Quantitative proteomic analysis

Quantitative proteomic analysis of Epac induced differentiation in human SH-SY5Y neuroblastoma cells
Neuroblastoma is a pediatric solid tumor, which is derived from precursor or immature cells of the sympathetic nervous system (SNS), and primary tumours can be found at any location of SNS structures [1]. Mutations in the receptor tyrosine kinase ALK (anaplastic lymphoma kinase) are the main cause of familial neuroblastoma. Moreover, oncogenic mutations of ALK are also found in primary tumours and in the human neuroblastoma cell line SH-SY5Y [2]. Despite that the noradrenergic SH-SY5Y cell line originates from a highly malignant tumour it has maintained the capacity to undergo neuronal differentiation under proper growth conditions. These characteristics together with a distinct sensitivity to oxidative stress makes the neuroblastoma SH-SY5Y cell line an excellent model system to study neuronal differentiation and neuroblastoma tumourigenesis, as well as several aspects of neuronal degenerative diseases.

The discovery of the exchange protein directly activated by cAMP (Epac) a novel cAMP receptor protein [3], opened up a new field of research in cAMP mediated cell signaling. Previously the belief was that all cAMP signaling was mediated through cAMP dependent protein kinase (PKA).

We use shRNA to knock out (KO) Epac 1. By subjecting both wild type and Epac KO SH-SY5Y cells to treatment with a selective Epac analog we are able to study the morphological changes induced by Epac. We also use quatitative proteomics. Some proteomic studies have been performed on differentiating neuron like cells, however none has been done on differentiating human neuroblastoma SH-SY5Y cells. We use 1D-SDS gradient gel electrophoresis and LC-Q-TOF-MS/MS to identify and quantify the effect of Epac on the proteome of differentiating neuroblastoma cells.