# Proteomic analysis of human vitreous humor

Krishna R Murthy<sup>1,2,3*†</sup>, Renu Goel<sup>1,4†</sup>, Yashwanth Subbannayya<sup>1</sup>, Harrys KC Jacob<sup>1</sup>, Praveen R Murthy<sup>3</sup>, Srikanth Srinivas Manda<sup>1,5</sup>, Arun H Patil<sup>1</sup>, Rakesh Sharma<sup>6</sup>, Nandini A Sahasrabuddhe<sup>1</sup>, Arun Parashar<sup>7</sup>, Bipin G Nair<sup>2</sup>, Venkatarangaiah Krishna<sup>4</sup>, TS Keshava Prasad<sup>1,2,5</sup>, Harsha Gowda<sup>1</sup> and Akhilesh Pandey<sup>8,9</sup>

**Abstract**

**Background:** The vitreous humor is a transparent, gelatinous mass whose main constituent is water. It plays an important role in providing metabolic nutrient requirements of the lens, coordinating eye growth and providing support to the retina. It is in close proximity to the retina and reflects many of the changes occurring in this tissue. The biochemical changes occurring in the vitreous could provide a better understanding about the pathophysiological processes that occur in vitreoretinopathy. In this study, we investigated the proteome of normal human vitreous humor using high resolution Fourier transform mass spectrometry.

**Results:** The vitreous humor was subjected to multiple fractionation techniques followed by LC-MS/MS analysis. We identified 1,205 proteins, 682 of which have not been described previously in the vitreous humor. Most proteins were localized to the extracellular space (24%), cytoplasm (20%) or plasma membrane (14%). Classification based on molecular function showed that 27% had catalytic activity, 10% structural activity, 10% binding activity, 4% cell and 4% transporter activity. Categorization for biological processes showed 28% participate in metabolism, 20% in cell communication and 13% in cell growth. The data have been deposited to the ProteomeXchange with identifier PXD000957.

**Conclusion:** This large catalog of vitreous proteins should facilitate biomedical research into pathological conditions of the eye including diabetic retinopathy, retinal detachment and cataract.

**Keywords:** Retina, SCX chromatography, OFFGEL electrophoresis, Proteome discoverer, Secreted proteins, Protein biomarkers, Body fluid proteomics

**Background**

The vitreous is a highly hydrated gelatinous mass that fills the space between the lens and the retina. The vitreous is adherent to the retina diffusely though the adhesion is strongest at the anterior border of retina, the macula, the optic nerve head, over lattice degenerations and areas of scars. The major function of the vitreous is to allow light to reach the retina and maintain the shape of the eyeball. The formation of vitreous occurs in two phases. Primary vitreous is formed by the third or fourth week of gestation, when the neural ectoderm separates from the surface ectoderm. The space between the two is the future

vitreous cavity. This space is bridged by fibrillar material which is thought to be collagenous in nature. By the time the fetus reaches the 10 mm stage, mesodermal cells enter the vitreous space via the fetal fissure and develop into hyaloid vessels which branch throughout the vitreous cavity [1]. In the adventitia surrounding the vessels, there are mononuclear phagocytes and fibroblasts which are thought to later differentiate into hyalocytes. This cellular vitreous is the primary vitreous. Acellular structures begin to appear by end of the sixth week of gestation between the retina and the hyaloid vasculature. This secondary vitreous is essentially extracellular matrix consisting primarily of type 2 collagen [2]. With the development of the secondary vitreous, the hyaloid vascular system regresses. Hyalocytes appear to be the most important cells and after birth, there is no new migration of these cells into the vitreous cortex. Thus, with an increase in globe size and vitreous cortex surface area, there is a decrease in the density of hyalocytes. Since the vitreous

<footnote>* Correspondence: krishna@viio.org</footnote>

<footnote>†Equal contributors</footnote>

<footnote n="1">¹Institute of Bioinformatics, International Technology Park, Bangalore 560 066, India</footnote>

<footnote n="2">²Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, Kerala 690 525, India</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
acts as a metabolic repository for the retina, hyalocytes and surrounding tissues [3], some of the proteins in vitreous humor could be contributed by these surround-ing tissues. Its viscosity is two to four times greater than water, which gives it a gelatinous consistency [4]. The human vitreous is composed of a complex network of cross-linked collagen fibres of types II, V, IX and XI of which type II is the most abundant. Non-collagenous structural proteins are less abundant as compared to collagen fibrils. Hyaluronic acid, a glycosaminoglycan is also found in abundance in the vitreous [5,6]. A significant amount of prealbumin and transferrin in the vitreous has also been reported [7-9]. Of the soluble proteins that con-stitute the vitreous, sialic acid containing glycoproteins constitute the largest fraction [10].

It is well understood that the changes occurring in the retina are closely linked to biochemical changes occurring in the vitreous humor [11]. Vitreous humor does not have any blood vessels but is nourished by vessels of the retina and the ciliary body. As the vitreous can be easily obtained during vitrectomy surgery, studying the changes occurring in the vitreous could provide valuable information about pathological changes occurring in the retina in disorders of the eye. Many researchers have studied the proteins of the normal vitreous using different techniques and some of them have also catalogued various proteins [12-16]. Nakanishi et al., have catalogued 51 proteins in the vitre-ous by Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry [12]. Kim et al. employed immunoaffinity depletion followed by nano-liquid chromatography-Matrix assisted laser desorption ionization (LC-MALDI-MS/MS) resulting in the identifi-cation of 346 proteins from non-diabetic controls [13]. In a subsequent study, Gao et al. identified 252 proteins from the vitreous employing sodium dodecyl sulphate polya-cylamide gel electrophoresis (SDS-PAGE) analyzed by LC-MS/MS [14]. Aretz et al have identified 1,111 distinct proteins by employing different protein prefractionation strategies such as liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both [16].

Here, we identified 1,205 proteins of which 682 proteins have been detected in the vitreous humor for the first time. A comprehensive proteomic profiling of normal vitreous would serve as an invaluable template for future studies that focus on protein dynamics in vitreous in pathological conditions.

## Results and discussion

Proteomic analysis of the vitreous humor was carried out by depleting the samples of abundant proteins and subjecting it to multiple fractionation techniques, such as In-gel digestion, SCX and OFFGEL fractionation. LC-MS/MS analyses of the different fractions were carried out on an LTQ-Orbitrap Velos (Thermo Electron, Bremen,

Germany) mass spectrometer. The corresponding MS data were searched using the search algorithms Mascot, SEQUEST and X! Tandem against NCBI RefSeq 59 human protein database containing 36,211 sequences with known contaminants (Figure 1). This resulted in the identification of 7,642 peptides from 1,205 proteins. A complete list of proteins identified in vitreous is provided in Additional file 1: Table S1 along with their known sub cellular localization, molecular function, biological process, domains/motifs, number of peptides and sequence coverage. Protein identifications include 41 proteins designated as 'missing proteins' by c-HPP and these are highlighted in Additional file 1: Table S1. A list of peptides identified from this study from all three searches is provided in Additional file 2: Table S2. When compared to previous reports, 682 proteins out of 1,205 were found to be uniquely identified in this study. A partial list of these proteins is provided in Table 1. The description of 3 novel proteins identified in this study and four previously reported proteins is provided in following sections.

