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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Proteomics Clin Appl. 2015 Sep 16;9(0):1105–1122. doi: 10.1002/prca.201500006

Priorities and trends in the study of proteins in eye research, 1924–2014

Richard D Semba 1, Maggie Lam 2, Kai Sun 1, Pingbo Zhang 1, Debra A Schaumberg 3,4, Luigi Ferrucci 5,*, Peipei Ping 2,*, Jennifer E Van Eyk 6,*
PMCID: PMC4695326  NIHMSID: NIHMS707297  PMID: 26123431

Abstract

Purpose

To identify the proteins that are relevant to eye research and develop assays for the study of a set of these proteins.

Experimental Design

We conducted a bibliometric analysis by merging gene lists for human and mouse from the National Center for Biotechnology Information FTP site and combining them with PubMed references that were retrieved with the search terms “eye”[MeSH Terms] OR “eye”[All Fields] OR “eyes”[All Fields].

Results

For human and mouse eye studies, respectively, the total number of publications was 13,525 and 23,895, and the total number of proteins was 4,050 and 4,717. For proteins in human and mouse eye studies, respectively, 88.7% and 81.7% had five or fewer citations. The top fifty most intensively studied proteins for human and mouse eye studies were generally in the areas of photoreceptors and phototransduction, inflammation and angiogenesis, neurodevelopment, lens transparency, and cell cycle and cellular processes. We proposed selected reaction monitoring assays that were developed in silico for the top fifty most intensively studied proteins in human and mouse eye research.

Conclusions and clinical relevance

We conclude that scientists engaged in eye research tend to focus on the same proteins. Newer resources and tools in proteomics can expand the investigations to lesser-known proteins of the eye.

Keywords: biological processes, eye, human proteome project, proteomics, mass spectrometry, mouse

1 Introduction

Proteomics is beginning to gain greater attention in the field of eye research, owing to recent advances that have been made in protein chemistry, mass spectrometry, and bioinformatics [1]. Although proteins are an essential link between genotype and phenotype, the mechanisms by which genomic variation is translated to disease phenotypes through proteins is not well understood in general [2]. The level of complexity between the genome and specific phenotypes increases tremendously at the protein level due to protein isoforms, single nucleotide polymorphisms, post-translational modifications (PTMs), and protein degradation.

The biology and disease oriented branch of the Human Proteome Project (B/D-HPP) was organized in 2010. The goal of the B/D-HPP is to support “the broad application of state-of-the-art measurements of proteins and proteomes by life scientists studying the molecular mechanisms of biological processes and human disease. This will be accomplished through the generation of research and informational resources that will support the routine and definitive measurement of the process or disease relevant proteins.” [2]. Specifically, the B/D-HPP seeks to identify proteins that are relevant to a particular field and generate assays and reagents for these proteins [2]. The dissemination of selected reaction monitoring (SRM) assays may help accelerate research in many different fields.

Our specific aims were to identify the proteins that have been most intensively studied in eye research and provide new tools for the investigation of the top fifty proteins in human and mouse eye research, respectively. The number of scientific publications was used as the indicator of how intensively a protein was studied in eye research.

2 Materials and Methods

In order to identify the proteins that have received the greatest attention in eye research, human and mouse gene information was retrieved from the National Center for Biotechnology Information FTP site. PubMed references with the search terms “eye”[MeSH Terms] OR “eye”[All Fields] OR “eyes”[All Fields] were downloaded from PubMed. The lists of human and mouse gene were then combined to creative respective lists of proteins for human and mouse eye research, respectively. The earliest publication on PubMed was from 1813, and the earliest reference to a gene was from 1924. There were few publications prior to 1970 (only 3 for human and 24 for mouse eye research).

PANTHER was used to classify protein function. For the top fifty proteins in human and mouse eye research, respectively, heat maps were used to show the number of publications per year, and STRING was used to examine functional protein networks. NeXtProt was used as the main reference for human proteins and their associated diseases, number of isoforms, variants, and PTMs using gold level criteria. UniProt was used as the main reference for mouse proteins and their associated diseases and number of isoforms. REACTOME and Gene Ontology were used to identify groups of proteins involved in specific biological pathways studied in human eye research: complement cascade, Wnt signaling, VEGF signaling, apoptosis, visual phototransduction, etc. We did not find published SRM assays for forty-eight of the top fifty proteins in human eye research and forty-nine of the top fifty proteins in mouse eye research in the peer-reviewed scientific literature. SRM assays were constructed in silico using Skyline (MacCoss Lab, University of Washington, Seattle, WA), a commonly used theoretical prediction and selection algorithm [3] and following the guidelines for SRM assay development of Kuzyk and colleagues [4]. None of the SRM assays have been applied in vivo.

3 Results

A total of 4,050 proteins were found in human eye studies (Supporting Information Table 1). A total of 4,717 proteins were found in mouse eye studies (Supporting Information Table 2). The total number of publications for human and mouse eye studies, respectively, was 13,525 and 23,895. PANTHER was used to classify protein function for the 4,050 proteins in human eye studies (Figure 1a) and 4,717 proteins in mouse eye studies (Figure 1b). The molecular functions and detection of the top fifty human eye proteins in the different tissues and biofluids of the human eye are presented in Supporting Information Table 3. The molecular functions of the top fifty mouse eye proteins are present in Supporting Information Table 4.

Figure 1.

Figure 1

Figure 1

Pie diagram of protein functions in (a) human and (b) mouse eye research classified by PANTHER.

PAX-6 was the top among the fifty most intensively studied proteins in both human (Table 1) and mouse (Table 2) eye research. Heat maps showing the frequency of publication per year for the top fifty proteins in human and mouse eye research are shown in Figures 2a and 2b, respectively. The functional protein networks of the top fifty most intensively studied proteins in human eye research revealed three clusters representing photoreceptors and phototransduction, inflammation and angiogenesis, and proteins involved in lens transparency (Figure 3a). The functional protein networks of the top fifty most intensively studied proteins in mouse eye research revealed three clusters that represented photoreceptors and phototransduction, neurodevelopment, and cell cycle and cellular processes (Figure 3b).

Table 1.

The fifty most studied proteins in human eye research1,2

UniProt ID Gene Protein name Citations Functions Associated diseases Isoforms, PTMs, variants
P26367 PAX6 paired box protein PAX-6 204 transcription factor aniridia; Peters anomaly; foveal hypoplasia 1; keratitis hereditary; coloboma of iris, choroid, and retina; coloboma of optic nerve; bilateral optic nerve hypoplasia; aniridia, cerebellar ataxia, and mental deficiency 2 isoforms
1 PTM
85 variants
Q99972 MYOC myocilin 198 regulates the activation of different signaling pathways; secreted glycoprotein glaucoma 1, open angle, A; glaucoma 3, primary congenital, A 1 isoform
2 PTMs
148 variants
P36955 SERPINF1 pigment epithelium-derived factor 180 neurotrophic protein; inhibits angiogenesis osteogenesis imperfecta 6 1 isoform
5 PTMs
38 variants
P15692 VEGFA vascular endothelial growth factor A 138 growth factor that plays a role in angiogenesis, vasculogenesis, and endothelial cell growth microvascular complications of diabetes 17 isoforms
6 PTMs
21 variants
P08603 CFH complement factor H 132 cofactor in alternative complement pathway basal laminar drusen; complement factor H deficiency; hemolytic uremic syndrome atypical 1; macular degeneration, age-related, 4 2 isoforms
51 PTMs
255 variants
O76090 BEST1 bestrophin-1 108 forms calcium-sensitive chloride channels vitelliform macular dystrophy 2; retinitis pigmentosa 50; adult onset vitelliform macular dystrophy; Bestrophinopathy, autosomal recessive; vitreoretinochoroidopathy, autosomal dominant 3 isoforms
0 PTMs
172 variants
P78363 ABCA4 retinal-specific ATP-binding cassette transporter 96 inward-directed retinoid flipase Stargardt disease 1; fundus flavimaculatus; macular degeneration, age-related, 2; cone-rod dystrophy 3; retinitis pigmentosa 19 1 isoform
10 PTMs
479 variants
Q92834 RPGR X-linked retinitis pigmentosa GTPase regulator 94 plays a role in ciliogenesis retinitis pigmentosa 3; retinitis pigmentosa and sinorespiratory infections with or without deafness; cone-rod dystrophy, X-linked 1; macular degeneration, X-linked, atrophic 6 isoforms
4 PTMs
155 variants
P0C7Q2 ARMS2 age-related maculopathy susceptibility protein 2 87 retina homeostasis macular degeneration, age-related, 8 1 isoform
0 PTMs
7 variants
P08100 RHO rhodopsin 87 key role in visual process retinitis pigmentosa 4; night blindness, congenital stationary, autosomal dominant 1 1 isoform
13 PTMs
111 variants
P02489 CRYAA alpha-crystallin A chain 77 plays a role in transparency and refractive index of the lens cataract 9, multiple types 1 isoform
13 PTMs
28 variants
Q15582 TGFBI transforming growth factor-beta-induced protein ig-h3 77 binds to type I, II, and IV collagens corneal dystrophy, epithelial basement membrane; corneal dystrophy, Groenouw type 1; corneal dystrophy, lattice type 1; corneal dystrophy, Thiel-Behnke type; corneal dystrophy, Reis-Bucklers type; corneal dystrophy, lattice type 3A; corneal dystrophy, Avellino type 1 isoform
33 PTMs
110 variants
Q16518 RPE65 retinoid isomerohydrolase 63 roles in production of 11-cis retinal and visual pigment regeneration Leber congenital amaurosis 2; retinitis pigmentosa 20; autosomal dominant retinitis pigmentosa with choroidal involvement 1 isoform
9 PTMs
106 variants
O15537 RS1 retinoschisin 62 involved in cell adhesion processes during retinal development retinoschisis juvenile X-linked 1 1 isoform
5 PTMS
99 variants
Q92743 HTRA1 serine protease HTRA1 60 serine protease macular degeneration, age-related 7; cerebral arteriopathy with subcortical infarcts and leukoencephalopathy, autosomal recessive 1 isoform
0 PTMs
34 variants
Q9NZN9 AIPL1 aryl-hydrocarbon-interacting protein-like 1 58 may be involved in protein trafficking and/or protein folding and stabilization Leber congenital amaurosis 4 5 isoforms
Q12948 FOXC1 forkhead box protein C1 54 regulator of cell viability and resistance to oxidative stress Axenfeld-Rieger syndrome 3; iridogoniodysgenesis anomaly; Peters anomaly 1 isoform
5 PTMs
37 variants
Q00604 NDP norrin 54 involved in canonical Wnt signaling pathway; role in retinal neovascularization Norrie disease; vitreoretinopathy, exudative 2 1 isoform
5 PTMs
75 variants
Q16678 CYP1B1 cytochrome C450 1B1 53 heme-thiolate monooxygenase Peters anomaly; glaucoma 3, primary congenital, A; glaucoma, primary open angle; glaucoma 1, open angle, A 1 isoform
2 PTMs
101 variants
Q04671 OCA2 P protein 52 role in melanin synthesis, melanosome maturation albinism, oculocutaneous, 2 3 isoforms
5 PTMs
209 variants
P06400 RB1 retinoblastoma-associated protein 51 tumor suppressor childhood cancer retinoblastoma; bladder cancer; osteogenic sarcoma 1 isoform
30 PTMs
201 variants
