Quantitative proteomic analysis found selleck chem Trichostatin A no significant difference in ApoE levels between control and clinically diagnosed AD platelet membrane fractions, as described below. Platelet isolation and membrane protein enrichment strategy Platelets from whole blood were isolated through centri fugation as previously published using a citrate buf fer, which significantly Inhibitors,Modulators,Libraries minimizes in vitro platelet activation. To assess the purity of the isolated platelets, flow cytometry was performed after double labeling with antibodies against the platelet specific mar ker, CD41, and a marker for white blood cells, CD45. Results demonstrate that the platelet enriched samples contained greater than 90% CD41 positive cells, whereas CD45 positive cells made up 1. 3% of the cells.

Platelets contain an extensive intracellular membrane, an open canalicular system that serves as a reservoir for plasma membrane proteins and membrane receptors and provides a passage for granule release after activation. There are also numerous membrane bound granules in platelets, the contents of which could be more easily identified in Inhibitors,Modulators,Libraries a membrane enriched sample. Differential centrifugation fractions obtained during enrichment of the membrane proteome prior to LC MS MS analysis were first visualized by silver stain of a representative sample indi cating altered protein complexity in the whole, soluble, wash and membrane fraction. Immunoblotting with Inhibitors,Modulators,Libraries antibodies against CD41, a platelet specific transmembrane protein, demonstrated an average of 2. 2 fold enrichment in the membrane fraction.

Conversely, immuno blotting Inhibitors,Modulators,Libraries with antibodies against actin, a cytoskeleton protein, demonstrated Inhibitors,Modulators,Libraries approximately 20 fold depletion in the membrane fraction compared to whole platelet lysate. To assess the global enrichment of proteins in the mem brane fraction, LC MS MS analysis was performed from equal amounts of whole platelet lysate and mem brane rich fraction. TMHMM 2. 0 was used to predict the number of transmembrane domains for each protein. In the membrane enriched fraction, 40% of proteins were predicted to have a TMD. This was a 2. 5 fold increase from an analy sis of the whole platelet proteome performed in parallel in which 17% of proteins identified contained a predicted TMD. The results are consistent with a similar previously published enrichment strategy for membrane proteins from plate lets.

The most significantly enriched and depleted proteins in the membrane fraction were determined by relative quantification using spectral counting. These included CD41 and beta actin, which were significantly enriched and depleted, respectively, in the membrane normally fraction consistent with immunoblot analysis. Together, these results indicate that the differential centrifugation approach was effective at both enriching membrane proteins and depleting solu ble cytoplasmic proteins.