Proteome/Proteomic analysis tissue samples

One of the most crucial considerations in proteomic analysis is sample preparation because this will ultimately dictate the number and type of proteins that can be processed. The first priority is to establish the precise protein system to be studied [e.g., will this be a comprehensive and exhaustive catalogue of every expressed protein within a tissue or cellular extract, or is only a small subset of a cellular proteome (e.g., only phosphoproteins or membrane-bound proteins) sufficient for analysis ]. Whether a full or partial proteome, or even a limited number of specific proteins is required for analysis, it is crucial that the extraction technique provide maximal protein recovery while preserving the integrity of the protein complex to be examined. Furthermore, the method of preparation must be totally compatible with the separation methods to be used. This is particularly important for separation technologies that are reliant on protein-protein interactions or drug/ligand/antibody, etc. interactions. Poor recovery of proteins is clearly... 

Stewart NA, Veenstra TD. Sample preparation for mass spectrometry analysis of formalin-fixed paraffin-embedded tissue proteomic analysis of formalin-fixed tissue. Methods Mol. Biol. 2008 425 131-138. 

Several proteomic studies using archival FFPE tissues have been reported in recent years. Some involve the analysis of very small tissue samples prepared... 

A study of by Palmer-Toy et al.,12 summarized in Table 19.1, provides further empirical evidence of the utility of techniques coupling heating with efficient protein extraction for the proteomic analysis of FFPE tissue. A specimen from a patient with chronic stenosing external otitis was divided in half and preserved as fresh-frozen tissue or FFPE. Ten micromolar sections of the FFPE tissue were vortexed in heptane to deparaffinize the tissue and were then co-extracted with methanol. The methanol layer was evaporated, and the protein residue was resuspended in 2% SDS/lOOmM ammonium bicarbon-ate/20mM dithiothreitol (DTT), pH 8.5 and heated at 70°C for lh. After tryptic digestion, 123 total confident proteins were identified in the FFPE tissue, compared to 94 proteins identified from the fresh-frozen tissue. Hwang et al. also reported up to a fivefold increase in protein extraction efficiency for samples extracted in a Tris-HCl/2% SDS/1% Triton X-100/1% deoxycholate solution at 94°C for 30 min versus samples extracted in 100 mM ammonium bicarbonate/30% acetonitrile at the same temperature.14... 

This is an important observation given that plasma membrane proteins are often used as markers of disease. This experiment demonstrated that shotgun proteomic analysis could be successfully performed on microdis-sected, formalin-fixed tissues using the antigen retrieval method with a sensitivity equal to that of analysis of the soluble fraction of a fresh-frozen sample. 

Archival tissues stored as FFPE tissue blocks represent the vast majority of tissue archives. To investigate the effect of storage on shotgun proteomic analysis of FFPE tissues, we analyzed 10 samples dating back to 1980.22 Nine leiomyomas (three from 1990, three from 1997, and three from 2002) and one... 

Traditional endoscopic and surgical procedures provide whole tumor samples well suited for microscopic examination and analysis in the pathology laboratory. The use of whole tissue tumor biopsies for proteomic studies has, however, raised several important issues that have been well demonstrated in CRC [9]. These include cellular heterogeneity in the different bowel parietal layers (mucosa, submucosa, muscularis mucosa, serosa) that may or may not be infiltrated, epithelial cell diversity in the mucosa itself, tissue infiltration by inflammatory cells such as lymphocytes, contamination with other body fluids, and protein degradation following tumor necrosis. In fact, epithelial cell content was found to vary between 9 and 67% in whole biopsies of normal mucosa and between 7 and 95% in tumor biopsies [10]. This study clearly demonstrates the likelihood of large cellular variation between tissue samples. 

The many attributes of proteins mentioned in Table 4.2 make complete global analysis of proteins from cells, tissues, organs, or organisms a formidable analytical challenge. A primary aim of proteomic analysis is to provide researchers with as much data as possible about one or more protein attributes. Flowever, to many researchers, proteomic analysis usually involves the three attributes of identification and quantitation of all proteins in a defined sample space. Only the highlights of each platform will be summarized here. A critical appraisal of capabilities, advantages, and drawbacks are summarized in Figure 4.13 (Section 4.5.6). 

Saravan, R.S. and Rose, J.K.C. (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues, Proteomics, 4, 2522-2532. 

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