Proteome Analysis of Human Vitreous Proteins*
Ken Yamane
,¶,
Atsushi Minamoto
,¶,
,
Hidetoshi Yamashita||,
Hiroshi Takamura||,
Yuka Miyamoto-Myoken**,
Katsutoshi Yoshizato
,
Takuji Nabetani
,
Akira Tsugita
and
Hiromu K. Mishima
From the
Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima; || Department of Ophthalmology, Yamagata University School of Medicine, Yamagata; ** Innovation Plaza Hiroshima, Japan Science and Technology Corporation, Hiroshima; 
Department of Biological Science, Graduate School of Science, Hiroshima University, Hiroshima; and the 
Proteomics Research Center, Fundamental Research Laboratories, NEC Laboratories, Tsukuba, Japan
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ABSTRACT
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Purpose: Various protein contents such as enzymes, growth factors, and structural components are responsible for biological activities in organs. We have created a map of vitreous proteins and developed a proteome analysis of human vitreous samples to understand the underlying molecular mechanism and to provide clues to new therapeutic approaches in eyes with proliferative diabetic retinopathy (PDR). Methods: Vitreous and serum samples were obtained from subjects with idiopathic macular hole (MH, 26 cases) and PDR (33 cases). The expressed proteins in the samples were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Protein spots were visualized by silver staining, and their expression patterns were analyzed. Some protein spots of concern were excised from the 2-D gels, digested in situ with trypsin, and analyzed by mass spectrometry. Results: More than 400 spots were detected on 2-D gels of MH cases, of which 78 spots were successfully analyzed. The spots corresponded to peptide fragments of 18 proteins, including pigment epithelium-derived factor, prostaglandin-D2 synthase, and interphotoreceptor retinoid-binding protein. These were not identified in the corresponding serum samples. These proteins were also expressed in PDR samples, with no distinct tendency to increase or decrease compared with the MH samples. More than 600 spots were detected on 2-D gels of PDR cases, of which 141 spots were successfully analyzed. The spots corresponded to peptide fragments of 38 proteins. Enolase and catalase were identified among four detected spots. Neither was found in MH vitreous or in PDR serum samples. Conclusion: A map of protein expression was made in human vitreous from eyes with MH and PDR. In the PDR eyes, the increased protein expression observed was due to barrier dysfunction and/or production in the eye. Proteome analysis was useful in systematic screening of various protein expression in human vitreous samples.
Hyperglycemia induces many abnormal changes, which are observed as diabetic retinopathy, in vascular and retinal cells in eyes with diabetes mellitus. The breakdown of the blood-retina barrier and new vessel formation are caused by hyperglycemia. In these processes, many hyperglycemia-induced biochemical changes occur, which cause vascular dysfunction. According to previous reports, various factors, including many kinds of proteins, are involved in the pathological processes of diabetic retinopathy. A breakdown of the blood-retina barrier is caused by an intraocular increase of vascular endothelial growth factor (VEGF)1 (17), interleukin-6, angiotensin II, and many other cytokines and/or growth factors. New vessel formation is a very complex multistep process and is regulated by many proteins including cytokines and/or growth factors. In addition to the factors mentioned, basic fibroblast growth factor (bFGF) (8, 9), insulin-like growth factor-1 (4), hepatocyte growth factor (HGF) (6, 7), and others are known to be involved during the destructive processes of endogenous ocular tissue. Changes in the expression levels of these factors have been described by the measurement of these substances by ELISA in aqueous and vitreous humor. In these studies, however, substances for measurement were targeted in advance, and the targets were limited because of the small amount of available sample material. Therefore, the interrelationship between various candidates for controlling disease processes has not yet been examined. To facilitate the research in the pathogenesis of diabetic retinopathy and to find new clues for developing new therapeutic agents, the database of the proteins expressed in eyes needs to be obtained. The problem has been that the amount of various proteins from human eyes derived by ELISA is very small. The recent development of proteome analysis, which consists of protein separation by two-dimensional (2-D) PAGE (10, 11), high-sensitivity mass spectrometry (MS), and the search for protein databases (12, 13), have made it feasible to analyze protein profiles in various cells, tissues, and body fluids. Originally described by OFarrell (14), 2-D PAGE is currently a reliable method of resolving protein components in various cells, tissues, and body fluids by separation according to their isoelectric point and molecular weight. Commercially available immobilized pH gradient (IPG) strips have further improved the resolution and the reproducibility of 2-D PAGE and allowed a higher level of sample load (15, 16). With these enhancements of 2-D PAGE, proteins in samples from human eyes are visible as separate protein spots. By adopting this technique, it would be possible to scrutinize whole protein profiles of vitreous humor in various destructive processes of ocular tissue. Few studies have evaluated human vitreous samples from normal and diseased eyes with this method, probably due to the technical difficulties. In this report, we present the protein profiles of human vitreous (the space inside of eyes), from eyes with proliferative diabetic retinopathy (PDR) with new vessel formation and idiopathic macular hole (MH) as a control, with a very silent and localized retinal region.
