The Extraction And Application Of Endogenous Serum Phosphopeptides From Lung Cancer Patients

Background:Cancer is a proteomics disease that a group of proteins change in the signal pathway and eventually abnormal occurs. It is great value of finding cancer biomarkers for early clinical diagnosis, pathological type, drug targets, drug intervention and treatment, therefore, it is a main direction of proteomics. Since half a century, lung cancer has become one of the first causes of death due to its fast increased morbidity and mortality. Currently iconography and sputum cytology are primary tools for screening early lung cancer, but there are certain degree of misdiagnosis and missed diagnosis existence. Pathological examination is confirmed as the clinical gold standard, but it has the disadvantage of patient's trauma. Therefore, most of patients with lung cancer can not get early detection, early treatment, and the quality of patients life was impaired. With the development of molecular biology, tumor markers detection has become a common mean of diagnosis of malignant tumors, and it has important clinical value for cancer screening. So, how to develop lung cancer tumor markers has got increasing interest in proteomics.Protein phosphorylation as one of the most extensive post-translational modification (Post Translational Modifications, PTMs), participated in and regulated the activities of almost all life processes, including cell proliferation, differentiation, muscle contraction, neural activity, metabolism and so on. Abnormal phosphorylation is one of the causes of human diseases, especially for cancer disease. Mass spectrometry has advantages of high sensitivity and rapid detection ability and often used as a tool for phosphorylated proteins analysis. According to the changes of phosphorylation in MS, it can predict, determine and diagnose disease. It is helpful for disease diagnosis accuracy, which is important to biological significance.Objective:In this experiment, we developed an endogenous serum phosphopeptides detection approach. Serum was precipitated with acetone, filtrated with ultrafiltration membrane, and then enriched by metal oxide (TiO2). The enriched phosphopeptides were detected by UPLC/Q-TOF. By examining the differences of endogenous serum phosphopeptides between lung cancer patients and normal human, we found the method was meaningful. The result was proved that the method provided a theoretical basis for the identification of tumor markers in lung cancer.Methods:We established a method which was standard sample filtrated with ultrafiltration membrane, enriched by metal oxide and detected with high performance liquid chromatography. Then 10 cases of lung cancer serums and 10 cases of normal serum were packed, precipitated with acetone and tested by the established method.Results:1. A new method of proteomics had been established after a comparative evaluation, that was standard samples filtrated with 10K ultrafiltration membrane, dissolved with 50%CH3OH/0.5FA and detected with 0.5μLα-casein adding 5μL serum as an experimental detection limit.2. Serum was detected with the established method: 14 endogenous phosphopeptides were detected from lung cancer patients and 10 endogenous phosphopeptides were detected from nomal human.Conclusion:It successfully established a proteomics method of endogenous serum phosphopeptides detection and identification with lung cancer serum and normal serum: 1. Established a theory method for processing and detection of complex proteomics samples (eg serum, urine, etc.).2.14 endogenous serum phosphopeptides from lung cancer and 10 endogenous serum phosphopeptides from normal human were successfully detected.3. The differences of endogenous serum phosphopeptides between lung cancer and normal human were verified, and it was contributed to identify lung tumor markers finally.