Proteomics Study Of Buffalo Follicular Fluids

This study was undertaken to evaluate protein expression differences between mature and immature follicular fluids of swamp buffalo in Guangxi. The research used two-dimensional gel electrophoresis (2DE) coupled to Matrix Assisted Laser Desorption/Ionization Time-of-flight Tandem Mass Spectrometry (MALDI-TOF/TOF) to analyze the different proteins. The thesis consists of three chapters:the first describes the establishment and optimization of the2DE system for buffalo follicular fluid proteomics, the second chapter describes the isolation and identification of the different proteins between mature and immature buffalo follicular fluids, and the third chapter describes the isolation and identification of these proteins purified using a ProteoExtractTM Albumin/IgG Removal Kit.The establishment of a suitable2DE system for separating buffalo follicular fluid proteins is described in Chapter1. This involved comparing and optimizing various parameters, such as methods for purifing buffalo follicular fluid proteins, IPG strips, and loading volume. The results indicated that acetone precipitation in a loading volume of350μg in24cm IPG strips (pH4-7) improved resolution and the quality of the2DE maps, and about280protein spots were detected using Image Master2D platinum software.Chapter two describes the separation of total proteins from mature and immature buffalo follicular fluid using the established and optimized2DE system. Image Master2D platinum software was used to compare and analyze the two2DE maps, and25different protein spots were obtained with a more than three-fold range of optical density values. Compared with the mature follicular fluid2DE map,13proteins were up-regulated in immature buffalo follicular fluid,6proteins were down-regulated,2proteins were deleted and4 new proteins were expressed in immature follicular fluid. Finally,10proteins were successfully identified by mass spectrometry; these are the zinc finger protein674, peroxiredoxin-2, lysosomal-trafficking regulator, KIFC1protein, aldose reductase, fibrinogen gamma-B chain precursor, transthyretin precursor, nucleoside diphosphate kinase, inhibin alpha chain precursor, and alpha-aminoadipic semialdehyde dehydrogenase.Chapter three first describes the purification of mature and immature buffalo follicular fluid samples using a ProteoExtractTM Albumin/IgG Removal Kit, It then describes the isolation and identification of proteins from these purified mature and immature follicular fluids using the established and optimized2DE system.21different expression protein spots were found with Image Master2D platinum software,10of these proteins were up-regulated in immature buffalo follicular fluid,7were down-regulated,3deleted and1protein specifically expressed. Finally,4proteins were successfully identified by mass spectrometry; they are sulfated glycoprotein-2, fibrinogen fragment D, transferrin and HP protein.In summary, an appropriate2DE system for separating buffalo follicular fluid proteins was established and optimized. This provided a good technological base for the subsequent research on buffalo follicular fluid proteomics. Different expression proteins from mature and immature buffalo follicular fluids were successfully analyzed and identified to provide new research insight into the developmental microenviroment and maturation mechanism for buffalo oocytes.