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EXPERIMENTAL PROCEDURES
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Sample Preparation
We obtained undiluted vitreous samples (0.30.8 ml volume) and corresponding serum samples from 51 patients (59 eyes) at the time of pars plana vitrectomy for PDR (33 eyes) and MH (26 eyes), after securing written permission from all patients. MH is, simply, a hole in the macula, a tiny oval area at the center of the retina. MH formation is thought to be caused by a spontaneous, usually abrupt, focal contraction of the perifoveal vitreous cortex, which elevates the retina in the foveal region (17). Diabetic retinopathy is a microangiopathy affecting the retinal precapillary arterioles, capillaries, and venules. Clinically, the three main types of diabetic retinopathy are background, preproliferative, and proliferative. PDR is a severe stage, with neovascularization and vitreous hemorrhage due to breakdown of neovascularization (18).
All subjects were informed of the purpose of the study and the nature of sampling procedures. Harvested vitreous and serum samples were collected in tubes, placed immediately on ice, centrifuged for 15 min to separate the cell contents, and stored at -80 °C until use. The protein concentration of each sample was measured on a V-1500 spectrophotometer (Hitachi, Tokyo, Japan) using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA).
2-D PAGE
2-D PAGE was performed as previously described (19). Briefly, 40 µl of vitreous sample and 3 µl of serum sample were diluted in a lysis buffer consisting of 7 mol/l urea, 2 mol/l thiourea, 4% 33-cholamidopropyldimethylammonio-l-propanesulflonate, 2% ampholine, pH 3.510 (Amersham Pharmacia Biotech, Uppsala, Sweden), and 1% dithiothreitol to a final volume of 400 µl. Protein samples were applied overnight to Immobiline Dry Strips (Amersham Pharmacia Biotech) (pH 47, 310, 18 cm) by in-gel rehydration (15, 16). For the first dimension, a Multiphor II electrophoresis chamber (Amersham Pharmacia Biotech) was used. Isoelectric focusing (IEF) was performed with the following voltage program: 500V for 2 min, 3,500V for 1.5 h, then 3,500V for 6 h. Immobiline Dry Strips were stored at -80 °C until the second-dimension electrophoresis was carried out. Second-dimension SDS-PAGE was performed in 918% acrylamide gradient gels (20 x 20 cm) using the Iso-Dalt system (Amersham Pharmacia Biotech).
The protein spots were visualized by silver staining (20), and the 2-D gels were scanned on an Epson ES 80000 scanner (Seiko Epson Corporation, Suwa, Japan). After scanning, the 2-D gels were sandwiched between two cellophane sheets and dried. Image analysis and 2-D gel proteome database management were done using the Melanie II 2-D PAGE software package (Bio-Rad).
Tryptic In-gel Digestion
In-gel digestion was performed as previously described (19). Briefly, protein spots were excised from the dried silver-stained gels by a gel cutter and rehydrated in 100 mmol/l ammonium carbonate. The gel pieces were destained in 30 mmol/l potassium ferricyanide and 100 mmol/l sodium thiosulfate, then rinsed a few times in Milli-Q (Millipore Co., Billerica, MA) water and once in 100 mmol/l ammonium carbonate. Dehydration was performed in acetonitrile until the gel pieces turned opaque white; they were subsequently dried in a vacuum centrifuge. The gel pieces were then rehydrated in a digestion buffer containing trypsin, and proteins underwent in-gel digestion overnight at 37 °C. The digestion was stopped by covering the gel pieces with 5% trifluoroacetic acid, and the peptides were extracted three times with 5% trifluoroacetic acid in 50% acetonitrile. The extracted peptides were pooled and dried in a vacuum centrifuge.
