Establishing A Method For Separation Of Proteins In Complex Biological System

Mass spectrometry (MS)-based proteomics has become a hot spot in the life science research. MS technology provides a rapidity and sensitivity to detect and identify the proteins in complex mixture of proteins. But the false positive rate of MS-based protein identification is high, which partly arise from the sample complexity before MS identification. Most commonly used method called "shotgun" is that the analytes before being introduced into the mass spectrometer are peptides generated by enzymatic cleavage of a mixture of proteins, which commonly results in consequence of the limited sequence coverage and high false positive rates. Based upon the above-mentioned fact, our aim is to establish a simple and effective method for separating a protein mixture from complex biological system, which can satisfy the need ofproteomics development.Our study mainly focuses on establishing a simple and efficient separation method on the molecular level of proteins. The investigations are as follows: 1)To determine a kind of new ion exchange packing materials superior over different kinds of chromatographic packing materials, such as C4, R2, strong cation exchange in the process of separating complex proteins in serum; 2) To optimize and get the best separative conditions by comparative experiments adopting different flow rates, the loaded quantity and the dimension of capillary; 3) To optimize the best gradient elution pattern for the separation of serum; 4) To evaluate the reproductivity of the separation method; 5) According to chromatographic peak and time, two fractions were selected randomly, followed by LC-MS/MS analysis, and gap junction beta 1 protein,ras associated protein32,ras associated protein10 and conserved hypothetical protein were identified with confidence of greater than 90%. Notablely, four proteins identified herein were all low-abundance proteins; 6) This separation method established herein highlights its availability by comparing the analytical results between the preoperative serum and the post-op serum from the same pancreatic cancer patient. A simple and effective method for separating proteins from complex biological system was established. It's not only to improve the confidence of the protein identification, but give a simple and potential pathway to find the enormous information about the pathologic mechanism, to screen early detection bomarkers and to evaluate prognosis assessment of disease.