 56 °C in 10 mM DTT/25 mM NH4HCO3 for 45 min and further incubated in dark at room temperature in 55 mM iodoacetamide/25 mM NH4HCO3 for another 45 min. After removal of the solution, ACN was used to wash the gel plugs before drying in a vacuum SpeedVac. Finally, the gel plugs were incubated in 10 ng/µL sequencing grade trypsin (Promega) in 25 mM NH4HCO3 at 37 °C overnight. Peptide extraction was performed with ACN/0.1% TFA (1:1 v/v) under sonication for three cycles. The collected “supernatant” was concentrated to 3-5 µL in a vacuum centrifuge. The sample (0.5 µL) was mixed with 2 µL of HCCA stock matrix solution [1 mg HCCA/500 µL ACN and then mixed with 0.2% TFA (1:1, v/v)]. They were allowed to air-dry on an AnchorChip (400 or 600 µm spot size) (Bruker Daltonics, Germany), a prestructured MALDI sample support which was proven to enhance the sensitivity of detection.18,19 Mass spectra were acquired using a Bruker Autoflex MALDI-TOF mass spectrometer under the positive ion reflectron mode. The time-of-flight was measured using the following parameters: 19 kV Ion source 1 (reflector), 16.55 kV Ion source 2 (reflector detector), 1400 V detector gain voltage offset, 5 Hz laser frequency, 80 ns plused ion extraction. Nonlinear external calibration was done with a calibrant mixture containing angiotensin II (m/z 1046.5418), angiotensin I (m/z 1296.6848), substance P (m/z 1347.7354), bombesin (m/z 1619.8223), ACTH (fragment 1-17, m/z 2093.0862), and ACTH (fragment 18-39, m/z 2465.1983). The spectra were further calibrated internally against the autoproteolytic trypsin fragments of m/z 842.509 and m/z 2211.104. The resulting peak lists from 800 to 3000 m/z for all samples were generated by the FlexAnalysis 2.0 software (Bruker Daltonik, Germany) and searched against the Swiss-Prot and NCBInr sequence databases via the Mascot search engine online (http://www. matrixscience.com) or through the m/z software, Biotools 2.0 (Bruker Daltonik, Germany) after removing all known contaminant peaks. For database entry, we restricted the taxonomy to Metazoa (animals) and allowed a maximum of 1 missed cleavage. The protonated molecule ion “MH+” and “Monoisotopic” were defined for the peak mass data input. The potential chemical modifications of a peptide such as the alkylation of

Figure 1. 2-D coomassie-stained chick retinal proteins profile (∼300 µg) resolved in 12% acrylamide gel (20 cm × 25 cm) between pH 3-6. The annotated spots were excised, and the proteins were identified by PMF using MALDI-TOF MS (Table 1).

a cysteine [Carbamidomethyl (C)] and the oxidation of a methionine residue [Oxidation (M)] were also considered in the search. For all mass lists, the peptide tolerance/error was at most 100 ppm and no restriction was applied for both the protein isoelectric point and molecular weight. 2.4.2. Processing Using the Robotic System. Protein spots identification of coomassie-stained gels were subjected to automatic processing using a robotic PROTEINEER Line (Bruker Daltonics, Germany). In brief, images of 2-D gels were first scanned, and the spots subjected for protein identification were located and assigned on screen as a picking list in the protein spot picking station. Relevant gel spots were then excised automatically by a robotic picker (PROTEINEER SP) into a 384well microtiter plate. Proteolytic peptide fragments of transferred protein spots were generated by a programmed protocol in the temperature-controlled PROTEINEER DP Digest&Prep station where the washing, in-gel digestion, and incubation steps were sequentially performed. The protein samples and calibrants were subsequently spotted and dried on an Anchor target plate (600 µm spot size) using the “thin-layer affinity preparation” approach.20 Automatic MALDI-TOF peptide acquisition was carried out in the Autoflex MS based on a realtime Fuzzy logic feedback control engine according to a predefined optimized protocol. Acquired peak lists were used as inputs for onsite intranet database search. The searching criteria were similar to that in the manual search except e150 ppm peptide tolerance/error was allowed. Protein identification was evaluated based on the predefined matching criteria similar to that of the manual search. A second phase of manual exam was carried out afterward to ensure that there was no missed laser hit due to any irregular matrix formation or system misalignment. Journal of Proteome Research • Vol. 5, No. 4, 2006 773

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Figure 2. Merged 2-D coomassie-stained chick retinal proteins profile (∼300 µg) resolved in 12% acrylamide gel (20 cm × 25 cm) between pH 5-8 with a separated section covering pH 8.5-10. The annotated spots were excised, and the proteins were identified by PMF using MALDI-TOF MS (Table 1.)

3. Results and Discussion 3.1. Development of Chick Retinal Proteome Database. The construction of a 2-D map is commonly the first step to allow a systematic study of a particular tissue in the proteomic approach. Master electronic gels (created using imaging software, Adobe Photoshop) of coomassie-stained 2-D chick retinal profiles covering a pH range from 3 to 10 are shown in Figure 1 and Figure 2. A total of 350 distinct protein spots were detected and most of them (∼71%) fell in the pH 5-8 range. Out of the 160 higher abundant spots picked within this range, 105 (66%) proteins could be identified by the robotic system as a significant match. For those unmatched spots, 32 of them (20%) failed with no peptide spectra generated after MALDITOF MS. This problem could be the result of insufficient trypsin in-gel digestion, uneven on-chip analyte/matrix mixture formation, or simply due to an insufficient amount of sample for identification. These unidentified proteins were trypsindigested again manually using gel plugs from coomassie- or silver-stained gels. All successful protein identifications after MALDI-TOF MS were annotated to the master gels. A total result of 155 retinal proteins identified are listed in Table 1 with their associated information and cross references in the protein sequencing databases. As expected, the average-matched amino acid sequence coverage was found to be higher for protein using gel plugs from coomassie-stained gel (∼36%) when 774


compared to that using silver-stained gel plugs (∼27%). There were 38 spots (∼25%) found with more than one unique significant match in the NCBI database (The National Center for Biotechnology Information search systems). Yet, those “parallel” matches were found either belonging to the multiple species of the same protein or fell into the same family of a particular group of protein. The major difficulty in automatic PMF identification using the robotic system was the large peptide m/z error encountered in using external spectra calibration alone. We found a significant improvement in the database match with the second phase exam by assigning the trypsin autoproteolytic fragments as internal calibration. The remaining identifications for those unmatched protein either require a database search later for more complete sequence libraries or the use of de novo microsequencing approaches such as ESI-MS/MS or PSD-MALDI-TOF. Success of protein identification using the proteomics approach relies heavily on the availability of the genomic data. Chicken has long been the primary model for embryology and development. In response to the growing need for a complete chicken genome, which was accelerated by the recent outbreaks of avian flu, a large-scale sequencing of the chicken genome finally began by a consortium of scientists in early 2003. With the help of whole genome shotgun approach, it was noted that the available genome data for chicken had rapidly