Proteomic analysis of the vitreous has been carried out by many groups using different techniques as summarized in Table 2. Some of the proteins not identified in the present study in comparison to previous studies may be due to differences in sample preparation methods or mass spectrometry platforms employed. Since obtaining normal vitreous from healthy individuals is not a possibility, clinically normal appearing vitreous samples from patients undergoing vitrectomy surgery for macular hole or traumatic cataract and cataract surgery for congenital cataract were pooled for the analysis. There is a potential possibility that the vitreous proteome in some cases is influenced by associated pathology in these subjects. However, this was close to normal vitreous that we could obtain for the study.

## Known proteins in the vitreous humor

Among the identified proteins in vitreous, we found many proteins that had been previously described in the vitreous, confirming the validity of the proteomic approach undertaken by us. Among the proteins previously reported in vitreous are aldolase A, fructose-bisphosphate (ALDOA), cystatin C (CST3), dickkopf homolog 3 (DKK3), orosomucoid 2 (ORM2), serpin peptidase inhibitor, clade C member 1 (SERPINC1), insulin-like growth factor binding protein 7 (IGFBP7), lipocalin 2 (LCN2), TIMP metallopeptidase inhibitor 2 (TIMP2), C-type lectin domain family 3, member B (CLEC3B), lectin mannose-binding 2 (LMAN2), opticin (OPTC), serpin peptidase inhibitor clade F (SERPINF1). Representative MS/MS spectra of some identified proteins in this study are shown in (Figure 2).

We identified several components of complement system including complement C1q subcomponent subunit A (C1QA), C1QB, C1QC, C1QL3, C1QTNF3, C1QTNF5,
<figure><img src="image_1.png" /><figcaption>**Figure 1** Experimental design for proteomic characterization of vitreous humor. Pooled vitreous humor samples were depleted of abundant proteins using Agilent's MARS 14 column followed by *in-gel* digestion, *in-solution* digestion and OFFGEL electrophoresis. The samples were analyzed on LTQ-Orbitrap Velos mass spectrometer.</figcaption></figure>

C1R, C1RL, C1S, C2, C3, C4A, C4B, C4BPA, C5, C6, C7, C8A, C8B, C8G, C9, CFB, CFD, CFH, CFHR1, CFHR2, CFHR3, CFI.

Activation of the complement pathways can initiate and accelerate thrombosis, apoptosis and leukostasis, all of which could be important steps in the development of diabetic retinopathy [20]. Some of the complement factors

are also associated with age-related macular degeneration (AMD). Raychaudhuri et al., have shown that compromised complement factor H (CFH) function contributes to pathogenesis of AMD [21]. CFH risk allele reveals a polymorphism representing a tyrosine to histidine change at amino acid 402, which is associated with AMD [22]. This region of CFH is crucial for binding of heparin and
<table><caption>Table 1 A partial list of novel proteins identified in this study</caption><thead><tr><td>Gene symbol</td><td>Description</td><td>Cellular component</td><td>Biological process</td><td>Molecular function</td></tr></thead><tbody><tr><td>LCN1</td><td>Lipocalin-1</td><td>Extracellular</td><td>Transport</td><td>Transporter activity</td></tr><tr><td>TIMP1</td><td>Metalloproteinase inhibitor 1</td><td>Extracellular</td><td>Cell growth</td><td>Extracellular matrix structural constituent</td></tr><tr><td>SPARC</td><td>Sparc</td><td>Extracellular</td><td>Cell growth and/or maintenance</td><td>Extracellular matrix structural constituent</td></tr><tr><td>CHRDL1</td><td>Chordin-like protein 1</td><td>Unknown</td><td>Cell communication, Signal transduction</td><td>Molecular function unknown</td></tr><tr><td>DCD</td><td>Dermcidin</td><td>Extracellular</td><td>Immune response</td><td>Molecular function unknown</td></tr><tr><td>C19orf10</td><td>Hypothetical protein loc56005</td><td>Unknown</td><td>Cell communication, Signal transduction</td><td>Growth factor activity</td></tr><tr><td>PI3</td><td>Elafin</td><td>Extracellular</td><td>Protein metabolism</td><td>Protease inhibitor activity</td></tr><tr><td>ADIPOQ</td><td>Adiponectin</td><td>Extracellular</td><td>Metabolism, Energy pathways</td><td>Molecular function unknown</td></tr><tr><td>SFRP2</td><td>Secreted frizzled-related protein 2</td><td>Extracellular</td><td>Cell communication, Signal transduction</td><td>Molecular function unknown</td></tr><tr><td>PTX3</td><td>Pentraxin-related protein</td><td>Extracellular</td><td>Immune response</td><td>Defense/immunity protein activity</td></tr><tr><td>PTPMT1</td><td>Protein-tyrosine phosphatase mitochondrial 1</td><td>Unknown</td><td>Cell communication, Signal transduction</td><td>Protein tyrosine/serine/threonine phosphatase activity</td></tr><tr><td>FLG</td><td>Filaggrin</td><td>Nucleus</td><td>Cell communication, Signal transduction</td><td>Calcium ion binding</td></tr><tr><td>ACTB</td><td>Actin, cytoplasmic 1</td><td>Cytoplasm</td><td>Cell growth and/or maintenance</td><td>Structural constituent of cytoskeleton</td></tr><tr><td>AZU1</td><td>Azurocidin</td><td>Cytoplasmic vesicle</td><td>Immune response</td><td>Defense/immunity protein activity</td></tr><tr><td>NENF</td><td>Neudesin</td><td>Extracellular</td><td>Biological_process unknown</td><td>Kinase regulator activity</td></tr></tbody></table>

C-reactive protein [23]. Ennis et al., have also shown that SNP in complement factor 1 is associated with risk of AMD [24].

Opticin (OPTC) is a member of the small leucine-rich repeat protein (SLRP) family. Opticin is expressed in the retina, skin, iris, vitreous humor, non-pigmented epithelium of the ciliary body, sclera, optic nerve, choroid, corneal epithelium, uveal tract and lens [25-27]. It is associated with age related macular degeneration and posterior column ataxia with retinitis pigmentosa, both of which are inherited eye diseases [28].

