I-MS/MS with the application of a distal 1.3 kV with heated capillary at the temperature of 200 C. Each cycle of one full scan mass spectrum was followed by three datadependent tandem mass spectra with the collision energy was set at 35%. 2.6. Database Search. For protein identification, Mascot software was used to search the human protein sequence database. For proteolytic cleavages, only tryptic cleavage was allowed, and the number of maximal internal cleavage sites was PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19828691 set to 2. Variable modifications of cysteine with carboxyamidomethylation, methionine with oxidation, and asparagine/glutamine with deamidation were allowed. The mass tolerances of the precursor peptide ion and fragment ion were set to 1 Da. Positive protein identifications were defined if the Mowse scores of greater than 50 were considered significant. Proteins were initially annotated by similar searches using UniProtKB/Swiss-Prot databases . 
3 and cell pathophysiological mechanisms. It is composed of proteins that are found in the extracellular growth medium. The cell secretome consists of proteins that are secreted, shed from the cell surface and intracellular proteins released into the supernatant due to cell lysis, apoptosis, and necrosis. The secretome which consists of proteins or peptides secreted from cells into the extracellular medium represents the major class of molecules involved in the intercellular communication in multicellular organisms. It constitutes an important class of proteins that control and regulate a multitude of biological and physiological processes and indicates a clinically relevant source for biomarker and therapeutic target discoveries. Thus, secreted proteins constitute an important category of active molecules that play crucial roles in a number of physiological and pathological processes and may reflect a broad variety of pathological conditions and thus represent a rich source of biomarkers. Proteomic characterization of proteins for identification of specific biomarkers provides a powerful tool to gain deep insights into disease mechanisms in which proteins play major roles. In this study, we have used gel electrophoresis associated with mass spectrometry for identification of the proteome and secretome of HNPE cell conditioned SF-medium samples. 3.1. RGC-5 Cell Differentiation. The differentiation system consisted of RGC-5 cells on coverslips inside 6-well plates, which were exposed to the conditioned medium from HNPE cells. RGC-5 cells proliferated rapidly with a doubling time of less than a day. Decreasing the percentage of serum in the medium may slow down proliferation. The control RGC-5 cells were heterogeneous in shape. Morphological changes of RGC-5 cells were induced by HNPE cell conditioned SF-medium and caused the shrinkage of the cell body with elongated neurite outgrowth ), which allows comparison with undifferentiated RGC-5 cells ). The overall morphology of RGC-5 cells after the treatment was similar to those seen in primary cultures of rat retinal ganglion cells. Moreover, the morphology of RGC-5 cells differentiated by our method was similar to the ones induced by a broad-spectrum protein kinase Pyrroloquinolinequinone disodium salt inhibitor staurosporine. Nevertheless, Frassetto and coworkers did not conclude this to be the possible differentiation mechanism. May play a role in regulation of cytoskeletal rearrangements involved in cytokinesis and cell migration Prevents motor-neuron apoptosis induced by a variety of signals Regulate the disassembl