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A Chick Retinal Proteome Database Table 1. A List of Chick Retinal Proteins Identified by MALDI-TOF MS with Their Annotations

spot NCBI no. GI no.a 1 2 3 4

3822553 45383562 63516 45382769

5 6

44969651 3023865

7

63739 2144546 21703694

8 9 10

52138699 71575 63070 50755475

11

45382339

12

50732409 138533 50804057

13 14 15 16 17

50732728 45382393 45382393 53130278 50804057

18 19

50732409 138533 1149509

20 21

86169 45383996

22

50753611

23 24

7960152 50053682 50734127

25

50754937

26

50758188

27

50758188

28

53131390 6756039

29

50758472

30

108451 53129746

31 32 33

45384332 46048866 50758681

34

7522621

35 36 37 38

45382251 53126513 481203 45382893 50807165

39

50809985

40

7689363

NREF entryb

Mascot protein name nuclear calmodulin-binding protein tumor rejection antigen (gp96) 1 heat shock protein 90 heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) calreticulin Guanine nucleotide-binding protein G(q), R subunit prolyl-4-hydroxylase (AA 5-494) protein disulfide-isomerase (EC 5.3.4.1) precursor cognin/prolyl-4-hydroxylase/protein disulfide isomerase tubulin, β 2 tubulin R chain, chicken (fragment) unnamed protein product predicted: similar to unc-84 homologue A chromatin assembly factor 1 p48 subunit predicted: similar to vimentin, chicken Vimentin L: similar to ATP synthase β chain, mitochondrial precursor, partial similar to Secernin 1 γ-subunit of enolase γ-subunit of enolase hypothetical protein predicted: similar to ATP synthase β chain, mitochondrial precursor, partial predicted: similar to vimentin, chicken Vimentin 37 kD Laminin receptor precursor/ p40 ribosomal associated protein actin type 5, cytosolic nucleophosmin; nucleolar phosphoprotein B23 predicted: similar to guanine nucleotide binding protein R oB leukemia-associated phosphoprotein p18 stathmin predicted: similar to Werner helicase interacting protein 1; Werner syndrome homologue (human) interacting protein predicted: similar to clathrin, light polypeptide isoform a; clathrin light chain LCB predicted: similar to isoform of 14-3-3 protein predicted: similar to isoform of 14-3-4 protein hypothetical protein tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, θ polypeptide predicted: similar to 3-monooxgenase /tryptophan 5-monooxygenase activation protein, γ polypeptide; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, γ polypeptide; 14-3-3 protein γ 14-3-3 protein, bovine hypothetical protein: predicted: similar to Proteasome subunit R type 3 (Proteasome component C8) (Macropain subunit C8) (Multicatalytic endopeptidase complex subunit C8) Calretinin peptide elongation factor 1-β predicted: similar to 14-3-3 protein β/R (Protein kinase C inhibitor protein-1) (KCIP-1) (Protein 1054) ubiquitin carboxy-terminal hydrolase-6 (EC 3.1.-.-) ubiquitin carboxyl-terminal hydrolase-6 hypothetical protein SNAP-25 protein CaBP-28 predicted: similar to ζ proteasome chain; PSMA5, partial predicted: similar to tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, ζ polypeptide, partial GTP-binding protein RAB3A

50751596 predicted: similar to RAB3C, member RAS oncogene family

MOWSE intensity seq. Mascot scorec coverage coverage pI d

Mascot MW (Da)e

organismf

NF00047248 NF00047483 NF00046697 NF00047880

122 118 203 237

64.1% 50.2% 93.1% 87.7%

15.9% 22.6% 21.2% 37.9%

4.68 4.81 5.01 5.12

84697 91446 84466 72088

G. gallus G. gallus G. gallus G. gallus

NF01896949 -

121 106

86.5% 85.9%

17.1% 26.3%

4.41 5.18

NF00047886 -

106 105

77.7% 77.7%

21.8% 20.8%

4.66 4.69

47079 G. gallus 41211 Mizuhopecten yessoensis 55174 G. gallus 57773 G. gallus

NF01028482

104

77.7%

20.3%

4.75

NF00050017 NF00047000 NF01957260

160 169 169 80

62.3% 64.1% 64.1% 72.1%

34.8% 43.3% 43.2% 18.2%

NF00050581

100

58.2%

NF01955811 NF01959240

83 82 150

NF01954659 NF00048684 NF00048684 NF02010384 NF01959240

Swiss-Prot/ Swiss-Prot/ TrEMBL accession UniProt entry nameh no.g Q9YHD2 P08110 P11501 Q90593

Q9YHD2 ENPL•CHICK HS9A•CHICK GR78•CHICK

Q6EE32 O15975

Q6EE32 GBQ•PATYE

P09102

PDI•CHICK

58896 G. gallus

-

-

4.78 5.00 4.96 5.55

50377 46256 46385 48372

P32882 P02552 -

TBB2•CHICK TBA1_CHICK -

23.1%

4.74

47862 G. gallus

Q9W7I5

Q9W7I5

86.0% 86.0% 73.6%

22.2% 22.2% 34.4%

5.24 5.09 5.16

53178 G. gallus 53167 G. gallus 50476 G. gallus

P09654 Q9PTY0

VIME•CHICK ATPB_CYPCA

136 232 232 148 137

87.3% 93.2% 83.9% 83.2% 72.1%

21.0% 59.2% 58.3% 35.5% 36.6%

4.86 4.84 4.84 5.59 5.16

69846 47621 47621 56650 50476

O57391 O57391 Q5ZLC5 -

ENOG•CHICK ENOG•CHICK Q5ZLC5 -

NF01955811 NF00046560

234 218 117

79.3% 82.7% 94.2%

52.1% 50.7% 33.8%

5.24 5.09 4.8


P09654 P50890

VIME•CHICK RSSA•CHICK

NF00050094

126 92

85.0% 82.6%

39.0% 19.7%

5.08 4.66

40352 G. gallus 32840 G. gallus

P53478 P16039

ACT5•CHICK NPM•CHICK


G. gallus G. gallus G. gallus G. gallus G. gallus

NF01954408

136

51.2%

30.4%

5.15

38942 G. gallus

P08239

GB01•BOVIN

NF00047962 NF00048773 NF01956527

94 86 82

80.2% 80.2% 67.0%

34.2% 25.7% 16.2%

6.41 6.18 7.88


Q9I895 P31395 -

Q9I895 STN1•CHICK -

NF01961108

128

20.4%

31.9%

4.6

23164 G. gallus

P08082

CLCB•RAT

NF01957009

97

69.7%

35.3%

4.67

29322 G. gallus

P62258

143E•HUMAN

NF01957009

143

72.7%

49.0%

4.67

29322 G. gallus

P62258

143E•HUMAN

NF02011439 NF00511802

123 125

67.5% 67.5%

47.8% 47.8%

4.68 4.69

28050 G. gallus 28046 Mus musculus

Q5ZKC9 P68254

Q5ZKC9 143T•MOUSE

NF01949829

90

54.6%

9.3%

5.29

P61981

143G•HUMAN

NF01947409

172 112

83.6% 74.3%

47.2% 26.7%

4.75 4.93

28272 Bos taurus 28691 G. gallus

P61981 Q5ZLI2

143G•HUMAN Q5ZLI2

NF00048535 NF00050035 NF01961224

158 95 190

85.2% 80.8% 100.0%

55.0% 24.2% 39.2%

5.10 4.54 4.82


P07090 Q9YGQ1 P68251

CLB2•CHICK Q9YGQ1 143B•SHEEP

-

107

89.9%

36.1%

4.91

26443 G. gallus

-

-

NF00048819 NF02011138 NF00050040 NF01963032

83 86 118 168 102

71.1% 81.4% 77.0% 90.3% 63.5%

27.4% 34.8% 42.6% 47.7% 33.8%

4.91 5.22 5.29 4.72 4.75

26469 23317 28835 30376 23632

Q9PW67 Q5ZMT1 P60878 P04354 P28066

Q9PW67 Q5ZMT1 SN25•CHICK CABV•CHICK PSA5•HUMAN

NF01956387

88

47.5%

49.5%

4.16

11817 G. gallus

P63103

143Z•BOVIN

NF00112272

131

83.2%

33.2%

4.8

P63012

RB3A•RAT

NF01951706

88

76.4%

14.3%

5.46

25198 Homo sapiens 44753 G. gallus

-

-

101356 G. gallus

G. gallus G. gallus G. gallus G. gallus GG. gallus

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Table 1 (Continued)

spot NCBI no. GI no.a 41 42 43 44 45 46 47 48 49 50 51 52 53 54

55 56 57 58

59 60 61 62 63 64 65 66 67

68

69 70 71 72

73 74 75 76

776

NREF entryb

Mascot protein name

227016 apolipoprotein AI 45382329 growth-related translationally controlled tumor protein 86482 visinin, chicken 45382903 calcium binding protein 45382773 β-synuclein 6440479 hippocalcin-like protein 3 45384366 calmodulin 50797888 predicted: similar to valosin precursor, partial 50746361 predicted: similar to Osmotic stress protein 94 (Heat shock 70-related protein APG-1) 50750447 predicted: similar to cytoplasmic dynein intermediate chain 2C 50747380 predicted: similar to Mitochondrial inner membrane protein (Mitofilin) (p87/89) 45384386 homogenin 50748790 predicted: similar to heat shock protein 70 45383179 collapsin response mediator protein-1B 24234686 heat shock 70kDa protein 8 isoform 2 45384370 heat shock cognate 70 50750318 predicted: similar to NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa precursor; NADH dehydrogenase (ubiquinone), Fe-S protein-1 (75 kD); NADH-coenzyme Q reductase; complex I, mitochondrial respiratory chain, 75-kD subunit; NADH dehydrogenase (ubiquinone...) 50732627 predicted: similar to Glycyl-tRNA synthetase (Glycine-tRNA ligase) (GlyRS) 50760643 predicted: similar to N-ethylmaleimide sensitive fusion protein 2996407 heat shock cognate 70 57524986 similar to Stress-70 protein, mitochondrial precursor (75 kDa glucose regulated protein) (GRP 75) (Peptide-binding protein 74) (PBP74) (Mortalin) (MOT) 53127632 hypothetical protein 882147 CRMP-62 33340025 collapsin response mediator protein-2B 37590083 heat shock protein Hsp70 882147 CRMP-62 33340025 collapsin response mediator protein-2B 50754075 predicted: similar to Lysyl-tRNA synthetase (Lysine-tRNA ligase) (LysRS) 50734929 predicted: similar to hypothetical protein MGC76252 50741749 predicted: similar to t-complex polypeptide 1 882147 CRMP-62 33340025 collapsin response mediator protein-2B 882147 CRMP-62 33340025 collapsin response mediator protein-2B 50758158 predicted: similar to chaperonin containing TCP1, subunit 6A (ζ 1); chaperonin containing T-complex subunit 6 45383890 58 kDa glucose regulated protein precursor; 1,25D3-MARRS protein; 1,25D3-membrane-associated, rapidresponse steroid-binding protein; glucose regulated thiol oxidoreductase protein 37695552 annexin 2 882147 CRMP-62 33340025 collapsin response mediator protein-2B 45383538 malic enzyme 1, NADP(+)-dependent, cytosolic 53130384 hypothetical protein 50750210 predicted: similar to 60 kDa heat shock protein, mitochondrial precursor (Hsp60) (60 kDa chaperonin) (CPN60) (Heat shock protein 60) (HSP-60) (Mitochondrial matrix protein P1) (P60 lymphocyte protein) (HuCHA60) 33340023 collapsin response mediator protein-2A 45383890 58 kDa glucose regulated protein precursor 50747490 predicted: similar to T-complex protein 1, η subunit (TCP-1-η) (CCT-η) (HIV-1 Nef interacting protein) 45384364 Rab-GDP dissociation inhibitor


