The application of proteomics to fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) human tissues is an important development spurred on by requests from stakeholder groups in clinical... Show moreThe application of proteomics to fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) human tissues is an important development spurred on by requests from stakeholder groups in clinical fields. One objective is to complement current diagnostic methods with new specific molecular information. An important goal is to achieve adequate and consistent protein recovery across and within large-scale studies. Here, we describe development of several protocols incorporating mass spectrometry compatible detergents, including Rapigest, PPS, and ProteaseMax. Methods were applied on 4 and 15 mu m thick FF tissues, and 4 mu m thick FFPE tissues. We evaluated sensitivity and repeatability of the methods and found that the protocol containing Rapigest enabled detection of 630 proteins from FF tissue of 1 mm(2) and 15 mu m thick, whereas 498 and 297 proteins were detected with the protocols containing ProteaseMax and PPS, respectively. Surprisingly, PPS-containing buffer showed good extraction of the proteins from 4 mu m thick FFPE tissue with the average of 270 protein identifications (1 mm(2)), similar to the results on 4 mu m thick FF. Moreover, we found that temperature increases during incubation with urea on 4 mu m thick FF tissue revealed a decrease in the number of identified proteins and increase in the number of the carbamylated peptides. Show less
Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues,... Show moreTissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh-frozen and formalin-fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue-sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom-up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys-C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue-specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm(2) obtained with laser capture microdissection to much larger amounts that weight several milligrams. Show less
Pathology laboratories throughout the world have compiled large archives of unique collections of tissue specimens. These tissue samples are used for patient diagnostics and research. Novel... Show morePathology laboratories throughout the world have compiled large archives of unique collections of tissue specimens. These tissue samples are used for patient diagnostics and research. Novel molecular insights into alterations in normal cellular function have led to the identification of targets for innovative therapies. Testing for biomarkers combined with molecular pathology has created the potential for __personalized medicine__ and improved diagnosis, treatment and prognosis. New technologies for molecular analysis in molecular tumor diagnostics and research must be developed and implemented to keep pace with the latest insights, resulting in a constant cycle of change. Such translational research can only progress if patient material can be accessed from the archives for further study. The resulting new insights and strategies will eventually be implemented in patient care. This thesis describes three important issues in this cycle of change with a focus on molecular pathology. Tissue preparation, method development and data analysis. Show less
Total mesorectal excision (TME) is the standard treatment for rectal cancer, while transanal endoscopic microsurgery (TEM) is a recently introduced surgical approach for the treatment of rectal... Show moreTotal mesorectal excision (TME) is the standard treatment for rectal cancer, while transanal endoscopic microsurgery (TEM) is a recently introduced surgical approach for the treatment of rectal adenomas. Incorrect preoperative staging before TEM is a problem. Therefore the aim of this thesis was to identify molecular differences between rectal tumors of different stages, using gene expression profiling and genomic analysis. First protocols and data analysis algorithms for a new type of SNP array were developed. These studies showed that reliable LOH and copy number changes could be obtained from both frozen and paraffin embedded material. Consequently SNP arrays were used to type groups of TEM and TME treated samples. Five genomic events were found which could make a clear discrimination between adenomas and carcinomas. Early carcinomas treated by TEM, which were not recognized preoperatively as carcinomas, showed already carcinoma-associated aberrations. Analysis of tree core biopsies per patient showed a large degree of intra- tumor heterogeneity; although a good correlation was obtained between the biopsy with the largest number of aberrations and its corresponding tumor fraction. Gene expression array analysis was performed on the same samples as the SNP array series. A high concordance between chromosomal aberrations and changes in gene expression was observed. Finally a clinical application of these data is discussed in the preoperative staging of rectal tumours. Show less
