Extraction Of Low Molecular Weight Proteins From Body Fluids By Disc PAGE

The interference from high abundance and high molecular weight (HWM) proteins are the main problems with which researchers have to be faced in the studies on low molecular weight (LMW) proteins/peptides in serum and pleural effusion. How to removing these proteins is also a hot topic. The successful removal of high abundance and HMW proteins are helpful for further identification of LMW proteins and low abundance proteins/peptides.In this thesis, a house-made disc tricine sodium dodecanesulphate polyacrylamide (SDS-PAGE) equipment was applied to extracting the LMW proteins/peptides from serum and pleural effusion, and the HMW and high abundance proteins (>30kDa)were successfully removed. Then, the extracted LMW proteins were digested by trypsin in solution, and identified by nano-HPLC/MS/MS. In addition, An rudiment exploration of non reduced disc tricine SDS-PAGE was discussed for further analysis of LMW proteins/peptides in serum.The traditional plate tricine SDS-PAGE method for LMW proteins/peptides was modified to tube SDS-PAGE, namely, disc SDS-PAGE by shorting the size of gel and reducing the amount of buffer solutions. The electrophoresis was performed at a constant voltage of 60 V. The SDS-protein complexes moved into cathode when the electrophoresis stared. The proteins whose molecular weights were more than 30kDa could not run into the resolving gel. Because only 1 ml of cathode buffer solution was used, the pH of the cathode buffer solution was lower than 3 after finishing the electrophoresis. The LMW proteins moved into the resolving gel were positively charged and retained in the gel. Finally, the LMW proteins were concentrated in about 2-3 mm long zone in resolving gel. we selected passive method to elute the proteins/peptides. Without staining, the gel containing LMW proteins/peptides was crushed and deionized water, instead of buffer solution, was used to elute the LMW proteins/peptides. In order to check whether the proteolysis of the proteins occurred during incubation, the LMW proteins in 4 aliquots of the gel were extracted under 4 conditions, ultrasonic treatment, freezing and thaw, at 4℃and at room temperature. There is not obvious difference in the proteins/peptides whose molecular weights are less than 14kDa obtained under the four incubation conditions, and the recoveries of the proteins whose molecular weight are higher than 14kDa obtained at room temperature overnight are higher than those obtained under the other three incubation conditions. The experimental results indicate that the proteolysis of the LMW proteins should not be severe under the incubation conditions. we performed a test that the serum was treated by 20%,50% and 70%(v/v) ACN and cold acetone respectively. The supernatant was analyzed by plate SDS-PAGE. The results showed that the proteins/peptides could be found in ACN solvent but can not in acetone solvent except for 50%acetone. Cytochrome C and reduced glutathione were used to examine the recoveries. The results showed that the recoveries of both cytochrome C and glutathione were more than 80%. The same seven serum samples were analyzed to examine the repeatability and the result was satisfactory. The data obtained by MALDI-TOF-MS and plate SDS-PAGE indicated that the LMW proteins/peptides located in from several hundreds to 30kDa could be extracted by this method and the HMW proteins (>30kDa) were removed successfully.The identification of LMW proteins/peptides was conducted by'shotgun'method. Before MS analysis, the mixture of LMW proteins must be degraded with trypsin. In order to degrade effectively, denaturation of proteins should be done thoroughly. The thermal denauration was applied. The HPLC analysis certified the feasibility. These peptide fragments obtained were then analyzed by nano-HPLC/MS/MS followed by a MASCOT search of protein database. Based on the scores and peptide sequencing coverage, the LMW proteins were identified. Compared with the method of 2-D electrophoresis-MS, the shotgun method need less time and get more information about proteins.Under denaturation condition, the disulphide bond of IgG was disrupted, the light chains whose molecular weight were less than 30kDa, of IgG could run into the resolving gel by the proposed method. So, we also made an exploration on non reduced disc tricine SDS-PAGE of serum. Plate SDS-PAGE and HPLC analysis showed that the interference from IgG light chains was reduced.