Mascot MW (Da)e

organismf

Swiss-Prot/ Swiss-Prot/ TrEMBL accession UniProt entry nameh no.g

NF00048630

156 102

92.2% 78.4%

31.7% 36.6%

5.45 4.9


P08250 P43347

APA1•CHICK TCTP•CHICK

NF00050222 NF00047770 NF00126893 NF00049513 NF01959101

127 127 192 70 93 181

96.5% 96.5% 99.0% 78.5% 54.3% 91.2%

53.6% 53.6% 54.1% 27.2% 45.6% 29.8%

5.01 5.01 4.38 5.23 4.1 4.93

22611 22621 14054 22293 16842 56857

P22728 Q9I9G9 O93410 P55072

VISI•CHICK Q9I9G9 O93410 TERA•HUMAN

NF01963408

89

71.2%

9.1%

7.80

127677 G. gallus

-

-

NF01956159

75

84.5%

15.6%

5.11

68758 G. gallus

Q13409

DYI2•HUMAN

NF01953040

98

89.8%

15.0%

5.53

81689 G. gallus

-

-

NF00048747 NF01301536

90 74

76.5% 72.8%

14.8% 12.9%

5.93 5.66


O93510 Q7SX63

GELS•CHICK Q7SX63

NF01548233 NF00123249 NF00048173 NF01962763

77 99 74 73

81.2% 85.7% 71.1% 84.7%

18.7% 23.1% 15.8% 12.8%

6.58 5.62 5.47 6.18

62766 53598 71011 78755

Q71SG3 O73885 -

Q71SG3 O73885 -

NF01962981

89

100.0%

7.9%

7.14

142786 G. gallus

P41250

SYG•HUMAN

NF01962060

176

100.0%

14.7%

9.11

126242 G. gallus

P18708

NSF•CRIGR

NF00048173 -

88 170

46.2% 92.0%

20.1% 28.3%

5.47 6.09


O73885 -

O73885 -

NF02011052 NF00049381 NF01548244 NF01301536 NF00049381 NF01548244 NF01962816

170 164 164 105 84 84 87

92.0% 100.0% 100.0% 76.4% 100.0% 100.0% 90.1%

28.3% 25.2% 25.2% 17.7% 18.4% 18.4% 16.1%

6.09 5.96 6.05 5.66 5.96 6.05 5.83

73432 62619 62691 70098 62691 62619 68334

Q5ZM98 Q90635 Q71SG1 Q7SX63 Q90635 Q71SG1 -

Q5ZM98 DPY2•CHICK Q71SG1 Q7SX63 DPY2•CHICK Q71SG1

NF01950044

139

85.1%

39.2%

5.53

60190 G. gallus

-

-

NF01947807

126

82.1%

28.9%

5.50

61057 G. gallus

-

-

NF00708442 NF01548244 NF00049381 NF01548244 NF01958869

137 137 298 298 126

93.9% 93.9% 94.9% 94.9% 69.6%

24.3% 24.3% 62.2% 62.2% 24.5%

5.96 6.05 5.96 6.05 6.36

62691 62619 62691 62619 58008

G. gallus G. gallus G. gallus G. gallus G. gallus

Q53837 Q71SG1 Q90635 Q71SG1 Q5ZJ54

Q53837 Q71SG1 DPY2•CHICK Q71SG1 Q5ZJ54

NF01096103

138

90.5%

25.5%

5.76

56546 G. gallus

Q8JG64

Q8JG64

NF01442401

136

63.0%

39.2%

6.92

Q6TEQ7

Q6TEQ7

NF00049381 NF01548244 NF00775272

78 78 100

88.1% 88.1% 72.4%

13.1% 13.1% 35.0%

5.96 6.05 6.45

38915 Canis familiariz 62691 G. gallus 62619 G. gallus 62531 G. gallus

Q90635 Q71SG1 Q90XC0

DPY2•CHICK Q71SG1 Q90XC0

NF02010491 NF01952005

140 92

89.9% 51.2%

33.9% 28.0%

5.72 5.06


Q5ZL72 -

Q5ZL72 -

NF01548252 NF01096103

210 209

76.3% 92.8%

39.2% 43.2%

6.03 5.76


Q71SG2 Q8JG64

Q71SG2 Q8JG64

NF01955500

129

77.3%

32.8%

5.58

53908 G. gallus

Q99832

TCPH•HUMAN

NF00047224

227

91.6%

52.9%

5.23

51107 G. gallus

O93382

O93382


G. gallus G. gallus G. gallus H. sapiens G. gallus G. gallus

G. gallus H. sapiens G. gallus G. gallus

G. gallus G. gallus G. gallus G. gallus G. gallus G. gallus G. gallus

research articles

A Chick Retinal Proteome Database Table 1 (Continued)

spot NCBI no. GI no.a

NREF entryb

Mascot protein name

77 50806920 predicted: similar to adenosinetriphosphatase (EC 3.6.1.3) B chain, chicken 8163560 vacuolar H-ATPase B subunit osteoclast isozyme 78 50806920 predicted: similar to adenosinetriphosphatase (EC 3.6.1.3) B chain, chicken 8163560 vacuolar H-ATPase B subunit osteoclast isozyme 1085256 adenosinetriphosphatase (EC 3.6.1.3) B chain 79 50762370 predicted: similar to heterogeneous nuclear ribonucleoprotein K isoform a; dC-stretch binding protein; transformation upregulated nuclear protein 80 22093559 E. coli Ras-like protein homologue 81 50728322 predicted: similar to chaperonin containing TCP1, subunit 2 (β); chaperonin containing t-complex polypeptide 1, β subunit 82 50762370 predicted: similar to heterogeneous nuclear ribonucleoprotein K isoform a; dC-stretch binding protein; transformation upregulated nuclear protein 50800999 predicted: similar to heterogeneous nuclear ribonucleoprotein K isoform a; dC-stretch binding protein; transformation upregulated nuclear protein 83 53136380 hypothetical protein 84 2119277 β-1 tubulin, chicken 52138699 tubulin, β 2 85 1706653 R enolase (2-phospho-D-glycerate hydro-lyase) (Phosphopyruvate hydratase) 86 46048768 enolase 87 1706653 R enolase (EC 4.2.1.11) 88 1706653 R enolase (2-phospho-D-glycerate hydro-lyase) (Phosphopyruvate hydratase) 89 1706653 R enolase (2-phospho-D-glycerate hydro-lyase) (Phosphopyruvate hydratase) 90 50746016 predicted: similar to septin 6 isoform D; septin 2 91 8922712 septin 11 92 211235 B-creatine kinase 93 211235 B-creatine kinase 6573492 Chain D, crystal structure of chicken brain-type creatine kinase at 1.41 Å resolution 45384340 B-creatine kinase 94 6573492 Chain D, crystal structure of chicken brain-type creatine kinase at 1.41 Å resolution 45384340 B-creatine kinase 95 6573492 Chain D, crystal structure of chicken brain-type creatine kinase at 1.41 Å resolution 45384340 B-creatine kinase 96 50758593 predicted: similar to adenine homocysteine hydrolase 97 226855 aldolase C 98 226855 aldolase C 99 226855 aldolase C 100 226855 aldolase C 101 45382781 Glutamine synthetase 102 45383392 dimethylarginine dimethylaminohydrolase 1 103 164543 malate dehydrogenase (EC 1.1.1.37) 104 53136570 hypothetical protein 50752793 predicted: similar to isocitrate dehydrogenase 3 (NAD+) R 105 45382147 syndesmos 106 50738541 predicted: similar to N-ethylmaleimide sensitive fusion protein attachment protein β; brain protein I47; brain protein 14; β-soluble NSF attachment protein 107 50749280 predicted: similar to heterogeneous nuclear ribonucleoprotein H3 isoform a 108 50759201 predicted: similar to β 1 subunit of heterotrimeric GTP-binding protein 109 1079456 actin-capping protein β chain, splice form 2 45382141 actin-capping protein Z (cap-Z) β subunit