Retinol-binding protein 3 (RBP3) is a soluble single subunit glycoprotein that is synthesized and secreted by

rod photoreceptor cells into the interphotoreceptor matrix [29-32]. It is believed to transport all trans retinol to the retinal pigment epithelium (RPE) and 11-cis retinal from the RPE to the bleached photoreceptors, thus playing an important role in the visual cycle [33]. A mutation in the RBP3 gene (Asp1080Asn) has been linked to autosomal recessive retinitis pigmentosa [34]. RBP3 is known to modulate the notch signal transduction by interacting with the phosphorylated intracellular domain of the notch receptor [35]. It is surprising that molecules such as RBP3, which appears to function at the back of the retina farthest from the vitreous, could still be found in the vitreous. The possible explanation for this

<table><caption>Table 2 A summary of some of the previously published proteomics studies on vitreous humor and the corresponding mass spectrometry platforms</caption><thead><tr><td></td><td>Study</td><td>Fractionation/separation method</td><td>Mass spectrometer</td><td>Number of proteins identified</td></tr></thead><tbody><tr><td>1.</td><td>Aretz S et al., [16]</td><td>SDS-PAGE, Liquid phase IEF</td><td>LTQ-ORBITRAP XL</td><td>1,111</td></tr><tr><td>2.</td><td>Simo et al., [17]</td><td>DIGE, Western blot</td><td>MALDI</td><td>2</td></tr><tr><td>3.</td><td>Gao et al. [14]</td><td>SDS-PAGE</td><td>LTQ</td><td>252</td></tr><tr><td>4.</td><td>Kim et al. [13]</td><td>2-DE<br>SDS-PAGE</td><td>(IS)/2-DE/MALDI-MS, LC-MALDI-MS/MS, and LC-ESI-MS/MS</td><td>346</td></tr><tr><td>5.</td><td>Ouchi et al. [15]</td><td>2D-PAGE</td><td>CapLC system with QTOF2</td><td>15</td></tr><tr><td>6.</td><td>Wu et al., [18]</td><td>SDS-PAGE</td><td>MALDI-TOF</td><td>12</td></tr><tr><td>7.</td><td>Yamane et al., [19]</td><td>2D-PAGE</td><td>Q-TOF</td><td>18</td></tr><tr><td>8.</td><td>Nakanishi et al. [12]</td><td>2D-PAGE</td><td>MALDI-TOF<br>LCQ DECA</td><td>51</td></tr></tbody></table>
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 2** Representative MS/MS spectra of known proteins in vitreous that were identified in this study. A. peptide YLAEFATGNDR belongs to 14-3-3 protein epsilon. B. Peptide EVPDEYEVGSFMEEVR belongs to dickkopf-related protein 3. C. Peptide TFHEASEDCISR belongs to tetranectin. D. Peptide IDLSNNLISSIDNDAFR belongs to opticin.</figcaption></figure>

could be that even though the intravascular contents are prevented from directly reaching the vitreous by the presence of the blood ocular barrier, there is no evidence of an effective barrier for proteins in the intercellular and interstitial spaces of retina and surrounding tissues.

Serpin peptidase inhibitor, clade F, member 1 (SERPINF1) is a 50 kDa secreted glycoprotein and reported to be expressed in many tissue and vitreous [36,37]. It belongs to a group of serine protease inhibitors with anti-angiogenic activities [38,39]. SERPINF1 concentration is found to be significantly lower in the vitreous fluid of subjects with PDR [40-42].

## Novel proteins identified in the vitreous humor

Representative MS/MS spectra of two unique proteins identified in this study –Interleukin 6 (IL-6) and transforming growth factor beta-2 (TGFB2) are shown in Figure 3. Interleukin-6 is a cytokine released in acute and chronic inflammation. This molecule has been extensively studied as an early indicator of inflammatory disorders such as acute pancreatitis and urticaria [43,44]. All insulin-like growth factor-binding proteins (IGFBP) are secreted and have a leader sequence. They are conserved at amino and carboxyl terminal with 18 cysteine residues. All these genes encode proteins with an IGFBP domain and a thyroglobulin type-I domain. These proteins bind to insulin-like growth factors (IGFs) I and II and are

found in the plasma in the form of glycosylated and non-glycosylated forms [45-48]. IGFBP4, IGFBP6 are O-glycosylated and IGFBP5 and IGFBP6 are N-glycosylated [49,50]. IGFBP6 is tumor suppressor, [51] extracellular protein [52] and preferentially binds to IGF-II [53]. IGFBP2 serves as an antiapoptotic biomarker [54]. We identified several members of the insulin-like growth factor-binding protein (IGFBP) family including IGFBP2, IGFBP4, insulin-like growth factor 2 (IGF2), insulin-like growth factor binding protein acid labile subunit (IGFALS). Other members of this family, IGFBP5, IGFBP6 and IGFBP7, have already been identified in previous studies.

We compared our results with other high throughput studies on vitreous humour [13,14,16]. We have identified 523 proteins identified by other studies. In addition, we have identified 682 novel proteins not described in previous studies. Of the 682 novel proteins, we identified proteins such as S-Arrestin(SAG) and transforming growth factor-beta 2 (TGF-B2) which are involved in the pathophysiology of ocular diseases. S Arrestin, also known as S antigen(S-Ag) is a major photoreceptor protein. It is a member of the beta-arrestin protein family which participate in agonist-mediated desensitization of G-protein-coupled receptors. Arrestin preferentially binds light activated phosphorylated rhodopsin and prevents further signaling by direct competition with transducin, a visual G-protein [55]. It is expressed in the retina and
A

Transforming growth factor beta-2
FAGIDGTSTYTSGDQK

<figure><img src="image_6.png" /></figure>

B

Interleukin-6 Precursor (IL-6)
YILDGISALR

<figure><img src="image_7.png" /><figcaption>**Figure 3** Representative MS/MS spectra of novel proteins identified in this study. A. Peptide FAGIDGTSTYTSGDQK belongs to Transforming growth factor beta-2 (TGFB2). B. Peptide YILDGISALR belongs to Interleukin-6 Precursor (IL-6).</figcaption></figure>

pineal gland. This protein is highly antigenic and has been implicated in experimental uveoretinitis. There is also recent evidence to suggest S-Ag specific T cells may be involved in the pathophysiology of Bechets disease, a chronic, relapsing, multisystem inflammatory disorder characterized by recurrent oral and genital ulcers, severe intraocular inflammation and skin lesions [56]. Mutations in the S Arrestin gene is also associated with an autosomal recessive form of night blindness known as Oguchi disease [57-59].