Electrospray Ionization (ESI) MS and Protein Identification
The peptides were resuspended in 1% formic acid in 4% methanol, and loaded to a laboratory-made OLIGO R3 column (PerSeptive Biosystems, Framingham, MA). After washing the column with 1% formic acid, the peptides were eluted with 1% formic acid in 70% methanol and subjected to mass analysis, as described below.
ESI mass spectrometry and protein identification were performed as previously described (19). Briefly, the eluted peptides were loaded into Au/Pd-coated nanoES spray capillaries (Protana, Odense, Denmark). The capillaries were inserted into the nano-flow Z-spray source of a quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, UK). Instrument operation, data acquisition, and analysis were performed by the MassLyny/Biolynx 3.2 software (Micromass, Manchester, UK). The Q-TOF was operated in two modes: MS and MS/MS. The MS mode was used to scan the sample for detectable peptides. The MS/MS mode was used to fragment individual peptides to obtain an amino acid sequence. Finally, the proteins were identified by matching the obtained amino acid sequences against the Swiss-Prot and GenBank databases using GenomeNet Internet server of Kyoto University, Japan (www.fasta.genome.ad.jp/).
Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI) MS and Protein Identification
The peptides were dissolves in 5 µl 0.1% trifluoroacetic acid and 50% acetonitrile. The matrix consisted of a mixture of
-cyano-4-hydroxy cinnamic acid (CHCA) (10 mg) and 1 ml mixture of 0.1% trifluoroacetic acid and 50% acetonitrile. The peptide mixture (1 µl) was deposited on the sample plate, the solvents were removed in air at room temperature, and then the matrix mixture (1 µl) was deposited on the sample plate.
MALDI MS was carried out on a Voyager-DE STR (PerSeptive Biosystems) in the reflector mode. The laser wavelength was 337 nm, and the laser repetition rate was 3 Hz. The MALDI spectra were averaged at 500 laser pulses. The calibration was performed with four peptides: des-Arg1-Bradykinin, angiotensin, Glu1-fibrinopeptide B, and ACTH (1839) with mono-isotopic (M+H)+ at m/z 904.4681, 1296.6853, 1570.6774, and 2465.1989. Peak lists were searched against the NCBInr protein sequence database using the MS-Fit search tool (MS tolerance 0.3 Da) in order to identify the proteins.
Western Blot Analysis
Briefly, 16 µl of vitreous sample from subjects with MH and 8 µl of vitreous sample from subjects with PDR were diluted with a half volume of 3x SDS buffer, and 0.9 µl of serum sample was diluted with 24 µl of 1x SDS. The samples were electrophoresed on 10% or 12.5% SDS-PAGE gel, and then electrophoretically transferred to polyvinylidene fluoride transfer membrane (Hybond-C; Amersham Biosciences Inc., Arlington Heights, IL) at 50 mV for 1.5 h. Membranes were blocked for 1 h at room temperature with 5% dry milk in Tris-buffered saline with 0.1% Tween and incubated for 1 h at room temperature with primary antibodies. Membranes were washed and incubated with 1:10,000 anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Biosciences) for 60 min at room temperature and washed again. Bound antibodies were detected with a Western blot analysis detection system (ECL Plus; Amersham Biosciences).
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RESULTS
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Idiopathic Macular Hole
The average protein concentration of vitreous samples for 2-D PAGE was 0.4668 µg/µl (ranging from 0.1020.998 µg/µl) in MH samples. As many as 371519 spots (average, 437 spots; Fig. 1) and 359580 spots (average, 463 spots; Fig. 2) were detected on silver-stained 2-D gels of vitreous protein in samples from eyes with MH, when IEF was performed using IPG 47 (23 of 26 eyes) and 310 (10 of 26 eyes), respectively. The patterns of protein expression profiles with MH were reproducible among vitreous samples, regardless of patients age, sex, and stage of MH, according to the Gass classification (21). A total of 18 proteins were identified from 78 spots; and most of them corresponded to serum proteins (Figs. 1 and 3). These spots were commonly detected in all vitreous samples with MH. Corresponding proteins between vitreous and serum samples included transferrin, serum albumin,
1-antitrypsin,
1-antichymotrypsin,
2-HG-glycoprotein, antithrombin III, hemopexin, fibrinogen
chain, haptoglobin-1, apolipoprotein J, apolipoprotein A-1, IgG heavy chain, IgG light chain, and transthyretin (Table I).