Mascot MW (Da)e organismf


NF01955352

259

88.0%

51.0%

5.34

55045

G. gallus

-

-

NF00050299

255

96.0%

48.8%

5.50

55357

G. gallus

Q9I8A2

Q9I8A2

NF01955352

277

94.8%

60.7%

5.34

54703

G. gallus

-

-

NF00050299

273

94.8%

60.5%

5.50

55015

G. gallus

Q9I8A2

Q9I8A2

-

273

94.8%

59.6%

5.50

55600

G. gallus

-

-

NF01954798

116

26.2%

37.7%

5.64

47467

G. gallus

P61979

ROK•MOUSE

NF01028477 NF01951848

76 117

100.0% 58.4%

11.9% 22.1%

9.5 6.01

51103 57794

G. gallus Q8JIF5 H. sapiens P78371

Q8JIF5 TCPB•HUMAN

NF01954798

127

47.8%

35.6%

5.64

47467

G. gallus

P61979

ROK•MOUSE

NF01957184

111

44.4%

35.6%

5.64

42666

G. gallus

P61980

ROK•MOUSE

NF02010807 NF00050017 -

130 151 151 128

84.8% 83.9% 83.9% 64.9%

31.1% 20.4% 20.4% 33.3%

6.02 4.78 4.78 6.17

50563 50333 50377 47617


Q5ZIC4 P09203 P32882 P51913

Q5ZIC4 TBB1•CHICK TBB2•CHICK ENOA•CHICK

NF00050013 -

72 115 124

62.8% 75.0% 71.4%

17.1% 40.8% 36.9%

6.17 6.17 6.17

47617 47617 47617

G. gallus G. gallus G. gallus

P51913 P51913 P51913

ENOA•CHICK ENOA•CHICK ENOA•CHICK

-

193

75.3%

47.9%

6.17

47617

G. gallus

P51913

ENOA•CHICK

NF01952054

105

92.8%

25.5%

6.53

49274

G. gallus

-

-

NF00131357 NF00046968 NF00046968 -

112 130 135 134

53.3% 87.9% 56.7% 56.7%

33.3% 33.8% 34.8% 34.5%

6.36 5.78 5.78 5.93

49367 42525 42525 42998

H. sapiens G. gallus G. gallus G. gallus

Q9NVA2 P05122 -

SE11•HUMAN KCRB•CHICK -

NF00047182 -

133 87

56.7% 55.7%

34.4% 20.5%

5.93 5.93

43129 42998

G. gallus G. gallus

P05122 -

KCRB•CHICK -

NF00047182 -

87 207

55.7% 86%

20.5% 46.8%

5.93 5.93

43129 42998

G. gallus G. gallus

P05122 -

KCRB•CHICK -

NF00047182 NF01958761

205 92

85.8% 88.6%

46.7% 15.6%

5.93 8.80

43129 77766

G. gallus G. gallus

P05122 -

KCRB•CHICK -

NF00050473 NF01301538

105 84 124 139 137 115

64.4% 47.2% 57.4% 58.9% 60.7% 51.0%

26.3% 36.6% 29.3% 51.1% 26.0% 33.1%

5.79 5.78 5.79 5.79 6.38 5.44

39023 39023 39023 39023 42747 31674

G. gallus G. gallus G. gallus G. gallus G. gallus G. gallus

P53449 P53449 P53449 P16580 Q7ZTS9

ALFC•CHICK ALFC•CHICK ALFC•CHICK GLNA•CHICK Q7ZTS9

NF00149009

85

76.0%

27.7%

6.15

31978

-

96 94

85.1% 85.1%

17.3% 17.6%

7.60 7.50

40727 51868

Sus scrofa G. gallus G. gallus

-

NF02011213 NF01953996

Q5ZI29 -

Q5ZI29 -

NF00046938 NF01955415

112 110

100.0% 55.2%

31.3% 41.6%

5.74 5.41

33978 33649

G. gallus G. gallus

Q9IAY5 Q9H115

Q9IAY5 SNAB•HUMAN

NF01950687

161

86.4%

49.7%

6.6

36690

G. gallus

-

-

NF01951415

117

62.4%

35.9%

5.60

38121

G. gallus

P79959

GBB1•XENLA

-

111

75.5%

25.7%

5.69

30820

G. gallus

-

-

NF00048709

110

75.5%

25.3%

5.36

31573

G. gallus

P14315

CAPB•CHICK


research articles

Lam et al.

Table 1 (Continued)

spot NCBI no. GI no.a

Mascot protein name

110 50730066 predicted: similar to pyridoxal kinase, partial 111 50730861 predicted: similar to esterase D 112 50738978 predicted: similar to Malate dehydrogenase, cytoplasmic 113 50753091 predicted: similar to Rlbp1-prov protein 114 50738541 predicted: similar to N-ethylmaleimide sensitive fusion protein attachment protein β; brain protein I47; brain protein 14; β-soluble NSF attachment protein 115 50755699 predicted: similar to RIKEN cDNA 1700012G19 116 1079456 actin-capping protein β chain, splice form 2 45382141 actin-capping protein Z (cap-Z) β subunit 117 17560844 cytochrome p450 2N1 family member (5C726) 118 50755168 predicted: similar to Voltagedependent anion-selective channel protein 1 (VDAC-1) (hVDAC1) (Outer mitochondrial membrane protein porin 1) (Plasmalemmal porin) 119 50750103 predicted: similar to methyltransferase 24 (37.8 kD) (3D495) 120 45384316 20S proteasome subunit C2 121 50760138 predicted: similar to Plateletactivating factor acetylhydrolase, isoform 1b, R2 subunit 122 50749899 predicted: similar to phosphoglycerate mutase (EC 5.4.2.1) B chain, rat 123 1334630 unnamed protein product 833606 carbonic anhydrase II (256 AA) (1 is 2nd base in codon) 46048696 Carbonic anhydrase II 124 833606 carbonic anhydrase II (256 AA) (1 is 2nd base in codon) 46048696 carbonic anhydrase II 125 833606 carbonic anhydrase II (256 AA) (1 is 2nd base in codon) 46048696 carbonic anhydrase II 126 53133098 hypothetical protein 127 50751030 predicted: similar to non-selenium glutathione phospholipid| hydroperoxide peroxidase 128 45382455 CLE7 protein 129 53133620 hypothetical protein 130 50748430 predicted: similar to Proteasome subunit R type 6 (Proteasome ι chain) (Macropain ι chain) (Multicatalytic endopeptidase complex ι chain) 131 53129115 hypothetical protein 50749899 predicted: similar to phosphoglycerate mutase (EC 5.4.2.1) B chain, rat 132 38566176 Phosphoglycerate mutase 1 49456447 PGAM1 133 1945745 chLAMP, g11-isoform 134 50732619 predicted: similar to Guk1 protein 135 45382061 Triosephosphate isomerase 136 45382061 Triosephosphate isomerase 137 50730713 predicted: similar to DNA segment, Chr 10, Johns Hopkins University 81 expressed 138 212347 myosin a1 light chain (partial) 139 45382561 GTP-binding protein 140 50740506 predicted: similar to glyoxylase 1; glyoxalase 1 141 50740506 predicted: similar to glyoxylase 1; glyoxalase 1 142 20380955 Pitpnc1 protein 143 50759776 predicted: similar to Proteasome subunit β type 2 (Proteasome component C7-I) (Macropain subunit C7-I) (Multicatalytic endopeptidase complex subunit C7-I) 144 50740506 predicted: similar to glyoxylase 1; glyoxalase 1 145 50745407 predicted: similar to nucleoside diphosphate kinase 146 42543522 Chain A, Arf1[δ1-17]-Gdp in complex with a Sec7 domain carrying the mutation of the catalytic glutamate to lysine 53127520 hypothetical protein

