On tissue digestion

FIGURE 12.8 On-tissue digestion aids the detection of high-molecular-weight species. Distribution of ion detected at in/z 1496.75 corresponding to a tryptic peptide of the 71 kDa protein, synapsin I. (Reproduced from Groseclose, M.R. et al., J. Mass Spectrom., 42, 254, 2007. With permission.)... 

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... 

FIGURE 3.9 On-tissue digestion process was enhanced by the heat-denaturation process and the use of detergent-supplemented trypsin solution. A. Mass spectra obtained from 10-pm mouse cerebellum sections of trypsin-digested position (see inset). B-D. Mass spectrum obtained from the tissue sections prepared with indicated treatment of denaturation and detergent. Asterisks represent the mass peaks at m/z 1198.7. E. Signal-to-noise ratio of peak at m/z 1198.7 obtained from each tissue section. Bar, 1 pm [19]. 

Key words Matrix-assisted laser desorption/ionization, time-of-fight, mass spectrometry, mass spectrometry imaging, formalin fixed and paraffin embedded, frozen tissue, on tissue digestion, proteins identification. 

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. 

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