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FIG. 1. Silver-stained 2-D gels of vitreous protein in samples from eyes with MH. IEF was performed using IPG 47. A total of 40 µl (containing 18.36 µg of protein) of vitreous sample was applied per IPG gel.
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FIG. 2. Silver-stained 2-D gels of vitreous protein in samples from eyes with MH. IEF was performed using IPG 310. A total of 40 µl (containing 18.36 µg of protein) of vitreous sample was applied per IPG gel.
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FIG. 3. Silver-stained 2-D gels of serum protein in samples from a patient with MH. IEF was performed using IPG 47. A total of 3 µl (containing 220.05 µg of protein) of serum sample was applied per IPG gel.
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Although the patterns of protein-expression profile of the vitreous and serum samples from patients with MH were similar, the number of spots was different. On average, there were 794 spots of serum and 437 spots of vitreous on silver-stained 2-D gels (IPG, 47). Most spots of the vitreous protein corresponded to spots on gels of the serum. However, 26 spots detected on gels of vitreous samples were not identified on gels of corresponding serum samples. Of these, 16 spots were identified as polypeptide fragments of pigment epithelium-derived factor (PEDF), prostaglandin-D2 synthase, plasma glutathione peroxidase, and interphotoreceptor retinoid-binding protein (IRBP) (Table I).
Proliferative Diabetic Retinopathy
The average protein concentration of vitreous samples for 2-D PAGE was 4.129 µg/µl (ranging from 1.2847.255 µg/µl) in PDR. As many as 511785 spots (average, 617 spots; Fig. 4) and 558965 spots (average, 779 spots; Fig. 5) were detected on silver-stained 2-D gels of vitreous protein in samples from eyes with PDR, when IEF was performed using IPG 47 (28 of 33 eyes) and 310 (12 of 33 eyes), respectively. The patterns of protein-expression profile were similar to those of corresponding serum protein. A total of 36 proteins were identified from 136 spots; and most of them corresponded to those of serum proteins (Table II). Among these, 18 proteins were also detected with the samples of MH, and the other 18 proteins not identified in vitreous samples of MH were in common with those in the serum. These 18 proteins included
2-macroglobulin, Ig
-1 chain C region,
1-B-glycoprotein,
1-microglobulin, C3
, ceruloplasmin, apolipoprotein E, complement C4, complement factor D, PRBP, complement factor I, IgM heavy chain, ß2-glycoprotein I, Zn-
2-glycoprotein, kininogen, apolipoprotein D, cathepsin D, and prothrombin (Figs. 4 and 5, Table II). The patterns of protein-expression profiles between the vitreous samples with diabetic retinopathy were reproducible, regardless of the presence of macular edema or the severity of PDR.

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FIG. 4. Silver-stained 2-D gels of vitreous protein in samples from eyes with PDR. IEF was performed using IPG 47. A total of 40 µl (containing 186.59 µg of protein) of vitreous sample was applied per IPG gel.
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FIG. 5. Silver-stained 2-D gels of vitreous protein in samples from eyes with PDR. IEF was performed using IPG 310. A total of 40 µl (containing 246.12 µg of protein) of vitreous sample was applied per IPG gel.
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Comparison Between Samples of MH and PDR
A total of 18 proteins were present in vitreous samples of PDR that were not identified in those of MH. In addition, the volume of spots in gels from vitreous samples of PDR increased when compared with identical spots in gels from vitreous samples of MH (Figs. 1, 2, 4, and 5). The spots with increased volume were all identified in serum samples as well (Figs. 15). There was no distinct tendency of spots to increase or decrease in volume of proteins specific to vitreous between MH and PDR vitreous samples, i.e. PEDF, prostaglandin-D2 synthase, plasma glutathione peroxidase, and IRBP. The spots identified in MH vitreous samples were retained in PDR vitreous samples.
The spots among the area of higher IPG (IPG > 6) with 2070 kDa molecular mass were overshadowed by polypeptide fragments of IgG, which were abundant and of great diversity in IPG and molecular weight over the basic region of 2-D gels. Therefore, as a separate series of analyses, we adopted IgG removal procedures in the sample preparation in advance of 2-D PAGE with IPG 310, with several samples of MH (three samples) and PDR (five samples).
Briefly, larger amounts of vitreous (200 µl) and serum (15 µl) samples were shaken at 4 °C overnight with 15 µl Protein A Sepharose 4 Fast Flow (Amersham Pharmacia Biotech). The Protein A bead was precipitated by centrifugation at 5,200xg, then clear supernatant liquid was collected. After the Protein A bead was washed five times with 500 ml PBS, clear supernatant liquid was collected. An UltraFree column (Millipore) was used to concentrate sample fluids.
With this modification of sample preparation, we could recognize five spots in PDR vitreous samples. Spots were not found in MH vitreous samples or in PDR serum samples. Among these five spots, four were identified as enolase and catalase (Fig. 6, AC).

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FIG. 6. Comparison between vitreous and corresponding serum sample with PDR. A, silver-stained 2-D gels of vitreous protein samples from eyes with PDR, from which IgG was removed. IEF was performed using IPG 310. A total of 200 µl (containing 1230.6 µg of protein) of vitreous sample was applied per IPG gel. B, silver-stained 2-D gels of plasma protein samples from patients with PDR, from which IgG was removed. IEF was performed using IPG 310. A total of 15 µl (containing 1050 µg of protein) of serum sample was applied per IPG gel. C, a clear up-regulation of enolase and catalase was seen in vitreous samples.
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Western Blot Analysis
To confirm 2-D gel findings of proteins specific to vitreous samples, we performed Western blot analysis for some of these proteins, i.e. PEDF, prostaglandin-D2 synthase, and enolase. Anti-pigment epithelium-derived factor (CHEMICON International, Temecula, CA) (1:500), anti- prostaglandin-D2 synthase (Cayman Chemical, Ann Arbor, MI) (1:250), anti-neuron-specific enolase (Abcam Limited, Cambridge, UK) (1:250), and normal rabbit IgG (ICN Pharmaceuticals, Aurora, OH) (1:500) were used as primary antibodies for Western blot analysis. These proteins were specifically detected in vitreous samples (Fig. 7, AC), as demonstrated during proteome analysis (Figs. 1, 3, 4, and 6C).

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FIG. 7. Western blot analysis. Lanes 1, serum; 2, vitreous, PDR; 3, vitreous, MH; 4, vitreous, MH (primary antibody: no immune rabbit IgG). A, western blot analysis of fragments of prostaglandin-D2 synthase, which was detectable in vitreous but not in serum samples. B, western blot analysis of fragments of PEDF, which was detectable in vitreous samples but not in serum samples. C, western blot analysis of NSE, which was detectable in vitreous samples of diabetic retinopathy, but not in MH vitreous and serum samples.
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DISCUSSION
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An analysis of vitreous protein profiles was successfully performed with the proteome analysis method. We detected hundreds of spots at a time on a single gel in vitreous samples. Previous reports of the protein content of diseased vitreous using the conventional ELISA method have identified only one or several proteins from vitreous samples (19), because the amount of available vitreous was limited compared with other body fluids such as serum, cerebrospinal fluid, and urine. Proteome analysis method can overcome this drawback because the whole-vitreous protein profile can be presented on a single gel. The mapping data we present here could become useful as a control for future studies of differentially expressed proteins in the vitreous from various vitreoretinal disorders.
More than 400 spots were detected on 2-D gels of MH, of which 78 spots were successfully analyzed as corresponding to peptide fragments of 18 proteins. The same protein appeared in multiple spots due to modifications and/or degradation. Although the patterns of protein-expression profile of the vitreous and serum samples with MH were very similar, four proteins of 18 identified proteins in MH vitreous samples were not identified in corresponding serum samples. Therefore, these peptides were likely to be fragments of proteins that were probably produced within the ocular tissue. These four proteins were identified as PEDF, prostaglandin-D2 synthase, plasma glutathione peroxidase, and IRBP.
More than 600 spots were detected on 2-D gels of PDR, of which 141 spots were successfully analyzed as corresponding to peptide fragments of 38 proteins. Of these, 32 proteins were detected from samples with corresponding serum, in which 18 proteins were also detected in MH samples but with increased volumes in PDR samples. In addition, there were serum proteins in PDR vitreous samples unidentified in MH vitreous samples. The presence of these proteins in vitreous was considered to be the result of increased permeability of retinal blood vessels and vitreous hemorrhage due to breakdown of new retinal vessels.