778

NREF entryb


Mascot MW (Da)e

organismf


NF01952121

77

100.0%

25.1%

5.68

31611 G. gallus

-

-

NF01959074 NF01950883

76 108

84.6% 84.8%

23.7% 16.4%

6.13 9.96


-

-

NF01951415

213

94.2%

51.9%

5.23

36682 G. gallus

-

-

NF01955415

195

96.3%

56.8%

5.41

33647 G. gallus

Q9H115

SNAB•HUMAN

NF01957150

97

54.6%

37.9%

4.43

22956 G. gallus

-

-

-

72

50.0%

25.7%

5.69

30820 G. gallus

-

-

NF00048709 NF00290512

71 78

50.0% 77.8%

25.3% 20.5%

5.36 6.35

NF01962011

131

97.2%

16.2%

10.03

NF01951594

127

49.3%

30.6%

6.00

30640 G. gallus

-

-

NF00048214 NF01951985

81 78

100.0% 92.4%

21.9% 39.4%

6.08 5.79


O42265 -

PSA1•CHICK -

NF01957940

102

37.8%

62.8%

8.94

23552 G. gallus

P18669

PMG1•HUMAN

NF00049577 NF00046508

111 111

93.3% 93.3%

36.9% 36.3%

6.65 6.51


-

-

NF00048355 NF00046508

111 146

93.3% 89.6%

35.8% 57.8%

6.56 6.51


P07630 -

CAH2•CHICK -

NF00048355 NF00046508

145 161

89.6% 88.6%

56.9% 67.2%

6.56 6.51


P07630 -

CAH2•CHICK -

NF00048355 NF02011821 NF01961051

161 94 114

88.6% 100.0% 73.9%

66.2% 20.8% 38.2%

6.56 5.56 8.29


P07630 Q5ZJY1 -

CAH2•CHICK Q5ZJY1 -

NF00048369 NF02011536 NF01961736

169 141 114

88.3% 49.6% 75.6%

53.6% 45.1% 37.0%

6.01 5.72 6.13


Q90706 Q5ZJF4 -

Q90706 Q5ZJF4 -

NF02011849 NF01957940

165 95

88.0% 41.5%

48.8% 38.6%

7.03 8.94


Q5ZLN1 -

Q5ZLN1 -

NF01564848 NF01838762 NF00048136 NF01960322 NF00049179 NF00049179 NF01958258

74 74 74 105 131 175 76

75.4% 75.4% 100.0% 23.3% 72.3% 84.4% 76.8%

21.3% 21.3% 20.6% 22.2% 39.1% 49.6% 33.8%

7.07 6.67 6.47 6.39 6.71 6.71 6.17

28828 28814 39145 45731 26832 26832 24486

Q6P6D7 Q6FHK8 O02869 P00940 P00940 -

Q6P6D7 Q6FHK8 O02869 TPIS•CHICK TPIS•CHICK -

NF00047871 NF00050313 NF01959509

83 74 113

79.9% 94.1% 70.8%

31.1% 14.6% 31.1%

4.72 5.98 6.1


P02604 Q90965 -

MLE1•CHICK Q90965 -

NF01959509

147

79.4%

52.8%

6.1

20654 G. gallus

-

-

NF01009299 NF01951144

87 77

68.3% 83.5%

60.6% 19.4%

8.82 9.18

11315 M. musculus 35325 G. gallus

Q8K165 -

Q8K165 -

NF01959509

111

92.4%

42.2%

6.10

20654 G. gallus

-

-

NF01959340

31573 G. gallus P14315 53839 Caenorhabditis O16672 elegans 101893 G. gallus P21796

H. sapiens H. sapiens G. gallus G. gallus G. gallus G. gallus G. gallus

CAPB•CHICK O16672 POR1•HUMAN

145

100.0%

41.8%

5.55

17440 G. gallus

-

-

-

98

79.2%

57.9%

5.63

18846 Bos taurus

P84077

ARF1•HUMAN

NF02011224

94

79.2%

52.5%

6.84

20850 G. gallus

Q5ZMA0

Q5ZMA0


research articles

A Chick Retinal Proteome Database Table 1 (Continued)

spot no. 147

148 149 150 151 152 153 154 155

NCBI GI no.a

NREF entryb

Mascot protein name

42543522 Chain A, Arf1[δ1-17]-Gdp in complex with a Sec7 domain carrying the mutation of the catalytic glutamate to lysine 53127520 hypothetical protein 45382979 destrin; actin depolymerizing factor 2134412 superoxide dismutase (EC 1.15.1.1) (Cu-Zn), chicken 56961659 eye-globin 45384320 cl. 453 fatty acid or lipid binding protein 45382717 protein kinase C inhibitor 45383752 creatine kinase, mitochondrial 1 1545942 mitochondrial creatine kinase 45384486 PGK protein 45384208 lactate dehydrogenase A

-


Mascot MW (Da)e organismf


89

75.4%

45.1%

5.63

18846

B. taurus

P84077

ARF1•HUMAN

NF02011224 NF00050525 -

85 105 85

75.4% 52.8% 78.8%

45.1% 43.6% 35.3%

6.84 7.52 6.12

20850 18920 15834

G. gallus G. gallus G. gallus

Q5ZMA0 P18359 -

Q5ZMA0 DEST•CHICK -

NF02189843 NF00049646 NF00047299 NF00048372 NF00047022 NF00048094 NF00046645

111 139 99 158 146 105 203

60.6% 93.7% 67.8% 88.7% 85.8% 90.7% 95.7%

39.7% 72.0% 56.3% 33.3% 39.2% 20.6% 41.0%

5.48 5.62 6.28 8.74 6.77 8.31 7.75

17353 14900 13864 47473 40774 45087 36776

G. gallus G. gallus G. gallus G. gallus G. gallus G. gallus G. gallus

Q05423 P70079 P51903 P00340

Q5QRU6 FABB•CHICK Q9I882 KCRU•CHICK PGK•CHICK LDHA•CHICK

a The GI number (GenInfo Identifier) is assigned to each nucleotide and protein sequence accessible through the The National Center for Biotechnology Information search systems. b The PIR (Protein Information Resource) is a nonredundant reference protein database located at Georgetown University Medical Centre. c Score for the protein with significant match calculated by Mowse scoring algorithm in the Mascot system. d ,eTheoretical values of the isoelectric point and molecular weight obtained in the database search using Mascot system. f The species identified with significant score for a particular protein. g Swiss-Prot is a protein knowledgebase maintained collaboratively by the Swiss Institute of Bioinformatics and the The European Bioinformatics Institute; TrEMBL is a computer-annotated supplement of Swiss-Prot that contains all the translations of EMBL nucleotide sequence entries not yet integrated in SwissProt. h The UniProt (Universal Protein Resource) is a comprehensive catalog of protein information created by the consortium members PIR, European Bioinformatics Institute, and Swiss Institute of Bioinformatics.

Figure 3. Classification of retinal proteins identified in Table 1 according to their molecular functions based on the Gene Ontology.

grown from only 1 (complete mitochondrion) to 31 (chromosomes 1-28, 32, m, and z) in a year. As a result, a huge increase in the translated proteins was also provided in the database (about 8300 proteins found in early 2004 to more than 29 000 proteins accumulated in early 2005). Database statistics are being continuously updated online at www.ncbi.nlm.nih.gov/ Taxonomy/Browser/wwwtax.cgi?id)9031. We observed a continuous improvement in the number of successful hits for our retinal protein identification in the past 24 months. Currently, only a few (∼ 5%) proteins in our protein list were identified based on homology with other species. In the functional aspect of the chick retinal proteome, we classified proteins identified into 10 groups according to their molecular functions based on the Gene Ontology at the AmiGO database (http://www.godatabase.org)21 as shown in Figure 3. Over 80% identified proteins fell into the grouping of three major molecular activities. These included “catalytic activity” (39%), “binding” (33%), and “transporter activity” (10%). Further classification for the subgroup of the major molecular

Figure 4. Subclassification of retinal proteins (of Figure 3) with catalytic function.

function in “catalytic activity” is shown in Figure 4. The major activity of the subgroup was “hydrolase activity” which functioned to hydrolyze various compounds. On the other hand, two major groups of retinal proteins were found dominating Journal of Proteome Research • Vol. 5, No. 4, 2006 779

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Table 2. Brief Functions of the Ten Abundant Chick Retinal Proteins Identified by MALDI-TOF MS NCBI GI no.

