An extracellular phosphoglycan (exPG), present in the culturem edium of the promastigote form L oefi shmania donovani, was purified and structurally characterized. The purification scheme included ethanol precipitation of the culture medium, anion exchange chromatography, hydrophobic chromatography on phenyl-Sepharose, and preparative polyacrylamgeild e electrophoresis. Structural analysis by ‘H-’H NMR, methylation linkage analysis, and glycosidase digestion revealed that the exPG consisted of thfoel lowing structure: (CAP)+[P04-6Galp@1-4Manpal]lo-11-POr6GalpB1-4Man. The capw as found to be ones eovf eral small, neutral oligosaccharides, the most abundant of which was the trisaccharide Galp@l-4(Manpal-2)Man. The results indicated structural analogy to the cellular-derived lipophosphoglycan (LPG) from L. donovani. The important exceptions are a lacko f the lipid anchor, the entire phosphosaccharide core, and several of the repeating disaccharide units. Although the function of exPGis presently unknowni,t may play a protective role for the promastigote in the insect vector or during infection of a mammalian host
Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes.