A Differential Proteomic Study On Secreted Proteins In Serum-free Medium Of Normal And Tumoral Bronchial Epithelial Cells From Lung Cancer Patients

The latest statistic data showed that the rates of incidence and death of lung cancer are all the highest among all malignant tumors. Much more concern is focused on probing the carcinogenesis mechanism of lung cancer at the molecular level and finding potential protein markers involved in the early phase of tumorigenesis. Many techniques are applied in this field, including the newly emerged proteome technique.Proteome means the total proteins expressed in a cell, tissue or organism. Proteome research can monitor the integrated change of the proteins happened in disease development, cell evolvement, and environment stress. In cancer research, differential proteome (or functional proteome) can be used in the finding of potential drug targets and markers for tumor classification, early stage diagnosis and intervention.In this study, normal and tumoral bronchial epithelial cells from lung cancerpatients were first cultured in serum-free medium. Then the secreted proteins in bothserum-free mediums were extracted and performed differential proteome analysis. Twostrategies from sample pretreatment, separation to identification were explored andemployed to screen potential protein markers. The results are as following:i. Two techniques were developed to separate and identify proteins in serum-freemedium. One was SDS-PAGE coupled with peptide mass fingerprinting (PMF)using matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS), the other was SDS-PAGE coupled with peptide sequencingusing nano-liquid chromatography electrospray ionization tandem massspectrometry (nano-LC-ESI-MS/MS);ii. Conditions for the removal of salts in serum-free medium were studied using dialysis tube and dialysis bag. Evaluation of dialysis effects using SDS-PAGE showed that the bag worked more effectively than tube. Both results from SDS-PAGE and 2-DE separation showed that dialysis of the serum-free mediumtook good effect.iii. An optimal in-gel digestion procedure for SDS-PAGE separated proteins was acquired via additional reduction and alkylation of the protein in gel slice.iv. An up-regulated protein, matrix metalloproteinases 1 (MMP-1) was identified with PMF and further verified by clinical immunohistochemistry assay.v. Matched normal and tumoral samples from same lung cancer patient were analyzed with nano-LC-ESI-MS/MS. 127 secreted proteins from serum-free medium of tumoral cell and 113 proteins from normal cells were identified respectively. 35 and 42 proteins were uniquely expressed in normal and tumor tissues respectively.vi. Functional analysis of the differentially secreted proteins in tumoral sample showed that many proteins are related with tumorigenesis, such as nm23-Hl, highly expressed in lung cancer and 14-3-3, a potential diagnosis marker.vii. Databases of secretory proteins were constructed, including 177 proteins expressed by cells from tumoral tissues and 150 from normal tissues.