P23942 PRPH2 peripherin-2 50 involved in outer segment disk morphogenesis retinitis pigmentosa 7; retinitis punctata albescens; adult-onset vitelliform macular dystrophy; patterned dystrophy of retinal pigment epithelium; choroidal dystrophy, central areolar 2; cone-rod dystrophy; retinitis pigmentosa; macular degeneration 1 isoform
2 PTMs
46 variants
P82279 CRB1 protein crumbs homolog 1 49 role in photoreceptor morphogenesis retinitis pigmentosa 12; Leber congenital amaurosis 8; pigmented paravenous chorioretinal atrophy 5 isoforms
82 PTMs
301 variants
P01137 TGFB1 transforming growth factor beta-1 46 multifunctional; involvement in proliferation, differentiation, etc. Camurati-Engelmann disease 1 isoform
12 PTMs
40 variants
Q8WWY3 PRPF31 U4/U6 small nuclear ribonucleoprotein Prp31 46 involved in pre-mRNA splicing retinitis pigmentosa 11 3 isoforms
9 PTM
40 variants
Q99697 PITX2 pituitary homeobox 2 46 involved in cell proliferation, morphogenesis Axenfeld-Rieger syndrome 1; iridogoniodysgenesis 2; Peters anomaly; ring dermoid of cornea 3 isoforms
1 PTM
47 variants
P07320 CRYGD gamma-crystallin D 44 component of lens cataract 4, multiple types 1 isoform
0 PTMs
21 variants
P14679 TYR tyrosinase 43 involved in formation of pigments albinism, oculocutaneous, 1A; albinism, oculocutaneous, 1B 2 isoforms
6 PTMs
225 variants
P30301 MIP lens fiber major intrinsic protein 43 water channel cataract 15, multiple types 1 isoform
4 PTMs
41 variants
P02511 CRYAB alpha-crystallin B chain 42 component of lens myopathy, myofibrillar, 2; cataract 16, multiple types; myopathy, myofibrillar, fata infantile hypertonic, alpha-B crystallin-related; cardiomyopathy, dilated 1II 1 isoform
7 PTMs
23 variants
P02649 APOE apolipoprotein E 42 mediates the binding, internalization, and catabolism of lipoprotein particles hyperlipoproteinemia 3; Alzheimer disease 2; sea-blue histiocyte disease; lipoprotein glomerulopathy; familial hypercholesterolemia 1 isoform
9 PTMs
46 variants
P48165 GJA8 gap junction alpha-8 protein 42 channel activity cataract 1, multiple types 1 isoform
0 PTMs
82 variants
P17302 GJA1 gap junction alpha-1 protein 39 gap junction protein that acts as regulator of bladder capacity oculodentodigital dysplasia; oculodentaldigital dysplasia, autosomal recessive; syndactyly 3; hypoplastic left heart syndrome 1; Hallermann-Streiff syndrome; atrioventricular septal defect 3; craniometaphyseal dysplasia, autosomal recessive 1 isoform
26 PTMs
113 variants
P51810 GPR143 G-protein coupled receptor 143 39 receptor for tyrosine, L-DOPA, and dopamine albinism ocular 1; nystagmus congenital X-linked 6 1 isoform
1 PTM
70 variants
Q02846 GUCY2D retinal guanylyl cyclase 1 39 rossible functional role in rod/cone photoreceptors Leber congenital amaurosis 1; cone-rod dystrophy 6 1 isoform
3 PTMs
128 variants
Q96CV9 OPTN optineurin 39 roles in maintaining Golgi complex, membrane trafficking, exocytosis glaucoma 1, open angle, E; glaucoma, normal pressure; amyotrophic lateral sclerosis 12 3 isoforms
8 PTMs
60 variants
O43186 CRX cone-rod homeobox protein 37 transcription factor, upstream of several photoreceptor-specific genes Leber congenital amaurosis 7; cone-rod dystrophy 2; retinitis pigmentosa 1 isoform
0 PTMs
47 variants
P04637 TP53 cellular tumor antigen p53 37 tumor suppressor esophageal cancer; Li-Fraumeni syndrome; squamous cell carcinoma of the head and neck; lung cancer; papilloma of the choroid plexus; adrenocortical carcinoma; basal cell carcinoma 7 9 isoforms
34 PTMs
1706 variants
P01375 TNF tumor necrosis factor 35 proinflammatory cytokine psoriatic arthritis 1 isoform
5 PTMs
18 variants
P35913 PDE6B rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta 34 role in transmission and amplification of the visual signal retinitis pigmentosa 40; night blindness, congenital stationary, autosomal dominant 2 3 isoforms
2 PTMs
104 variants
O75695 RP2 protein XRP2 33 GTPase-activating protein retinitis pigmentosa 2 1 isoform
4 PTMs
51 variants
Q08397 LOXL1 lysyl oxidase homolog 1 33 active on elastin and collagen substrates exfoliation syndrome 1 isoform
7 PTMs
34 variants
P05231 IL6 interleukin-6 32 multifunctional cytokine; induces acute phase response rheumatoid arthritis systemic juvenile 1 isoform
3 PTMs
32 variants
P61812 TGFB2 transforming growth factor beta-2 32 suppresses interleukin-2 dependent T-cell growth Loeys-Dietz syndrome 4 2 isoforms
8 PTMs
47 variants
Q92781 RDH5 11-cis retinol dehydrogenase 32 catalyzes final step in biosynthesis of 11-cis retinaldehyde, the universal chromophore of visual pigments retinitis punctata albescens 1 isoform
1 PTM
50 variants
O95343 SIX3 homeobox protein SIX3 31 transcriptional regulator holoprosencephaly 2 1 isoform
0 PTMs
30 variants
Q9GZR5 ELOVL4 elongation of very long chain fatty acids protein 4 31 elongates saturated and monosaturated very long chain fatty acids Stargardt disease 3 1 isoform
1 PTM
38 variants
Q9NZR4 VSX1 visual system homeobox 1 31 binds to the locus core region of the red/green visual pigment gene cluster corneal dystrophy, posterior polymorphous, 1; keratoconus 1; craniofacial anomalies and anterior segment dysgenesis syndrome 8 isoforms
0 PTMs
39 variants
Q9UI36 DACH1 Dachshund homolog 1 31 transcription factor involved in regulation of organogenesis -- 4 isoforms
6 PTMs
84 variants
O60313 OPA1 dynamin-like 120 kDa protein, mitochondrial 30 role in mitochondrial fusion and regulation of apoptosis optic atrophy 1; dominant optic atrophy plus syndrome 2 isoforms
4 PTMs
142 variants
1

Protein function(s), disease states, isoforms, PTMs, and variants based upon NeXtProt entries using gold level criteria. Disease states, isoforms, PTMs, and variants reported for NeXtProt curated entries.

2

It should be noted that although this list comprises fifty proteins, there are actually many more proteins due to isoforms and variants noted in the last column

Table 2.

The fifty most studied proteins in mouse eye research1,2

UniProt ID Gene Protein name Citations Functions Associated diseases in mice Isoforms
P63015 Pax6 paired box protein Pax-6 653 transcription factor involved in development of eye and other organs defects in Pax6 cause condition of small eye (Sey) with lack of eyes and nasal primordial 3 isoforms
P23440 Pde6b rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta 298 role in transmission and amplification of visual signal defects in Pd36b are cause of a retinal degeneration 2 isoforms
P15409 Rho rhodopsin 244 photoreceptor required for vision at low light intensity -- 1 isoform
Q61412 Vsx2 visual system homeobox 2 162 role in specification and morphogenesis of sensory retina defects in Vsx2 are cause of ocular retardation (OR(J)), a disease with microphthalmia, retinal destruction, and absence of optic nerve 1 isoform
Q91ZQ5 Rpe65 retinoid isomerohydrolase 144 role in visual pigment regeneration defects in Rpe65 cause light damage susceptibility (LDS) of the retina 1 isoform
Q9ERD7 Tubb3 tubulin beta-3 chain 137 major constituent of microtubules -- 1 isoform
Q08874 Mitf microphthalmia-associated transcription factor 132 transcription factor for genes that play essential roles in cell differentiation, proliferation, and survival defects in Mitf cause microphthalmia (mi) 9 isoforms
P61372 Isl1 insulin gene enhancer protein ISL-1 129 regulates promoters of insulin, glucagon, and somatostatin genes -- 2 isoforms
Q63934 Pou4f2 POU domain, class 4, transcription factor 2 127 transcription factor -- 1 isoform
P32114 Pax2 paired box protein Pax-2 126 transcription factor renal-coloboma syndrome 2 isoforms
Q62226 Shh sonic hedgehog protein 117 intercellular signal essential for various patterning events during development -- 1 isoform
P12658 Calb1 calbindin 115 buffers cytosolic calcium -- 1 isoform
P24622 Cryaa alpha-crystallin A chain 113 contributes to transparency and refractive index of the lens -- 2 isoforms
P70677 Casp3 caspase-3 112 role in activation cascade of caspases involved in apoptosis -- 1 isoform
P15499 Prph2 peripherin-2 111 may function as adhesion molecule in outer segment disks responsible for retinal degeneration slow (Rds) 1 isoform
Q00731 Vegfa vascular endothelial growth factor A 104 growth factor involved in angiogenesis, vasculogenesis, and endothelial cell growth -- 6 isoforms
P48432 Sox2 transcription factor SOX-2 95 transcription factor for some genes involved in embryonic development -- 1 isoform
P48437 Prox1 Prospero homeobox protein 1 89 transcription factor involved in developmental processes -- 1 isoform
P54846 Nrl neural retina-specific leucine zipper protein 88 transcription factor involved in expression of several rod-specific genes -- 1 isoform
P11344 Tyr tyrosinase 87 involved in formation of pigments such as melanins defects in Tyr results in various forms of albinism 1 isoform
Q62233 Six3 homeobox protein SIX3 87 transcriptional regulator -- 2 isoforms
O54751 Crx cone-rod homeobox protein 87 transcription factor that transactivates a sequence upstream of several photoreceptor-specific genes -- 1 isoform
P20612 Gnat1 guanine nucleotide-binding protein G(t) subunit alpha-1 86 modulator or transducer of various transmembrane signaling systems -- 1 isoform
Q9QXZ9 Opn4 melanopsin 85 photoreceptor required for regulation of circadian rhythm -- 2 isoforms
Q08331 Calb2 calretinin 84 calcium-binding protein -- 1 isoform
Q9Z2E5 Atoh7 protein atonal homolog 7 83 transcription factor involved in the differentiation of most ganglion cells -- 1 isoform
P03995 Gfap glial fibrillary acidic protein 81 class-III intermediate filament -- 2 isoforms
P21275 Bmp4 bone morphogenetic protein 4 81 induces cartilage and bone formation -- 1 isoform
P25322 Ccnd1 G1/S-specific cyclin-D1 78 phosphorylates and inhibits members of the retinoblastoma (RB) protein family 1 isoform
P08553 Nefm neurofilament medium polypeptide 78 component of neurofilaments -- 1 isoform
