Analysis Of Differentially Expressed Proteome In Urinary EXOSOMES From Non-small Cell Lung Cancer Patients

Objective: Urine provides an alternative to blood plasma as a potential source of disease biomarkers. Urine is non-invasive source and could get a large of samples, so urinary proteomics gets more attendations.. The exosome, as the urine of a special body, which composition and function may be have great significance for diagnosis, prevention and treatment in diseases, This study aimed to screen out exosome-specific proteins for offering some help in treatment and early diagnosis of patients by comparing differentially expressed proteome between NSCLC patients and normal human subjects in urinary exosome.Methods: In this study, We separated exosomes by ultracentrifuge at 200000×g in normal person and non-small cell lung cancer (NSCLC) patients'urine. For proteomic analysis of urinary exosome, 1D SDS-PAGE was carried out , then the software of Quantity One was used for screening differentially proteins NSCLC patients and normal human subjects in urinary exosome.Get the differentially proteins band, Proteins in the gel blocks were sequentially reduced, alkylated with iodoacetamide, trypsinized, and got a mixture of peptides which being analyzed by HPLC-chip-MS/MS, mass spectrometry data which obtained into UniProtKB / SWISS -PORT database to identify the relevant proteins.Results: The result of 1D SDS-PAGE analysis showed that normal group and disease group differences mainly in the 90kDa, 66kDa and 31kDa ~ 20kDa bands in urinary exosome proteome. The 31kDa~20kDa band was cut from the gel band for mass spectrometry analysis.Twenty four kinds of proteins were searched and finally were identified with the MS/MS data into UniProtKB/WISS-PORT database, of which 8 proteins appeared justly in urinary exosome of patients with NSCLC, including three fragments of the immunoglobulinκ, two kinds of Ras related proteins, glutathione S-transferase A2 (GSTA2), Serum amyloid P-component precursor and Phosphatidylethanolamine-binding protein 1(PEBP1).Discussion: 1D SDS-PAGE analysis showed that normal group and disease group differences mainly in the 90kDa, 66kDa and 31kDa ~ 20kDa bands in urinary exosome proteome. The 90kDa band is Tamm-Horsfall protein (THP); and more than 60kDa proteins in the plasma can not pass glomerular filtration, so they are secreted by the kidney epithelial cells into the urine. Therefore, only proteins of 31kDa ~ 20kDa band were from plasma and this study focused on this band for proteomic analysis. Through UniProtKB/SWISS-PORT database,which found 8 differentially expressed proteins only in NSCLC patients urinary exosomes, including three fragments of immune-related immunoglobulinκthe; two kinds of Ras-related protein, Ras gene family mutations are the most common human tumor matter and a common pathway on tumorigenesis and tumor development; glutathione S-transferase A2, glutathione S-transferase phenotype changes are found in tumors; serum amyloid P component precursor, has used as diagnostic index of lung pathology in process of development in report; phosphatidylethanolamine-binding protein 1 (PEBP1), PEBP is the serine protease inhibitor, PEBP is the serine protease inhibitor, which found that PEBP was high expression in secreted proteins of non-small cell lung cancer cell line A549. It suggests that PEBP which may come from lung cancer tissues in urinary exosome. PEBP may provide an new candidate molecule for biomarkers of NSCLC.