title: Proteomic profiling of retinoblastoma by high resolution mass spectrometry.
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.
authors: 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.
authors: Ravikanth
authors: R
authors: Danda
authors: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India.
authors: Kalaivani
authors: K
authors: Ganapathy
authors: Institute of Bioinformatics, International Technology Park, Bangalore, Karnataka 560066 India.
authors: Gajanan
authors: G
authors: Sathe
authors: Institute of Bioinformatics, International Technology Park, Bangalore, Karnataka 560066 India.
authors: Anil K
authors: AK
authors: Madugundu
authors: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India.
authors: Sharavan
authors: S
authors: Ramachandran
authors: Centre for Nanotechnology and Advanced Biomaterials, Shanmugha Arts, Science, Technology and Research Academy University, Tanjore, Tamilnadu India.
authors: Uma Maheswari
authors: UM
authors: Krishnan
authors: Shri Bhagwan Mahavir Vitreoretinal Services and Ocular Oncology Services, Medical Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India.
authors: Vikas
authors: V
authors: Khetan
authors: Shri Bhagwan Mahavir Vitreoretinal Services and Ocular Oncology Services, Medical Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India.
authors: Pukhraj
authors: P
authors: Rishi
authors: Institute of Bioinformatics, International Technology Park, Bangalore, Karnataka 560066 India.
authors: T S
authors: TS
authors: Keshava Prasad
authors: 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.
authors: Akhilesh
authors: A
authors: Pandey
authors: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India.
authors: Subramanian
authors: S
authors: Krishnakumar
authors: Institute of Bioinformatics, International Technology Park, Bangalore, Karnataka 560066 India.
authors: Harsha
authors: H
authors: Gowda
authors: Department of Nano-Biotechnology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamilnadu 600006 India.
authors: Sailaja V
authors: SV
authors: Elchuri