Transforming growth factor-beta 2(TGF-B2) belongs to the TGFB family of cytokines. These proteins bind to their transmembrane receptors, which in turn activate

their downstream effectors like SMAD proteins which are known to regulate cell proliferation, apoptosis and differentiation [60]. Primary open angle glaucoma is a disease of the eye which is characterized by elevated intra ocular pressure due to increased resistance to the outflow of aqueous humor through the trabecular meshwork. TGF-B2 has been shown to be elevated in the aqueous humor of patients with primary open angle glaucoma. Its role in glaucoma is thought to be due to the increased production of extracellular matrix in the trabecular meshwork [61]. A genetic defect in the gene that codes for this protein is associated with Peter's anomaly which is a congenital defect of the anterior chamber of the eye [62].
## Gene ontology analysis

We carried out a bioinformatics analysis of subcellular localization, molecular function and biological processes by searching the identified proteins against the manually curated Human Protein Reference Database (HPRD; http://hprd.org) and Human Proteinpedia (http://humanproteinpedia.org) [63-65]. Of the 1,205 proteins identified in this study, 599 proteins possess signal peptides and 318 proteins are reported to be localized in extracellular compartment. As illustrated in Figure 4A, the majority of the proteins reported in our study were localized to extracellular space (24%), cytoplasm (20%) or plasma membrane (14%). We also classified proteins based on molecular functions and biological processes as shown in Figure 4B, C. Classification based on molecular function showed that the large majority of the proteins are involved in catalytic activity (27%), structural activity (10%), binding activity such as calcium ion binding, receptor binding and complement binding (10%), cell adhesion molecule activity (4%), and transporter activity (4%). A large group (19%) of proteins are still unclassified in terms of their molecular function. Further categorization was done for biological processes which constitute metabolism (28%), cell communication (19%), cell growth (12%), and immune response (7%).

## Conclusions

Our study provides a comprehensive proteomics profile of the human vitreous humor. Many of the pathologic changes occurring in the retina are likely to be reflected in the vitreous because of its close proximity to the retina and also because of the breakdown of the blood retinal barrier. Hence, a proteomic study of the vitreous in related retinal diseases such as diabetic retinopathy, retinal detachment and central or branch retinal vein occlusions would provide valuable insights about the pathophysiology of these diseases. The information from our study could serve as a baseline for future studies especially those aimed at identifying biomarkers for retinal disorders.

## Materials & methods

### Vitreous sample collection

The vitreous samples for the proteomic analysis were obtained from five patients undergoing vitrectomy for macular hole, three patients with congenital cataract who underwent cataract surgery with intra-ocular lens implantation and primary posterior capsulotomy and two samples from patients with traumatic cataract with undisturbed vitreous and intact lens capsule but who also needed vitreous surgery due to zonular damage. All samples were collected by pars plana vitrectomy, were centrifuged at 13,000 rpm at 4°C for 15 minutes and

A

<figure><img src="image_8.png" /></figure>

B

<figure><img src="image_9.png" /></figure>

C

<figure><img src="image_10.png" /></figure>

**Figure 4** Subcellular localization and functional annotation of proteins identified in vitreous humor. **A.** Gene Ontology analysis for subcellular localization of identified proteins. **B.** Molecular functions of identified proteins. **C.** Biological processes of the identified proteins. The data regarding proteins was obtained from Human Protein Reference Database (HPRD: http://www.hprd.org).
archived at -80°C until further use. Informed consent was
obtained from all the subjects and the research adhered
to the tenets of Declaration of Helsinki. The study was
approved by the Ethics committee "Science for Health"
under the approval number 20080713/SFH-014/010.
Pooled samples were concentrated by 3 kDa filter through
low-adsorption membranes (Amicon, Milliore, Billerica,
MA). Protein estimation was carried out for pooled concen-
trated sample using Lowry's assay (BioRad Laboratories). A
total of 4 mg protein was subjected to Agilent's Multiple
Affinity Removal System 14 (MARS 14) for depletion of
abundant proteins. MARS 14 column is routinely used for
depletion of abundant proteins in serum/plasma. This step
allows us to deplete highly abundant albumin, IgG, trans-
ferrin, heptoglobin, IgM, IgA, fibrinogen, alpha antitryp-
sin, apolipoprotein A1, alpha 1 acid glycoprotein, alpha2
macroglobin, transthyretin, complement C3 and apolipo-
proteins. We had to optimize our procedures for depletion
of abundant proteins from vitreous humor. For each de-
pletion cycle, Agilent recommends 20 µl of serum that is
approximately equivalent to 1 mg of total protein. During
depletion of abundant proteins from vitreous, we also
used 1 mg of protein for each depletion cycle. As we had
4 mg protein, we carried out 4 independent depletion cy-
cles and pooled the flow through fractions. After passing
the vitreous sample through MARS 14 column, 460 µg of
protein was recovered in the flow through and the
remaining 3.54 mg of protein accounted for the bound
fraction. 60 µg of depleted vitreous sample was resolved
on SDS-PAGE and subjected to in-gel digestion prior to
mass spectrometry analysis. The remaining 400 µg of pro-
tein was reduced (5 mM DTT), alkylated (20 mM iodoa-
cetamide) and digested using trypsin 1:20 w/w (Promega,
Madison, WI) overnight at 37°C. The samples were acid-
ified by adding 20% trifluoro acetic acid (TFA) to a final
concentration of 0.1% and desalted using C₁₈ macro-spin
columns (Harvard apparatus, catalog no. 74-4101). 200 µg
peptide digest was used for SCX fractionation as well as
OFFGEL fractionation.

In-gel digestion

Sixty micrograms of depleted vitreous sample was resolved
by SDS-PAGE and stained using colloidal Coomassie
stain. The lane was excised into 16 pieces and destained
with 40 mM ammonium bicarbonate in 50% acetonitrile
(ACN). Trypsin digestion was carried out essentially as de-
scribed previously [66,67]. Briefly, reduction was carried
out using 5 mM dithiothreitol (DTT, 60°C for 45 minutes)
followed by alkylation using 20 mM iodoacetamide (room
temperature for 10 min). Sequencing grade modified por-
cine trypsin (Promega, Madison, WI, US) in ammonium
bicarbonate was added to the gel pieces at 4°C and incu-
bated for 45 minutes. Excess trypsin was removed and the
gel pieces were immersed in ammonium bicarbonate and

incubated overnight at 37°C. The peptides were extracted
from the gel bands using 0.4% formic acid in 3% ACN
twice, once using 0.4% formic acid in 50% ACN and once
using 100% ACN. The extracted peptides were dried using
speedvac and stored at -80°C until LC-MS/MS analysis.

