On-tissue digestion method

On-Tissue Digestion Method Traditional MALDI is used to generate intact molecular ions of proteins up to m/z 100,000. In the current IMS technology, however, the upper mass limit of protein detection is approximately 40 kDa because the detection sensitivity severely falls at higher mass [15]. This is a considerable limitation which narrows the application capability of this technology. As another important problem, insoluble proteins to the matrix solution, such as membrane proteins, which is a protein molecule that is attached to the membranes, are difficult to be extracted into the applied matrix solutions and thus hardly crystallize with matrix, and they were in turn difficult to detect. On this... 

In this chapter, we describe methods and protocols for direct analysis of frozen and FFPE tissue samples, including on tissue digestion for both frozen and FFPE samples for protein identification. 

A rapid and simple MW-assisted digestion method with alkaline solution (TMAH or methanolic KOH solution) was developed for speciation analysis of inorganic Hg and methyl-Hg in biological tissues [41]. Extracts with quantitative recoveries of Hg species after the alkaline dissolution of the sample were directly analyzed by an automated on-line hyphenated system incorporating aqueous HG, cryogenic trapping, GC, and detection by A AS. The proposed method was validated by the analysis of biological CRMs (CRM 463, DORM-1, TORT-1) and one BCR sample from an interlaboratory study (Tuna Fish 2). 

Results obtained using these digestion methods are reported on a wet basis. To report a dry-weight basis, a subset of the tissue or fish to be analyzed can be oven-dried and a wet-to-dry weight ratio calculated. The ratio can be used to report metal concentrations in terms of dry weight. 

The method outlined here uses a modification of the Hedley technique (9,10) to prepare nuclear suspensions from the paraffin-embedded tissue samples. Microtome sections are dewaxed, hydrated, and incubated in pepsin with intermittent vortexing and mechanical disruption to release the nuclei. After completion of the tissue digestion, the nuclei are either suspended in 70% ethanol for storage or stained with propidium iodide (PI) for FCM analysis. There are three alternative techniques for preparation of the nuclei on microslides, in tea bags, or in test tubes. 

The mode of sample pretreatment depends on the analytical method used. If a mineralization of the specimen is required, the high volatility of mercury must always be kept in mind. If necessary, acid digestion at elevated temperatures should only be performed in sealed vessels (e.g., Teflon-or quartz-lined pressure bombs) and never in open vials. A digestion of blood or tissues with hydrochloric acid at room temperature for 15 hr extracts all mercury species sufficiently from the matrix. The supernatant is, for example, suitable for the flow injection technique of cold vapor atomic absorption spectrometry (CV-AAS) [111]. Furthermore, this cold digestion does not destroy organomercury compounds. Therefore the supernatant is suitable for a speciation [110]. 

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... 

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