protein name

52138699

tubulin

86482

visinin

45384332, 45384366 45382773

calretinin, calmodulin beta-synuclein

45382781

glutamine synthetase

1706653

enolase

211235

B-creatine kinase

226855

aldolase

833606

carbonic anhydrase II

predicted/known functions

It is a cytoskeletal protein which is the major constituent microtubules and is found highly expressed in neuronal cells. An abundant calcium-binding protein in the eye. It is found expressed in retinal cone cells for phototransduction function. Both are calcium-binding proteins. They are homologous proteins which localized in many neurons of the CNS. It is expressed primarily in neural tissue, specifically in synapses around neurons, but not in glial cells. A retinal Muller glial-specific enzyme that amidates glutamate, a well-known retinal neurotransmitter, to glutamine. It is inducible only when Muller cells are closely associated with retina neurons. It is an essential glycolytic enzyme that catalyses the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. There are 3 different tissue-specific isoenzymes (R, β, and γ) currently known. It catalyzes the reversible transfer of high energy phosphate from ATP to creatine, generating phosphocreatine and ADP. It plays a central role in energy transduction in the brain and retina. It is one of the ubiquitous glycolytic enzymes found in the brain for glycolysis. It is one of the cytosolic isozymes of carbonate dehydratase which is responsible for the reversible hydration of carbon dioxide. It is found widespread in brain tissue.

in terms of their biological processes. Among the 100 annotated proteins according to the database (QuickGO database, http:// www.ebi.ac.uk/ego/), 30% was found to be involved in glycolysis or metabolism actions, while 20% was related to transport or signal transduction. These findings reflected the energetic property of retina as a highly differentiated neuroectodermal tissue to perform its task. In addition to common housekeeping proteins, such as heat shock proteins and the actin-related proteins, a number of high abundant retinal proteins that had been reported were readily observed in our retinal proteomic maps (Figure 1 and Figure 2). These included tubulin (spots 39, 40);22,23 visinin (spot 33);24 calretinin (spot 34), calmodulin (spot 146);25-27 β-synuclein (spot 145);28,29 glutamine synthetase (spot 61);30,31 enolase (spots 64, 65, 67, 70, 108);32 B-creatine kinase (spots 66, 71, 82, 109);33,34 aldolase C (spots 63, 84, 98),35 and carbonic anhydrase II (spots 58, 59),36 among others. A brief summary of these proteins is listed in Table 2. In the 2-DE profile, Enolase, B-creatine kinase and aldolase C showed several isoelectric variants. These suggested the existence of a number of physiological post-translational protein modifications such as protein phosphorylation where the most basic spot was the dominant one (not phosphorylated). For a better understanding of these physiological changes and to confirm the exact locations of such PTMs, further protein sequence study using LC-MS/MS will provide better hints. This study provided complement reference data to previous human RPE database37 as well as a more global profile than the previous available retinal photoreceptors38 and retinal Muller glia cells maps.39 In view of the common embryological origin between the retina and CNS, it is not surprising to see that the major retinal profile shared good similarity with that of the brain or cerebellum according to the published proteome databases.40-42 3.2. Differential Protein Expression Studies in Normal Chick Retinal Growth. It is well-known that vertebrates’ eyes of many species, such as human43,44 and chicken,45 are born 780


Mascot pI

Mascot MW (Da)

4.78

50377

5.01

22611

5.10 4.10 4.38

31171 16842 14054

6.38

42747

6.17

47617

5.78

42525

5.79

39023

6.56

28989

hyperopic (long-sighted) and they grow toward emmetropia (normal refractive status) during the early postnatal period. Our data showed that the mean refractive error (in equivalent sphere) of postnatal growth approached emmetropic from +1.88 ( 0.30 D at PN3 to +0.79 ( 0.28 D at PN20 (mean ( SEM where n)6 in each group). The above results agreed with the findings in previous studies of postnatal chick growth very well.45,46 Applying an equal amount of retinal protein extract from an individual chick (n)3 in each group), we also studied the protein profiles of the pooled retinae from PN3, PN10, and PN20 chicks using broad range pH 3-10 IPG strips and silver staining. The gel images from the left eyes were studied and shown in Figure 5A. Protein profiles among these 3 time-points were very similar to most of the proteins distributed within the pH 5-8 range. Using the gel analysis software, we detected over 500 (524 ( 31, mean ( SEM, n)3) distinct protein spots within that range. There were six spots (#D1, #D2, #U1, two spots in #U2, and #U3) found to be differentially expressed during the period investigated. The related gel image sections were enlarged and shown in Figure 5B. Similar differential protein changes were also found in the contralateral eyes, and the overall findings are summarized in Figure 5C based on the calculated “spot volume” of each specific spot [“spot volume” is the integration of optical density over the spot area where the value is calibrated against the gel background value adjacent to the spot]. To closely study the retinal protein profile within the central region of the gel where most of the protein spots were located, the pooled protein samples of PN3 and PN20 for both eyes were further separated using pH 5-8 IPG strips (Figure 6). Apart from the six differentially expressed spots detected previously, two more differentially expressed spots (#D3 and #U4) were found in the 2-D gels using this narrow pH range. Out of the 8 proteins, 6 of them could be successfully identified by MALDI-TOF MS, while very weak peptide signals could be detected in the other two spots. In Figure 5B,C, two spots showed down-regulation during the retinal growth


research articles

Figure 5. (A) Two-dimensional maps of pooled chick retinal proteins at three postnatal time points of 3-day old (PN3), 10-day old (PN10), and 20-day old (PN20). A total of 60 µg (20 µg from each individual, 3 chicks in each group) soluble proteins in each time point was separated on the IPG strips covering pH 3-10, followed by 12% SDS-PAGE. The gels were stained with MS-compatible silver nitrate solution (2-D gels for the left eye were shown as an example). Differentially expressed protein areas were shown in blocks. (B) Relevant gel sections in blocks were magnified, and the differentially expressed proteins were marked by arrows or circles. Two spots were found to be down-regulated (#D1 and #D2), whereas four spots were found to be up-regulated (#U1, #U2a, #U2b, and #U3). (C) The amount of protein changes in term of spot volume (mean ( SEM, n ) 2 for right and left eyes, each consists of a pool of three chicks), which is the integration of optical density over the spot area, for the six differentially expressed spots.

process. #D1 (Apolipoprotein AI) showed a gradual decrease in protein amount. #D2 (not identified) showed a very rapid protein down-regulation, and it could not be detected at PN20. For the up-regulated proteins, four spots included #U1 (PREDICTED: similar to glyoxylase 1; glyoxalase 1), two spots at #U2 (both were identified as phosphoglycerate mutase type B subunit, PGM-B), and #U3 (destrin; actin depolymerizing factor, ADF) showed increasing protein amount with time. The #U1

spot (PREDICTED: similar to glyoxylase 1; glyoxalase 1) was undetectable at PN3 which might be due to the limited protein expression for this particular protein at an earlier retinal differentiation stage. To validate the result of the pool protein profiles, 2-D gels of the single chick of the pool were also generated to check for the differential protein expressions. This was done to ensure that the findings were in agreement in the 3 individual chicks in the same group (data not shown). Journal of Proteome Research • Vol. 5, No. 4, 2006 781

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Figure 6. Enlarged panels showing the differential retinal protein expressions (in arrows or circles) during normal postnatal retinal growth in both eyes between PN3 and PN20. Two-dimensional gels were generated using narrower pH 5-8 IPG strips. Two down-regulated proteins (#D1 and #D2) and four up-regulated proteins (#U1, #U2a, #U2b, and #U3) were found similar to the findings in Figure 5. Three new and additional differentially expressed proteins (#D3, #U4, and arrows indicated in #U1) were found with narrow pH range (5-8).