**Strong cation exchange chromatography**

The peptide digest equivalent to 200 µg was reconstituted with 10 mM potassium phosphate buffer containing 30% ACN, pH 2.7 (solvent A). SCX fractionation was carried out using Polysulfoethyl A column (PolyLC, Columbia, MD) (300 Å, 5 µm, 100 × 2.1 mm) using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, USA) containing a binary pump, UV detector and a fraction collector [68,69]. The peptides were eluted using a linear salt gradient (0 to 35%) of solvent B (10 mM potassium phosphate buffer containing 30% ACN, 350 mM KCl, pH 2.7) at a flow rate of 200 µl/min. The fractions were completely dried and reconstituted in 0.1% TFA. They were desalted using stage-tips and dried on speedvac.

OFFGEL fractionation

The peptide digest equivalent to 200 µg in-solution digest
was used for OFFGEL fractionation [70,71]. Agilent 3100
OFFGEL fractionator (Agilent Technologies, Santa Clara,
USA) was used for pI based separation of peptides. As per
the protocol, peptides were separated using pH 3-10
Immobilized pH gradient (IPG) strip, 13 cm. The peptides
were focused at 50 kVh with maximum current of 50 µA
and maximum voltage set to 4000 V. Twelve fractions
were collected and acidified to obtain a final concentration
of 0.1% TFA prior to sample cleaning using stage-tip
protocol [72].

LC-MS/MS analysis

LC-MS/MS analyses of the samples were carried out using
high resolution Fourier transform mass spectrometer, LTQ-
Orbitrap Velos (Thermo Electron, Bremen, Germany). The
mass spectrometer was interfaced with a nano-LC system
to a trap column (2 cm × 75 µm, C18 material 5 µm,
120 Å) and an analytical column (10 cm × 75 µm, C18 ma-
terial 5 µm, 120 Å). Electrospray source was fitted with an
8 µm emitter tip (New Objective, Woburn, MA) and was
applied a voltage of 2000 V. Peptide samples were loaded
onto trap column in 3% solvent B (90% ACN in 0.1% for-
mic acid) and washed for 5 minutes before peptide elution
using a gradient of 3-35% solvent B for 60 minutes at a
constant flow rate of 0.4 µl/min. Xcalibur 2.1 (Thermo
Electron, Bremen, Germany) was used for data acquisi-
tion. The MS spectra were acquired in a data-dependent
manner targeting the twenty most abundant ions in each
survey scan in the range of m/z 350 to 1,800. Precursor
ions selected for MS/MS fragmentation were dynamically
excluded for 30s. Target ion quantity for FT full MS and
MS2 were 5 × 10⁵ and 2 × 10⁵ respectively. Higher-energy collisional dissociation (HCD) was used for precursor fragmentation. MS and MS/MS data were acquired at a resolution of 60,000 and 15,000 at 400 m/z, respectively. Internal calibration was enabled using polydimethylcyclo-siloxane (m/z, 445.1200025) ions and lock mass was used for accurate mass measurements.

## Data analysis

Mass spectrometry data was processed using the Proteome Discoverer software (Version 1.4.1.14, Thermo Fisher Scientific, Bremen, Germany). Mascot, SEQUEST and X!Tandem search engines were employed to maximize the peptide identification. The mass spectrometry data was searched against NCBI RefSeq 59 human protein database containing 36,211 sequences with known contaminants. Carbamidomethylation of cysteine was used as the fixed modification and oxidation of methionine and protein N-terminal acetylation as variable modifications. Peptide mass and fragment mass tolerance were set as 20 ppm and 0.1 Da, respectively with 1 missed cleavage. Peptide identifications were filtered by setting 1% target false discovery rate (FDR). Subcellular localization, molecular function and biological process of identified proteins were analyzed using gene ontology (GO) compliant databases - Human Protein Reference Database (HPRD: http://www.hprd.org) and Human Proteinindex [63,73].

## Data availability

The raw data obtained from vitreous proteome are submitted to public data repositories. The peptide identifications and MS/MS spectra are available on Human Proteinindex [63], (https://www.humanproteinindex.org) as accession number HuPA_00682. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [74] via the PRIDE partner repository with the dataset identifier PXD000957.

## Additional files

**Additional file 1: Table S1.** A complete list of proteins identified in the vitreous humor. This table provides a list of all the proteins identified in vitreous including their gene symbol, RefSeq accession, protein description, number of peptides, sequence coverage, reported subcellular localization, molecular function, biological process and domains/motifs.

**Additional file 2: Table S2.** A list of all peptides identified in the vitreous humor.

## Competing interest

All authors have expressed no conflict of interest.

## Authors' contributions

KRM, PRM, HG, TSKP and AP conceptualized and designed the study. KRM and PRM provided the samples. KRM, RG and HKCJ contributed to sample preparation. NAS and RS performed the mass spectrometry experiments SMS, YS, AHP and A Parashar contributed to data analysis. KRM and RG wrote the manuscript. KRM, BGN, VK, TSKP, HG and AP designed the

experiments, supervised the experiments and data analysis and critically reviewed the manuscript. All the authors have read and approved the final manuscript.

## Acknowledgements

We thank the Department of Biotechnology (DBT) of the Government of India for research support to the Institute of Bioinformatics, Bangalore. Nandini A. Sahasrabuddhe and Harrys K.C. Jacob are recipients of Senior Research Fellowship from the Council for Scientific and Industrial Research (CSIR), India. Srikanth Srinivas Manda is a recipient of Senior Research Fellowship from University Grants Commission (UGC), India. Rakesh Sharma is a Research Associate supported by DBT. Harsha Gowda is a Wellcome Trust/DBT India Alliance Early Career Fellow. Dr. T. S. Keshava Prasad is the recipient of a research grant on "Development of Infrastructure and a Computational Framework for Analysis of Proteomic Data" from DBT.

## Author details

¹Institute of Bioinformatics, International Technology Park, Bangalore 560 066, India. ²Amrita School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, Kerala 690 525, India. ³Vittal International Institute Of Ophthalmology, Bangalore, Karnataka 560085, India. ⁴Department of Biotechnology, Kuvempu University, Shankaraghatta, Karnataka 577 451, India. ⁵Centre of Excellence in Bioinformatics, Bioinformatics Centre, School of Life Sciences, Pondicherry University, Puducherry 605 014, India. ⁶Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bangalore 560 006, India. ⁷Armed Forces Medical College, Pune 411 040, India. ⁸Department of Biological Chemistry, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore 21205, MD, USA.

⁹Department of Oncology and Pathology, Johns Hopkins University School of Medicine, Baltimore 21205, MD, USA.

Received: 19 February 2014 Accepted: 16 May 2014

Published: 14 July 2014

## References

1. Jack RL: Ultrastructure of the hyaloid vascular system. *Arch Ophthalmol* 1972, **87**(5):555-567.

2. Linsenmayer TF, Gibney E, Little CD: Type II collagen in the early embryonic chick cornea and vitreous: immunoradiochemical evidence. *Exp Eye Res* 1982, **34**(3):371-379.