O35602 Rax retinal homeobox protein Rx 77 regulates initial specification of retinal cells and/or their subsequent proliferation -- 1 isoform
Q02248 Ctnnb1 catenin beta-1 73 downstream component of canonical Wnt signaling pathway -- 1 isoform
P02340 Tp53 cellular tumor antigen p53 72 tumor suppressor -- 1 isoform
P17208 Pou4f1 POU domain, class 4, transcription factor 1 71 probable transcription factor -- 1 isoform
Q02067 Ascl1 Achaete-scute homolog 1 70 transcription factor -- 1 isoform
P20444 Prkca protein kinase C alpha type 70 calcium-activated, phospholipid- and diacylglycerol (DAG)-dependent serine/threonine-protein kinase expression of mutant form UV25 causes malignant transformation of cells 1 isoform
P23927 Cryab alpha-crystallin B chain 69 may contribute to transparency and refractive index of lens -- 1 isoform
P46414 Cdkn1b cyclin-dependent kinase inhibitor 18 67 regulator of cell cycle progression -- 1 isoform
Q60867 Neurod1 neurogenic differentiation factor 1 64 transcriptional activator Neurod1 null mice are deaf and die shortly after birth 1 isoform
P09803 Cdh1 cadherin-1 63 calcium-dependent cell adhesion protein -- 1 isoform
P70447 Neurog2 neurogenin-2 62 transcriptional regulator -- 1 isoform
P49919 Cdkn1c cyclin-dependent kinase inhibitor 1C 57 inhibitor of several G1 cyclin/CDKcomplexes and mitotic cyclin B-CDC2 2 isoforms
Q08481 Pecam1 platelet endothelial cell adhesion molecule 56 cell adhesion molecule needed for leukocyte transedothelial migration -- 4 isoforms
P51180 Mip lens fiber major intrinsic protein 55 water channel defects in Mip cause autosomal dominant cataract 1 isoform
P15105 Glul glutamine synthetase 53 essential for proliferation of fetal skin fibroblasts -- 1 isoform
P97474 Pitx2 pituitary homeobox 2 52 involved in cell proliferation and morphogenesis mice embryos lacking isoform Ptx2c show left-right patterning defects and severe development abnormalities 5 isoforms
P28236 Gja8 gap junction alpha-8 protein 52 component of gap junction -- 1 isoform
Q9CXV0 Isl2 insulin gene enhancer protein ISL-2 52 transcriptional factor -- 1 isoform
P51491 Opn1sw short-wave-sensitive opsin 1 50 visual pigment -- 1 isoform
P20443 Sag S-arrestin 50 binds to photoactivated-phosphorylated rhodopsin -- 1 isoform
1

Protein function(s), disease states, and isoforms based upon UniProt entries.

2

It should be noted that although this list comprises fifty proteins, there are actually many more proteins due to isoforms and variants noted in the last column

Figure 2.

Figure 2

Figure 2

(a) Heat map of the fifty most studied proteins in human eye research, 1974–2014. The first publication associated with both a gene and eye for human research in PubMed appeared in 1974. (b) Heat map of the fifty most studied proteins in the mouse eye research, 1924–2014. The first publication associated with both a gene and eye for mouse research in PubMed appeared in 1924. The heat map does not represent a full year for 2014, but only what was published on PubMed by 10/20/14.

Figure 3.

Figure 3

Figure 3

Functional protein networks among the top 50 most studied proteins for (a) human and (b) mouse eye.

We further examined the overlap between the fifty most intensively studied proteins in human and mouse eye research. There were fifteen proteins that were common to both human and mouse eye studies: paired box protein PAX-6, vascular endothelial growth factor A, rhodopsin, alpha-crystallin A chain, retinoid isomerohydrolase, peripherin-2, pituitary homeobox 2, tyrosinase, lens fiber major intrinsic protein, alpha-crystallin B chain, gap junction alpha-8 protein, cone-rod homeobox protein, cellular tumor antigen p53, rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta, and homeobox protein SIX3.

The least-studied proteins comprised a large proportion of the proteins in both human and mouse eye studies, as mentioned above. Of the 4,050 proteins in human eye studies, the percentages of proteins with 5, 4, 3, 2, or 1 citation(s) were 2.5%, 3.8%, 7.6%, 17.6%, and 57.2%, respectively. Of the 4,717 proteins in mouse eye studies, the percentages of proteins with 5, 4, 3, 2, or 1 citation(s) were 3.2%, 4.4%, 8.3%, 16.0%, and 49.8%, respectively. In other words, 88.7% of proteins in human eye studies and 81.7% of proteins in mouse eye studies had five or fewer citations.