The presence of two proteins (enolase and catalase) in PDR vitreous samples, which were undetected in corresponding serum samples and MH vitreous samples, suggested that newly produced proteins exist within the diabetic eye and therefore may play some crucial roles in the process of development and exacerbation in vitreoretinal disorders with PDR; or that they may be related to the very severe damage of retinal tissue. We could obtain the data only by proteomics. Enolase, which was detected in vitreous samples from eyes with PDR after modification of vitreous samples with IgG removal, should be the neuron-specific enolase (NSE), as confirmed by Western blot analysis using anti-NSE (Fig. 7C). NSE may be used as a marker of acute neuronal damage in humans with neurological disorders. Cerebral ischemia in rats and humans induce release of NSE to the extracellular matrix (22, 23). Retinal neuron injury in retinal detachment releases sufficient NSE to be detected in subretinal fluid, aqueous, and even serum (24). Catalase, an antioxidant enzyme, was detected from samples of PDR, possibly serving as a protective substance within the diseased eye. Its presence implicates this substance as a candidate for the treatment of acute ischemic diseases of the retina (25).
PEDF is present in aqueous humor, interphotoreceptor matrix, and vitreous (26), and has been reported to play a role as a potent inhibitor of angiogenesis (27, 28). In our study, there was no distinct tendency of PEDF spots to increase or decrease in volume between samples of PDR and MH. This finding suggests that the balance between angiogenetic stimulators and inhibitors should be chiefly regulated by the increase or decrease of angiogenic stimulators such as VEGF, and PEDF as an angiogenic inhibitor should support the effect of increased angiogenic stimulation to a certain level. Previous reports have indicated that the level of PEDF in the vitreous varies far less than that of VEGF between PDR and nonproliferative diabetic retinopathy or MH (29, 30).
At present, to complete the reference library of proteins expressed in eyes is very difficult because performing proteomics using vitreous fluid as samples creates several problems. It has been reported that albumin and immunoglobulin account for over 80% of whole-vitreous protein. Because the large spots of albumin and immunoglobulin overlap small spots, less abundant proteins might be unrecognized. It is known that silver staining has been shown to be 100 times more sensitive than Coomassie blue, with detection limited to the picogram level (31) and the sensitivity of MS limited to approximately the low femtomole level (32). Also, it has been reported that VEGF, bFGF, HGF, and other factors that exacerbate diabetic retinopathy are detectable at the picogram level even at a severely diseased stage (19). We cannot detect such scant proteins or peptides with currently available proteomics methods. These limitations resulted from using small amounts of vitreous fluid as samples.
Further progress in gel electrophoresis technique, improvement in staining sensitivity, and identification by MS spectrometry should contribute to the more detailed analysis of vitreous protein profiles, thereby deepening our knowledge of the pathological features in sight-threatening vitreoretinal disorders.
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FOOTNOTES
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Received, May 2, 2003, and in revised form, September 11, 2003.
Published, MCP Papers in Press, September 15, 2003, DOI 10.1074/mcp.M300038-MCP200
1 The abbreviations used are: VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; HGF, hepatocyte growth factor; MS, mass spectrometry; IPG, immobilized pH gradient; PDR, proliferative diabetic retinopathy; MH, idiopathic macular hole; 2-D, two-dimensional; ESI, electrospray ionization; IEF, isoelectric focusing; Q-TOF, quadrupole time-of-flight; MALDI, matrix assisted laser desorption/ionization; MS, mass spectrometry; PEDF, pigment epithelium-derived factor; IRBP, interphotoreceptor retinoid-binding protein; NSE, neuron-specific enolase. 
* This work was supported by a Health Science Research Grant, no. 12120101 (to Drs. Yamashita, Mishima, Minamoto, Takamura, and Yoshizato), from the Japanese Ministry of Health and Welfare for Research on Eye and Ear Sciences, Immunology, Allergy and Organ Transplantation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
¶ K. Y. and A. M. contributed equally to this work. 
To whom correspondence should be addressed. Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, Hiroshima University, 123 Kasumi, Minami-ku, Hiroshima, Japan. E-mail: amina{at}hiroshima-u.ac.jp.
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