3. Walker F, Patrick RS: Constituent monosaccharides and hexosamine concentration of normal human vitreous humour. *Exp Eye Res* 1967, **6**(3):227-232.

4. Locke JC, Morton WR: Further studies of the viscosity of aspirated human vitreous fluid: with special reference to its use in retinal detachment surgery. *Trans Am Ophthalmol Soc* 1965, **63**:129-145.

5. Scott JE: The chemical morphology of the vitreous. *Eye* 1992, **6**(Pt 6):553-555.

6. Bishop PN, Crossman MV, McLeod D, Ayad S: Extraction and characterization of the tissue forms of collagen types II and IX from bovine vitreous. *Biochem J* 1994, **299**(Pt 2):497-505.

7. Ramakrishnan S, Sulochana KN, Parikh S, Punitham R: Transthyretin (prealbumin) in eye structures and variation of vitreous-transthyretin in diseases. *Indian J Ophthalmol* 1999, **47**(1):31-34.

8. Eichenbaum JW, Zheng W: Distribution of lead and transthyretin in human eyes. *J Toxicol Clin Toxicol* 2000, **38**(4):377-381.

9. Clausen R, Weller M, Wiedemann P, Heimann K, Hilgers RD, Zilles K: An immunochemical quantitative analysis of the protein pattern in physiologic and pathologic vitreous. *Graefes Arch Clin Exp Ophthalmol* 1991, **229**(2):186-190.

10. Jacobson B: Identification of sialyl and galactosyl transferase activities in calf vitreous hyalocytes. *Curr Eye Res* 1984, **3**(8):1033-1041.

11. Yoshimura T, Sonoda KH, Sugahara M, Mochizuki Y, Enaida H, Oshima Y, Ueno A, Hata Y, Yoshida H, Ishibashi T: Comprehensive analysis of inflammatory immune mediators in vitreoretinal diseases. *PLoS One* 2009, **4**(12):e8158.

12. Nakanishi T, Koyama R, Ikeda T, Shimizu A: Catalogue of soluble proteins in the human vitreous humor: comparison between diabetic retinopathy and macular hole. *J Chromatogr B Analyt Technol Biomed Life Sci* 2002, **776**(1):89-100.
13. Kim T, Kim SJ, Kim K, Kang UB, Lee C, Park KS, Yu HG, Kim Y: Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients. *Proteomics* 2007, **7**(22):4203-4215.

14. Gao BB, Chen X, Timothy N, Aiello LP, Feener EP: Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy. *J Proteome Res* 2008, **7**(6):2516-2525.

15. Ouchi M, West K, Crabb JW, Kinoshita S, Kamei M: Proteomic analysis of vitreous from diabetic macular edema. *Exp Eye Res* 2005, **81**(2):176-182.

16. Aretz S, Krohne TU, Kammerer K, Warnken U, Hotz-Wagenblatt A, Bergmann M, Stanzel BV, Kempf T, Holz FG, Schnolzer M, Kopitz J: In-depth mass spectrometric mapping of the human vitreous proteome. *Proteome Sci* 2013, **11**(1):22.

17. Simo R, Higuera M, Garcia Ramirez M, Canals F, Garcia-Arumi J, Hernandez C: Elevation of apolipoprotein A-1 and apolipoprotein H levels in the vitreous fluid and overexpression in the retina of diabetic patients. *Arch Ophthalmol* 2008, **126**(8):1076-1081.

18. Wu CW, Sauter JL, Johnson PK, Chen CD, Olsen TW: Identification and localization of major soluble proteins in human ocular tissue. *Am J Ophthalmol* 2004, **137**(4):655-661.

19. Yamane K, Minamoto A, Yamashita H, Takamura H, Miyamoto-Myoken Y, Yoshizato K, Nabetani T, Tsugita A, Mishima HK: Proteome analysis of human vitreous proteins. *Mol Cell Proteomics* 2003, **2**(11):1177-1187.

20. Zhang J, Gerhardinger C, Lorenzi M: Early complement activation and decreased levels of glycosylphosphatidylinositol-anchored complement inhibitors in human and experimental diabetic retinopathy. *Diabetes* 2002, **51**(12):3499-3504.

21. Raychaudhuri S, Iartchouk O, Chin K, Tan PL, Tai AK, Ripke S, Gowrisankar S, Vemuri S, Montgomery K, Yu Y, Reynolds R, Zack DJ, Campochiaro B, Campochiaro P, Katsanis N, Daly MJ, Seddon JM: A rare penetrant mutation in CFH confers high risk of age-related macular degeneration. *Nat Genet* 2011, **43**(12):1232-1236.

22. Klein RJ, Zeiss C, Chew EY, Tsai JY, Sackler RS, Haynes C, Henning AK, SanGiovanni JP, Mane SM, Mayne ST, Bracken MB, Ferris FL, Ott J, Barnstable C, Hoh J: Complement factor H polymorphism in age-related macular degeneration. *Science* 2005, **308**(5720):385-389.

23. Rodriguez de Cordoba S, Esparza-Gordillo J, Goicoechea de Jorge E, Lopez-Trascasa M, Sanchez-Corral P: The human complement factor H: functional roles, genetic variations and disease associations. *Mol Immunol* 2004, **41**(4):355-367.

24. Ennis S, Gibson J, Cree AJ, Collins A, Lotery AJ: Support for the involvement of complement factor I in age-related macular degeneration. *Eur J Hum Genet* 2010, **18**(1):15-16.

25. Reardon AJ, Le Goff M, Briggs MD, McLeod D, Sheehan JK, Thornton DJ, Bishop PN: Identification in vitreous and molecular cloning of opticin, a novel member of the family of leucine-rich repeat proteins of the extracellular matrix. *J Biol Chem* 2000, **275**(3):2123-2129.

26. Ramesh S, Bonshek RE, Bishop PN: Immunolocalisation of opticin in the human eye. *Br J Ophthalmol* 2004, **88**(5):697-702.

27. Friedman JS, Faucher M, Hiscott P, Biron VL, Malenfant M, Turcotte P, Raymond V, Walter MA: Protein localization in the human eye and genetic screen of opticin. *Hum Mol Genet* 2002, **11**(11):1333-1342.

28. Friedman JS, Ducharme R, Raymond V, Walter MA: Isolation of a novel iris-specific and leucine-rich repeat protein (oculoglycan) using differential selection. *Invest Ophthalmol Vis Sci* 2000, **41**(8):2059-2066.

29. Liou GI, Ma DP, Yang YW, Geng L, Zhu C, Baehr W: Human interstitial retinoid-binding protein. *Gene structure and primary structure. J Biol Chem* 1989, **264**(14):8200-8206.