To facilitate the use of mass spectrometry for the quantification of these top proteins, we have proposed SRM assays for the top fifty proteins in human and mouse eye research as presented in Supporting Information, Tables 5 and 6. The list of the top proteins as characterized by a bibliometric approach, corresponds to what Van Eyk has called “popular proteins.” A complementary approach is to identify “priority proteins” based upon biological pathways. Biological pathways that are currently under intensive investigation in eye research include the complement cascade, Wnt signaling, VEGF signaling, apoptosis, visual phototransduction, degradation of extracellular matrix, cell response to hypoxia, oxidative stress-induced senescence, ERK activation, signaling by the TGF-beta receptor complex, and the inflammasome. A provisional list of 1416 “priority proteins” is shown in Supporting Table 7. Only 16 of the top 50 most intensively studied human eye proteins overlapped with the provisional list of priority proteins.

4 Discussion

In the present study, we identified over 4000 proteins that have been studied in human eye research and over 4700 proteins that have been studied in mouse eye research. There were nearly 80% more scientific publications for proteins in eye research for mouse than for humans. The underlying reason for the difference is not clear, but one could speculate that mouse eye proteins have been more frequently studied due to the greater available of eye tissues from mice than from humans. The ten most intensively studied proteins in human and mouse eye research are discussed below.

Paired box protein PAX-6 (PAX6) has been the most intensively studied protein in both human and mouse eye studies. PAX6 plays a multi-level role in the morphogenesis of the eye, especially in the development of the lens, cornea, and retina [5]. PAX6 is a transcriptional factor that binds with DNA through interactions with two N- and C-terminal domains, termed PAI and RED, respectively. Three isoforms of PAX6 are produced via alternative splicing. The ratio between the canonical form, isoform 1, and isoform 5a varies among tissue types [5]. PTMs of PAX6 include phosphorylation and ubiquitination. Multiple variants have been reported in PAX6. Mutations in Pax-6 are associated with small eye (Sey) in mouse [6] and aniridia (partial or complete absence of the iris) in humans [7].

Myocilin is a 504 amino acid glycoprotein that was initially identified because it is induced in the eye by glucocorticoid treatment [8,9]. Myocilin is found in the trabecular meshwork, cornea, lamina cribosa, ciliary body, iris, vitreous, retina, optic nerve, and aqueous humor [10]. The structure of myocilin includes a signal peptide sequence for cleavage as a secreted protein and a C-terminal olfactomedin domain. Over 70 glaucoma-associated variants have been identified in myocilin, of which >90% are located in exon 3 that codes for the olfactomedin domain [9]. Most myocilin variants that contain an amino acid substitution are not secreted but accumulate within the endoplasmic reticulum as homo- or heterodimers [9]. The function of myocilin is not well understood [9,10].

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein that belongs to the serpin (serine protease inhibitor) family [11]. PEDF has heparin- and collagen-binding sites, and an unusual asymmetric distribution of charged amino acid residues, with basic and acidic regions on the opposite poles of the protein [12,13]. PEDF has neurotrophic and anti-angiogenic effects [11] and provides protection against oxidative stress in diabetic retinopathy [14]. PEDF inhibits retinal neovascularization induced by vascular endothelial growth factor [15].

Vascular endothelial growth factor-A (VEGF-A), a member of the vascular permeability factor/VEGF family, is a disulfide-bonded dimeric glycoprotein that plays a central role in angiogenesis [16]. VEGF-A has 17 isoforms that arise from alternative promoter usage, alternative splicing, and alternative initiation. The VEGF-A164/165 isoform, named after the total number of amino acid residues in mouse and human proteins, respectively, has been most intensively studied because of its role in angiogenesis [16]. VEGF-A binds with VEGF receptors 1 and 2, two high affinity tyrosine kinase receptors [17], and with neuropilin-1 [18]. Neutralization of VEGF-A with ranibizumab, a recombinant monoclonal antibody, was shown to prevent visual loss in neovascular age-related macular degeneration (AMD) [19]. Other antibodies against VEGF-A have shown similar effects in treatment of AMD.

Complement factor H (CFH) is a 1213 amino acid glycoprotein that plays a central role in the complement system. The complement system of innate immunity is involved in cellular integrity, microbial killing, immune surveillance, tissue homeostasis, and mediation of inflammatory responses [20]. Complement is involved in the recognition of diseased or damaged host cells, regulation of cellular immune responses, and interaction with the coagulation cascade. CFH plays a role in limiting complement-mediated damage to healthy host cells [21]. CFH has multiple binding sites, including those for C3b, heparin, C-reactive protein, and sialic acid. Two variants of CFH, Y402H and I62V, are strongly associated with the risk of AMD [22]. Immunohistochemical studies have demonstrated that CFH is present within vascular lumens and perivascular spaces around large blood vessels, in the choriocapillaris, intercapillary septa, Bruch’s membrane, and in large choroidal vessels and stroma in eyes with AMD [23].

Bestrophin-1 (BEST1) is a 585 amino acid transmembrane protein that is involved in intracellular calcium signaling [24]. There are three isoforms that arise through alternative splicing. BEST1 is most strongly localized in the cytosol close to the basolateral membrane of the retinal pigment epithelium (RPE) [24]. Mutations in BEST1 cause a variety of retinal degenerations, the best known being Best’s vitelliform macular dystrophy, or Best’s disease. Mutations in BEST1 are associated with increased accumulation of lipofuscin, a yellow aging-associated pigment, in the RPE, but the underlying pathophysiology is not well understood [25].

