Proteomic profiling of retinoblastoma by high resolution mass spectrometry

Ravikanth Danda; Kalaivani Ganapathy; Gajanan Sathe; Anil K Madugundu; Sharavan Ramachandran; Uma Maheswari Krishnan; Vikas Khetan; Pukhraj Rishi; T S Keshava Prasad; Akhilesh Pandey; Subramanian Krishnakumar; Harsha Gowda; Sailaja V Elchuri

doi:10.1186/s12014-016-9128-7

Proteomic profiling of retinoblastoma by high resolution mass spectrometry

Clin Proteomics. 2016 Oct 26:13:29. doi: 10.1186/s12014-016-9128-7. eCollection 2016.

Affiliations

¹ Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India ; Centre for Nanotechnology and Advanced Biomaterials, Shanmugha Arts, Science, Technology and Research Academy University, Tanjore, Tamilnadu India.
² Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India.
³ Institute of Bioinformatics, International Technology Park, Bangalore, Karnataka 560066 India.
⁴ Centre for Nanotechnology and Advanced Biomaterials, Shanmugha Arts, Science, Technology and Research Academy University, Tanjore, Tamilnadu India.
⁵ Shri Bhagwan Mahavir Vitreoretinal Services and Ocular Oncology Services, Medical Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India.
⁶ McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA ; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA ; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA ; Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
⁷ Department of Nano-Biotechnology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India.

Abstract

Background: Retinoblastoma is an ocular neoplastic cancer caused primarily due to the mutation/deletion of RB1 gene. Due to the rarity of the disease very limited information is available on molecular changes in primary retinoblastoma. High throughput analysis of retinoblastoma transcriptome is available however the proteomic landscape of retinoblastoma remains unexplored. In the present study we used high resolution mass spectrometry-based quantitative proteomics to identify proteins associated with pathogenesis of retinoblastoma.

Methods: We used five pooled normal retina and five pooled retinoblastoma tissues to prepare tissue lysates. Equivalent amount of proteins from each group was trypsin digested and labeled with iTRAQ tags. The samples were analyzed on Orbitrap Velos mass spectrometer. We further validated few of the differentially expressed proteins by immunohistochemistry on primary tumors.

Results: We identified and quantified a total of 3587 proteins in retinoblastoma when compared with normal adult retina. In total, we identified 899 proteins that were differentially expressed in retinoblastoma with a fold change of ≥2 of which 402 proteins were upregulated and 497 were down regulated. Insulin growth factor 2 mRNA binding protein 1 (IGF2BP1), chromogranin A, fetuin A (ASHG), Rac GTPase-activating protein 1 and midkine that were found to be overexpressed in retinoblastoma were further confirmed by immunohistochemistry by staining 15 independent retinoblastoma tissue sections. We further verified the effect of IGF2BP1 on cell proliferation and migration capability of a retinoblastoma cell line using knockdown studies.

Conclusions: In the present study mass spectrometry-based quantitative proteomic approach was applied to identify proteins differentially expressed in retinoblastoma tumor. This study identified the mitochondrial dysfunction and lipid metabolism pathways as the major pathways to be deregulated in retinoblastoma. Further knockdown studies of IGF2BP1 in retinoblastoma cell lines revealed it as a prospective therapeutic target for retinoblastoma.