When the narrow pH range wss used, the 6 differential expressions could be repeated again. In addition, we found an extra down-regulated spot #D3 (Fatty acid-binding protein, retina; R-FABP) as well as another up-regulated spot #U4 (not identified). Moreover, when the same region at #U1 (PREDICTED: similar to glyoxylase 1; glyoxalase 1) between gel separations using IPG strips pH 3-10 (Figure 5B) and pH 5-8 (Figure 6) was compared, an extra spot (arrows) which showed a shifting in location could only be resolved with narrow IPGs. All differential expressions detected here at least had a 2-fold difference in the calculated protein volume. With respect to the gel images, they clearly showed that a narrower “zoom-in” gel was more powerful in resolving the closely packed protein

spots in the 2-D gel analysis. The general documented functions for all identified proteins are listed in Table 3. Although a similar investigation using the proteomic approach was not yet available, we could find indirect evidence supporting our observations in some studies. Apolipoprotein AI (apo-AI) is a primary protein constituent of high-density lipoprotein (HDL). It was found that the HDL concentration in chick liver and skeletal muscle increased sharply around hatching and then decreased after 1 week of postnatal time.47,48 The Apo-AI mRNA level and its synthesis showed a similar trend.49,50 Apo-AI was also found to be developmentally regulated in the chick sciatic nerve, in parallel with the process of active myelination.51 R-FABP is the avian counterpart of murine brain FABP implicated in glial cell differentiation and neuronal cell migration. A study on chick mRNAs level also confirmed the elevation of FABPs in undifferentiated retina from embryonic day 3.5 and a subsequent decrease (50-100-fold) upon tissue maturation through day 7 to day 19. They suggested that the encoded protein/s may play an important role in the early retinal development.52,53 Localization of FABP during the early stages has also been described elsewhere.54 PGM-type B has not been well-characterized in chick tissue. Studies have shown PGM’s involvement in muscle differentiation in human muscle culture55 and rat facial development.56 It was shown to increase with age in cultured dermal fibroblasts.57 Therefore, PGM is likely to be important in the growth of retina. Glyoxalase I is not only vital in the earliest stages of embryogenesis, through maturation and development, but is also involved in aging and death.58 Although the developmental change of glyoxalase has not been previously found in the ocular system, its activity was increased in liver, spleen, and kidney of aging mice.59 Moreover, its role in the regulation of growth and differentiation in plant has been documented.60 Destrin is important for a variety of cellular processes involving actin remodeling.61 In the eye system, a close relationship between the mice destrin gene and the proliferation rate of the corneal epithelium has been found.62 ADF was believed to be the essential regulator necessary for neurite outgrowth, and its role in nervous system development was recently reviewed.63 We investigated the chick retinal protein changes in the first 3 weeks after hatching when ocular growth and development is the greatest. Matsumoto, H., et al. was one of the first groups to recognize the role of mass spectrometry in molecular biosciences using Drosophilia eye as an example.64 Cavusoglu, N., et al. had also applied the proteomic approach to analyze

Table 3. Brief Functions of the Five Differentially Expressed Proteins (Six Spots) in the Normal Postnatal Chick Retinal Growth NCBI GI no.

227016

45384320

50749899

50740506 45382979

782

expression (spot abbreviation)

protein name

down-regulation (#D1)

predicted/known functions

Apolipoprotein AI It is the major protein component of the serum high-density lipoprotein (HDL). Plasma lipoproteins serve many functions in lipid metabolism & cholesterol homoeostasis in the CNS. down-regulation R-FABP It is responsible for the intracellular transport of hydrophobic (#D3) metabolic intermediates and acts as a carrier of lipids between membranes. up-regulation PGM-type B It is the brain isoform of this enzyme for interconversion of (#U2a and #U2b) 3-phosphoglycerate and 2-phosphoglycerate during glycolysis and gluconeogenesis. up-regulation Glyoxalase I It belongs to the glyoxalase system and can be found in the (#U1) cytosol of all cells for detoxification function. up-regulation Destrin It is a mammalian actin-binding protein belongs to (#U3) superfamily of “ADF/cofilin” that rapidly depolymerizes F-actin in a stoichiometric manner. (#D2, #U4) not identified -


Mascot pI

Mascot MW (Da)

5.45

28790

5.62

14900

8.94

23552

6.10

20654

7.52

18920

-

-


the degeneration of photoreceptors using a mouse model.65 With the proteomic approach, we have not only confirmed those known proteins involved in the chick growth but also reported new proteins that may play roles in the retinal growth. Additional knowledge of the gene products as well as their temporal change may shed light on the molecular mechanisms involved in the tissue maturation. One of the aims of developmental biology is to prevent disease developed with aging. Without a more thorough understanding of pathophysiology, it is difficult to treat degenerative diseases such as glaucoma, retinitis pigmentosa, and age-related macular degeneration.

4. Concluding Remarks While a conventional approach of in situ hybridization (reviewed in refs 66, 67) or immunocytochemistry (reviewed in ref 68) allows study at the single cell level, a massive expansion of the field of proteomics has recently offered the opportunity to assess the whole tissue extracts at the cellular and subcellular levels. Proteomics approach with 2-DE allows separation and comparison of the expression profiling of a complex protein mixture. The current study has profiled the overall protein population in retinal tissue using the proteomics approach. We have also studied the differential protein expressions which are believed to be involved in the retinal maturation or the emmetropization process in the early development of chick eyes. The recent completion of the chicken (Gallus gallus) genome sequence (first draft in March 2004; The National Human Genome Research Institute, http://www. nhgri.nih.gov/11510730) has definitely facilitated the 2-DEbased protein identification by MALDI-TOF MS. Our current dataset of retinal proteome may provide a foundation for global differential proteome analysis and comparison so that targeted studies on specific group of proteins could be undertaken. By the application of a similar approach, the proteomic investigations employing chick as a myopic model are recently being explored and presented in the conference of Association for Research in Vision and Ophthalmology (E-Abstract 3144 and E-Abstract 3331).69,70 A number of the candidate proteins suggested at the preliminary finding could be matched to the current proteome database. The strength of applying “comparative proteomics” in eye research is apparent because of the presence of two bilaterally symmetrical sensory systems. Different manipulations of the two eyes of a single animal, with the same genetic make up, are possible. The same approach may not be applicable to many other cellular tissues. Therefore, the proteomics application in retina, as well as eye research, is set to provide novel and valuable information on both the normal physiology and disease processes of the eye.

Acknowledgment. This work was supported partly by a grant from “The HKPU Area of Strategic Development, Centre for Myopia Research (A360)” funding and partly by a grant G-YD29 from the Research Committee, The Hong Kong Polytechnic University. Mr. Thomas C. Lam was supported by a Postgraduate Studentship (Account No.: G-W080). Supporting Information Available: List of Chick retina proteins identified by MALDI-TOF MS with their annotations. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Dwek, M. V.; Rawlings, S. L. Current perspectives in cancer proteomics. Mol. Biotechnol. 2002, 22 (2), 139-152.

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PR050280N

A Chick Retinal Proteome Database and Differential Retinal Protein

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