Protein extraction studies Liquid Tissue™ method

In one study by Hood et al., 282 of 1153 identified proteins were identified by at least 2 unique tryptic peptides from FFPE prostate cancer (PCa) tissue.9 According to the gene ontology classification of the proteins identified, -65% of proteins were predicted to be intracellular proteins, while -50% of the total human proteome is predicted to be located in the intracellular compartment. Additionally, 20% of the proteins identified in the PCa tissue were classified as membrane proteins, which is significantly less than the predicted 40% for the human proteome. This relative disparity is not unexpected, considering the Liquid Tissue sample preparation kit lacks specific protocols for membrane protein extraction. The Liquid Tissue method has also been used for proteomics studies of a variety of FFPE tissue samples, including pancreatic tumors,28 squamous cell carcinoma,4 and oral human papillomavirus lesions.27... 

A number of proteomic studies on archival material have utilized Liquid Tissue (Expression Pathology, Inc., Gaithersburg, MD), a commercial protein extraction kit for FFPE tissue.4,9,25-28 This kit is also based upon HIAR techniques and shares a similar work flow to the methods already discussed. Thin, typically 5-10pM, sections are cut from paraffin tissue blocks, the paraffin is removed, and the tissue deparaffinized and rehydrated in alcohols and distilled water before microdissection. The cellular material is then suspended in Liquid Tissue buffer and heated at 95°C for 90 min. Trypsin is added, and the material is digested overnight at 37°C prior to reduction with DTT and analysis by LC-MS/MS.26... 

It is unlikely that the unchanged nerve agent would be detected in the blood or tissues of a casualty unless samples were collected very soon after the exposure. A number of methods have been reported for the analysis of nerve agents in blood, for application to animal studies. These involve simple liquid or SPE extraction, for example, using chloroform (sarin, soman) (47), C18 SPE (sarin, soman) l48 49 , ethyl acetate (VX) (50), usually after precipitation of proteins, and analysis by GC/MS or gas chromatography/nitrogen-phosphorus detection (GC/NPD). Sarin bound to cholinesterase and displaced with fluoride ion was extracted by C18 SPE (see Part B) (51). 

---