30. Taniguchi T, Adler AJ, Mizuochi T, Kochibe N, Kobata A: The structures of the asparagine-linked sugar chains of bovine interphotoreceptor retinol-binding protein. *Occurrence of fucosylated hybrid-type oligosaccharides. J Biol Chem* 1986, **261**(4):1730-1736.

31. Hollyfield JG, Fliesler SJ, Rayborn ME, Bridges CD: Rod photoreceptors in the human retina synthesize and secrete interstitial retinol-binding protein. *Prog Clin Biol Res* 1985, **190**:141-149.

32. Barnstable CJ, Tombran-Tink J: Neuroprotective and antiangiogenic actions of PEDF in the eye: molecular targets and therapeutic potential. *Prog Retin Eye Res* 2004, **23**(5):561-577.

33. Pepperberg DR, Okajima TL, Wiggert B, Ripps H, Crouch RK, Chader GJ: Interphotoreceptor retinoid-binding protein (IRBP). Molecular biology and physiological role in the visual cycle of rhodopsin. *Mol Neurobiol* 1993, **7**(1):61-85.

34. Den Hollander AI, McGee TL, Ziviello C, Banfi S, Dryja TP, Gonzalez-Fernandez F, Ghosh D, Berson EL: A homozygous missense mutation in the IRBP gene (RBP3) associated with autosomal recessive retinitis pigmentosa. *Invest Ophthalmol Vis Sci* 2009, **50**(4):1864-1872.

35. Foltz DR, Nye JS: Hyperphosphorylation and association with RBP of the intracellular domain of Notch1. *Biochem Biophys Res Commun* 2001, **286**(3):484-492.

36. Ortego J, Escribano J, Becerra SP, Coca-Prados M: Gene expression of the neurotrophic pigment epithelium-derived factor in the human ciliary epithelium. *Synthesis and secretion into the aqueous humor. Invest Ophthalmol Vis Sci* 1996, **37**(13):2759-2767.

37. Meyer C, Notari L, Becerra SP: Mapping the type I collagen-binding site on pigment epithelium-derived factor. Implications for its antiangiogenic activity. *J Biol Chem* 2002, **277**(47):45400-45407.

38. Fan W, Crawford R, Xiao Y: The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization. *Differentiation* 2011, **81**(3):181-191.

39. Park K, Jin J, Hu Y, Zhou K, Ma JX: Overexpression of pigment epithelium-derived factor inhibits retinal inflammation and neovascularization. *Am J Pathol* 2011, **178**(2):688-698.

40. Garcia-Ramirez M, Canals F, Hernandez C, Colome N, Ferrer C, Carrasco E, Garcia-Arumi J, Simo R: Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of proliferative diabetic retinopathy. *Diabetologia* 2007, **50**(6):1294-1303.

41. Noma H, Funatsu H, Mimura T, Eguchi S, Shimada K, Hori S: Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in macular edema with central retinal vein occlusion. *Curr Eye Res* 2011, **36**(3):256-263.

42. Konson A, Pradeep S, D'Acunto CW, Seger R: Pigment epithelium-derived factor and its phosphomimetic mutant induce JNK-dependent apoptosis and p38-mediated migration arrest. *J Biol Chem* 2011, **286**(5):3540-3551.

43. Khanna AK, Meher S, Prakash S, Tiwary SK, Singh U, Srivastava A, Dixit VK: Comparison of Ranson, Glasgow, MOSS, SIRS, BISAP, Apache-II, CTSI scores, IL-6, CRP and Procalcitonin in predicting severity, organ failure, pancreatic necrosis and mortality in acute pancreatitis. *HPB Surg* 2013, **2013**:367581.

44. Ucmak D, Akkurt M, Toprak G, Yesilova Y, Turan E, Yildiz I: Determination of dermatology life quality index, and serum C-reactive protein and plasma interleukin-6 levels in patients with chronic urticaria. *Postepy Dermatol Alergol* 2013, **30**(3):146-151.

45. Clemmons DR: Role of insulin-like growth factor binding proteins in controlling IGF actions. *Mol Cell Endocrinol* 1998, **140**(1-2):19-24.

46. Clemmons DR: Insulin-like growth factor binding proteins and their role in controlling IGF actions. *Cytokine Growth Factor Rev* 1997, **8**(1):45-62.

47. Clemmons DR, Busby W, Clarke JB, Parker A, Duan C, Nam TJ: Modifications of insulin-like growth factor binding proteins and their role in controlling IGF actions. *Endocr J* 1998, **45**(Suppl):S1-S8.

48. Ceda GP, Fielder PJ, Henzel WJ, Louie A, Donovan SM, Hoffman AR, Rosenfeld RG: Differential effects of insulin-like growth factor (IGF)-I and IGF-II on the expression of IGF binding proteins (IGFBPs) in a rat neuroblastoma cell line: isolation and characterization of two forms of IGFBP-4. *Endocrinology* 1991, **128**(6):2815-2824.

49. Shimasaki S, Gao L, Shimonaka M, Ling N: Isolation and molecular cloning of insulin-like growth factor-binding protein-6. *Mol Endocrinol* 1991, **5**(7):938-948.

50. Li Z, Picard F: Modulation of IGFBP2 mRNA expression in white adipose tissue upon aging and obesity. *Horm Metab Res* 2010, **42**(11):787-791.

51. Koyama N, Zhang J, Huqun, Miyazawa H, Tanaka T, Su X, Hagiwara K: Identification of IGFBP-6 as an effector of the tumor suppressor activity of SEMA3B. *Oncogene* 2008, **27**(51):6581-6589.

52. Ehrenborg E, Zazzi H, Lagercrantz S, Granqvist M, Hillerbrand U, Allander SV, Larsson C, Luthman H: Characterization and chromosomal localization of the human insulin-like growth factor-binding protein 6 gene. *Mamm Genome* 1999, **10**(4):376-380.

53. Neumann GM, Marinaro JA, Bach LA: Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6. *Biochemistry* 1998, **37**(18):6572-6585.
54. Migita T, Narita T, Asaka R, Miyagi E, Nagano H, Nomura K, Matsuura M, Satoh Y, Okumura S, Nakagawa K, Seimiya H, Ishikawa Y: Role of insulin-like growth factor binding protein 2 in lung adenocarcinoma: IGF-independent antiapoptotic effect via caspase-3. *Am J Pathol* 2010, **176**(4):1756-1766.

55. Huang T, Chen Q, Cho MK, Vishnivetskiy SA, Iverson TM, Gurevich VV, Sanders CR: Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin. *Proc Natl Acad Sci U S A* 2013, **110**(3):942-947.

56. Zhao C, Yang P, He H, Lin X, Li B, Zhou H, Huang X, Kijlstra A: S-antigen specific T helper type 1 response is present in Behcet's disease. *Mol Vis* 2008, **14**:1456-1464.