Retinal-specific ATP binding cassette transformer (ABCA4) is in the family of ABC transporters, a ubiquitous set of integral membrane proteins present in all living organisms [26]. ABCA4 has two transmembrane domains, two nucleotide-binding domains (ATP-binding cassettes), and two extracellular domains [26]. ABCA4 is located in the disk margins of photoreceptor outer segments. The reason for the restricted localization of ABCA4 within the rod outer segments is not clear [26]. ABCA4 seems to play a role in the clearance of all-trans-retinal from disk membranes after photoexcitation of rhodopsin [26]. Mutations in ABCA4 are associated with Stargardt disease [27], fundus flavimaculatus [28], cone-rod dystrophy, and a form of retinitis pigmentosa [26].

X-linked retinitis pigmentosa GTPase regulator (RPGR) is a 1020 amino acid protein that has six isoforms arising from alternative splicing. Isoform 6, or RPGRORF15 is highly expressed in photoreceptors and is implicated in retinal disease. RPGR contains a glycine/glutamic-acid rich domain near the C-terminal end that accounts for up to 80% of RPGR mutations [29]. RPGR is found in centrioles, ciliary axonemes, and microtubular transport complexes [29]. RPGR plays a role in microtubular transport through the ciliary structures that connect the inner and outer segments of photoreceptors [29,30]. X-linked forms of cone-rod dystrophy, cone dystrophy, and macular atrophy have been associated with RPGRORF15 mutations.

Age-related maculopathy susceptibility protein 2 (ARMS2) is a 107 amino acid protein that has been implicated in AMD. ARMS2, a recent gene in evolution, is present only in humans and higher primates [31]. No homologous gene has been annotated in lower vertebrates or other organisms [32]. ARMS2 has nine predicted phosphorylation sites but no remarkable structural motifs. Recent studies show there are two isoforms of ARMS2: isoform A, the canonical form and isoform B that arises as a splice variant [33,34]. The function of ARMS2 is not well understood. ARMS2 has been localized to retina and RPE [35]. The A69S risk variant of ARMS2 is strongly associated with AMD [22]. Since ARMS2 is in strong linkage disequilibrium with serine protease HTRA1, it is unclear whether ARMS2, HTRA1, or both proteins are involved in the pathogenesis of AMD.

Rhodopsin (RHO), a visual pigment found in rod photoreceptors in the retina, is essential for the process of vision. RHO is a member of the G-protein-coupled-receptor family. The structure of RHO includes a transmembrane protein moiety, opsin, which contains a ligand-binding site for retinal on the extracellular side of the transmembrane bundle [36]. The absorption of photons causes the isomerization of 11-cis retinal to all-trans retinal, conformation changes in rhodopsin, and downstream signal transduction [36]. Mutations in rhodopsin are associated with congenital stationary night blindness and retinitis pigmentosa (RP) [37].

Rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta (Pde6b) has been extensively studied in mouse models for autosomal recessive retinitis pigmentosa [38]. Rod phosphodiesterase (PDE) is a membrane-associated protein that consists of two catalytic subunits, rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit alpha (Pde6a) and Pde6b, and two gamma inhibitory subunits [38]. PDE plays a role in phototransduction by hydrolyzing the cGMP second messenger. Natural mouse models with the Pde6b mutations have been used to evaluate pharmacological treatments and gene therapy for protecting photoreceptors from apoptosis [38].

Visual system homeobox 2 (Vsx2) is a transcription factor that controls the morphogenesis of the eye [39,40]. Vsx2 is a 361 amino acid protein that contains a 60 amino acid homeodomain, or DNA binding module composed of three alpha helices [39]. Mutations in Vsx2 are associated with microphthalmia in humans [39,40] and mice [41].

Retinoid isomerohydrolase (Rpe65) is an RPE-specific protein that plays an important role in the visual cycle by converting all-trans retinyl esters to 11-cis-retinol [42]. Rpe65 is bound to smooth endoplasmic reticulum in RPE cells, but the exact mechanism of this binding is unclear [42]. In humans, mutations of Rpe65 are associated with Leber’s congenital amaurosis, recessive RP, fundus albipunctatus, and autosomal dominant RP with choroidal involvement [43].

Tubulin beta-3 chain (Tubb3) is a component of microtubules. Microtubules form the cytoskeleton and consist of heterodimers of alpha- and beta-tubulin. Tubulin has a wide range of PTMs, including acetylation, phosphorylation, detyrosination, polyglycylation, and polyglutamylation [44]. Tubb3, one of six tubulins found in mammals, has expression mainly limited to neurons [45]. In humans, TUBB3 mutations are associated with congenital oculomotor nerve hypoplasia and later-onset peripheral axon degeneration [45].

Microphthalmia-associated transcription factor (Mitf), a member of the family of basic helix-loop-helix leucine-zipper microphthalmia-related transcription factors, is a regulator of melanocytes and has pleiotrophic roles in RPE cells, mast cells, and osteocytes [46]. There are nine isoforms of Mitf. PTMs of Mitf include phosphorylation, sumoylation, and ubiquitination. The target genes for Mitf include those involved in pigmentation, cell cycle, survival, motility and invasion, metabolism, and oxidative stress [47]. Defects in Mitf cause microphthalmia in mice.

Insulin gene enhancer protein ISL-1 (Isl1), a transcription factor of the LIM-homeodomain protein family, is a 349 amino acid that is essential in development of many cell types, including retina [48]. Isl1 has two tandemly arrayed LIM domains near the N terminus that mediate protein-protein interactions and an adjacent homeodomain that binds DNA [49]. ISl1 regulates promoters of insulin, glucagon, and somatostatin genes.

