Proteomic analysis of differentially expressed proteins in vitreous humor of patients with retinoblastoma using iTRAQ-coupled ESI-MS/MS approach

Abstract

There is close proximity of vitreous humor with the tumor bulk in eyes with retinoblastoma. This renders vitreous humor a promising source to evaluate disease-specific protein targets in retinoblastoma. We studied the differential proteome of vitreous fluid in retinoblastoma tumors (n = 4) as compared to controls (n = 4). The vitreous humor was depleted off the high abundant fraction using MARS-6 affinity column. Subsequently, the tryptic peptides were derivatised with iTRAQ labels. The labelled peptides were pooled and subjected to fractionation using bRPLC. This was followed by protein identification and quantification using electrospray ionisation mass spectrometry (ESI-MS/MS) approach. The identified proteins were subjected to bioinformatics analysis utilizing PANTHER 7.0 and IPA software. Four hundred and thirty-one non-redundant (362 upregulated and 69 downregulated) proteins (≥2 unique peptides, ± 1.5 folds, p < 0.05) were identified. The majority of the proteins were cytoplasmic (40 %), majorly involved in catalytic (32.7 %) and binding activities (26.3 %). Highly deregulated proteins included MMP2, TNC, CD44, SUZ12 and CRABP1. The protein expression of GFAP, CRABP1, MMP2 and TNC was validated by western blotting. Pathway and network analyses revealed p38MAPK and Akt signalling to be the most significantly regulated pathways in retinoblastoma. This is the first report of differential vitreous proteome of retinoblastoma and highlights novel protein targets, such as MMP2, TNC and CRABP1. Further investigations into unravelling the biological role of the proteins and their prospects of being utilised as potential candidates in therapeutics are warranted.

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“…[78] Another group conducted iTRAQ-coupled ESI-MS/MS technology to analyze vitreous humor in patients with retinoblastoma, finding several proteins to be highly dysregulated including matrix metalloproteinase 2 (MMP2), tenascin C (TNC), CD44, polycomb protein SUZ12 (SUZ12), and cellular retinoic acid binding protein 1 (CRABP1). [79] Subjects with glaucoma were found to have significant downregulation of crystallins (which have also been implicated in neuroprotection), antioxidant proteins, and upregulation of the complement cascade, however it remains unclear if these alterations have clinical significance or simply reflect efflux of proteins from the retina. [81]…”