57. Huang L, Li W, Tang W, Zhu X, Ou-Yang P, Lu G: A Chinese family with Oguchi's disease due to compound heterozygosity including a novel deletion in the arrestin gene. *Mol Vis* 2012, **18**:528-536.

58. Fujinami K, Tsunoda K, Nakamura M, Oguchi Y, Miyake Y: Oguchi disease with unusual findings associated with a heterozygous mutation in the SAG gene. *Arch Ophthalmol* 2011, **129**(10):1375-1376.

59. Fuchs S, Nakazawa M, Maw M, Tamai M, Oguchi Y, Gal A: A homozygous 1-base pair deletion in the arrestin gene is a frequent cause of Oguchi disease in Japanese. *Nat Genet* 1995, **10**(3):360-362.

60. Shi Y, Massagué J: Mechanisms of TGF-beta signaling from cell membrane to the nucleus. *Cell* 2003, **113**(6):685-700.

61. Wordinger RJ, Fleenor DL, Hellberg PE, Pang IH, Tovar TO, Zode GS, Fuller JA, Clark AF: Effects of TGF-beta2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma. *Invest Ophthalmol Vis Sci* 2007, **48**(3):1191-1200.

62. David D, Cardoso J, Marques B, Marques R, Silva ED, Santos H, Boavida MG: Molecular characterization of a familial translocation implicates disruption of HDAC9 and possible position effect on TGFbeta2 in the pathogenesis of Peters' anomaly. *Genomics* 2003, **81**(5):489-503.

63. Mathivanan S, Ahmed M, Ahn NG, Alexandre H, Amanchy R, Andrews PC, Bader JS, Balgley BM, Bantscheff M, Bennett KL, Bjorling E, Blagoev B, Bose R, Brahmachari SK, Burlingame AS, Bustelo XR, Cagney G, Cantin GT, Cardasis HL, Celis JE, Chaerkady R, Chu F, Cole PA, Costello CE, Cotter RJ, Crockett D, DeLany JP, De Marzo AM, DeSouza LV, Deutsch EW, et al: *Human Proteinindex enables sharing of human protein data*. *Nat Biotechnol* 2008, **26**(2):164-167.

64. Mishra GR, Suresh M, Kumaran K, Kannabiran N, Suresh S, Bala P, Shivakumar K, Anuradha N, Reddy R, Raghavan TM, Menon S, Hanumanthu G, Gupta M, Upendran S, Gupta S, Mahesh M, Jacob B, Mathew P, Chatterjee P, Arun KS, Sharma S, Chandrika KN, Deshpande N, Palvankar K, Raghavnath R, Krishnakanth R, Karathia H, Rekha B, Nayak R, Vishnupriya G, et al: *Human protein reference database-2006 update*. *Nucleic Acids Res* 2006, **34**(Database issue):D411-D414.

65. Goel R, Harsha HC, Pandey A, Prasad TS: Human Protein Reference Database and Human Proteinindex as resources for phosphoproteome analysis. *Mol Biosyst* 2012, **8**(2):453-463.

66. Harsha HC, Molina H, Pandey A: Quantitative proteomics using stable isotope labeling with amino acids in cell culture. *Nat Protoc* 2008, **3**(3):505-516.

67. Goel R, Murthy KR, Srikanth SM, Pinto SM, Bhattacharjee M, Kelkar DS, Madugundu AK, Dey G, Mohan SS, Venkatarangaiah K, Prasad TSK, Chakravarti S, Harsha HC, Pandey A: Characterizing the normal proteome of human ciliary body. *Clin Proteomics* 2013, **10**(1):9.

68. Chaerkady R, Harsha HC, Nalli A, Gucek M, Vivekanandan P, Akhtar J, Cole RN, Simmers J, Schulick RD, Singh S, Torbenson M, Pandey A, Thuluvath PJ: A quantitative proteomic approach for identification of potential biomarkers in hepatocellular carcinoma. *J Proteome Res* 2008, **7**(10):4289-4298.

69. Yang Y, Chaerkady R, Kandasamy K, Huang TC, Selvan LD, Dwivedi SB, Kent OA, Mendell JT, Pandey A: Identifying targets of miR-143 using a SILAC-based proteomic approach. *Mol Biosyst* 2010, **6**(10):1873-1882.

70. Barbhuiya MA, Sahasrabuddhe NA, Pinto SM, Mutjusamy B, Singh TD, Nanjappa V, Keerthikumar S, Delanghe B, Harsha HC, Chaerkady R, Jalaj V, Gupta S, Srivastav BR, Tiwari PK, Pandey A: Comprehensive proteomic analysis of human bile. *Proteomics* 2011, **11**(23):4443-4453.

71. Kelkar DS, Kumar D, Kumar P, Muthusamy B, Yadav AK, Srivastava P, Marimuthu A, Anand S, Sundaram H, Kingsbury R, Harsha HC, Nair B, Prasad TS, Chauhan DS, Katoch K, Katoch VM, Kumar P, Chaerkady R, Ramachandran S, Dash D, Pandey A: Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry. *Mol Cell Proteomics* 2011, **10**(12):M111.011627. doi: 10.1074/mcp.M111.011445. Epub 2011 Oct 3.

72. Rappsilber J, Mann M, Ishihama Y: Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. *Nat Protoc* 2007, **2**(8):1896-1906.

73. Keshava Prasad TS, Goel R, Kandasamy K, Keerthikumar S, Kumar S, Mathivanan S, Telikicherla D, Raju R, Shafrreen B, Venugopal A, Balakrishnan L, Marimuthu A, Banerjee S, Somanathan DS, Sebastian A, Rani S, Ray S, Harrys Kishore CJ, Kanth S, Ahmed M, Kashyap MK, Mohmood R, Ramachandra YL, Krishna V, Rahimian BA, Mohan S, Ranganathan P, Ramabadran S, Chaerkady R, Pandey A: Human Protein Reference Database-2009 update. *Nucleic Acids Res* 2009, **37**(Database issue):D767-D772.

74. Vizcaíno JA, Deutsch EW, Wang R, Csordas A, Reisinger F, Ríos D, Dianes JA, Sun Z, Farrah T, Bandeira N, Binz PA, Xenarios I, Eisenacher M, Mayer G, Gatto L, Campos A, Chalkley RJ, Kraus HJ, Albar JP, Martinez-Bartolomé S, Apweiler R, Omenn GS, Martens L, Jones AR, Hermjakob H: ProteomeXchange provides globally co-ordinated proteomics data submission and dissemination. *Nature Biotechnol* 2014, **30**(3):223-226.

doi:10.1186/1559-0275-11-29

Cite this article as: Murthy et al.: Proteomic analysis of human vitreous humor. *Clinical Proteomics* 2014 **11**:29.

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