POU domain, class 4, transcription factor 2 (Pou4f2) is a 411 amino acid transcription factor that is expressed in developing and adult retinal ganglion cells [50]. Pou4f2 is one of three members of the POU4F family, all of which are expressed only in ganglion cells of the retina [51]. Selective ablation of Pou4f2 had no impact on long-term survival of retinal ganglion cells in adult mice [51]. Pouf4f2 has been of interest in glaucoma research, since glaucomatous optic atrophy is characterized by a progressive loss of retinal ganglion cells.

Paired box protein Pax-2 (Pax2) is a transcription factor that is required for optic fissure closure in the developing eye [52]. Astrocytes, the earliest glial cell population in optic nerve development, play a role in retinal angiogenesis and formation of the brain-retinal barrier [53]. Pax2 mutations are associated with a renal-coloboma syndrome that involves the eye, ear, central nervous system, and urogenital tract in humans and mice [54,55].

A bibliometric analysis conducted in 2011 showed that about three-quarters of protein research focuses on the 10% of proteins that were known before the human genome was mapped [56]. Most of the diseases or processes associated with the ten most intensively studied proteins of the human eye are related to development or single gene disorders such as retinitis pigmentosa. Some of the proteins that are being investigated, such as ARMS2, have gained recent attention mainly because of strong disease associations at the genetic level. As noted by the investigators of the 2011 bibliometric analysis, scientists have an apparent reluctance to work on unknown or lesser-known proteins. The reasons for this are unclear but may possibly have to do with greater risk in grant applications, as it is harder to explain rationale and significance for proteins that have unknown functions [56]. In addition, the intensity with which certain proteins were studied was related to the availability of chemical probes for the particular protein [56]. The present analysis corroborates the observation that most proteins of the eye have not been well studied: over 57% of proteins in human eye research and nearly 50% of proteins in mouse eye research had one citation only. A limitation of this study is that many older references may have had less stringent quality control than are currently used in claiming identification of proteins. Another limitation is that the proteins identified as “priority proteins” for human eye research will likely grow and change in the future. What we have proposed here is a starting point based upon some of the most intensely studied biological pathways in human eye research.

Recent advances in proteomics, bioinformatics, and mass spectrometry instrumentation should help expand scientific investigations to lesser-known proteins in eye research. Discovery work on proteomes of specific tissues and cell types has been greatly facilitated by data-dependent acquisition approach using Orbitrap mass spectrometers [57] or data-independent approaches (e.g. SWATH) [58]. Targeted methods for selective protein quantitation such as SRM most often use triple quadrupole mass spectrometers. SRM assays were recently used for quantification of a large number of human tear proteins [59]. The SRM assays proposed in the present paper for human and mouse eye proteins can be applied for protein quantification. All instruments can quantitate proteins without the need for antibodies or specific chemical probes, although these can employed when increased sensitivity is required to deal with dynamic range constraints [60,61]. Protein interaction studies can be used to determine binding partners and infer their functional networks [62,63]. Many strategies are available to identify and quantify different PTMs, such as phosphorylation, O-GlcNAcylation, and glycosylation [6466]. Specific isoforms and variants arising from single nucleotide polymorphisms can be targeted by SRM assays. Application of these new advances should help both discovery and hypothesis-based research about the rich diversity of proteins involved in biological processes of the eye and vision and in health and disease. These newer tools will help scientists investigate proteins that remain “hidden in plain sight” [56].

Supplementary Material

supporting table 1
supporting table 2
supporting table 3
supporting table 4
supporting table 5
supporting table 6
supporting table 7

Statement of clinical relevance.

Research on the biology of the eye and underlying molecular mechanisms of eye disease can be advanced through the larger application of state-of-the-art quantification and characterization of protein and proteomes. This study utilized a bibliometric analysis to identify the most intensively studied proteins in human and mouse eye research. Selected reaction monitoring assays have been developed in silico for the top fifty most intensively studied proteins in human and mouse eye research.

Acknowledgments

This work was supported by the National Institutes of Health grants R01 EY024596, R01 AG027012, the Joint King Khaled Eye Specialist Hospital and Wilmer Eye Institute Research Grant Program, the Edward N. & Della L. Thome Memorial Foundation, and Research to Prevent Blindness.

List of abbreviations

ABCA4

retinal-specific ATP binding cassette transformer

AMD

age-related macular degeneration

ARMS2

age-related maculopathy susceptibility protein 2

B/D-HPP

Biology/Disease - Human Proteome Project

BEST1

bestrophin-1

CFH

complement factor H

Isl1

insulin gene enhancer protein ISL-1

Mitf

microphthalmia-associated transcription factor

Pax2

paired box protein Pax-2

PAX6

paired box protein PAX-6

Pde6a

rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit alpha

Pde6b

rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta

PEDF

pigment epithelium-derived factor

PDE

phosphodiesterase

Pou4f2

POU domain, class 4, transcription factor 2

RHO

rhodopsin

RP

retinitis pigmentosa

RPE

retinal pigment epithelium

RPE65

retinoid isomerohydrolase

RPGR

X-linked retinitis pigmentosa GTPase regulator

SRM

selected reaction monitoring

Tubb3

tubulin beta-3 chain

VEGF-A

vascular endothelial growth factor-A

Vsx2

visual system homeobox 2

Footnotes

The authors have declared no conflict of interest.

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