Abstract
Most tissue biopsies from patients in hospital environments are formalin-fixed and paraffin-embedded (FFPE) for long-term storage. This fixation process produces a modification in the proteins called “crosslinks”, which improves protein stability necessary for their conservation. Currently, these samples are mainly used in clinical practice for performing immunohistochemical analysis, since these modifications do not suppose a drawback for this technique; however, crosslinks difficult the protein extraction process. Accordingly, these modifications make the development of a good protein extraction protocol necessary. Due to the specific characteristics of each tissue, the same extraction buffers or deparaffinization protocols are not equally effective in all cases. Therefore, it is necessary to obtain a specific protocol for each tissue. The present work aims to establish a deparaffinization and protein extraction protocol from FFPE kidney samples to obtain protein enough of high quality for the subsequent proteomic analysis. Different deparaffination, protocols and protein extraction buffers will be tested in FFPE kidney samples. The optimized conditions will be applied in the identification by LC-MS/MS analysis of proteins extracted from 5, 10, and 15 glomeruli obtained through the microdissection of FFPE renal samples.
Highlights
Chronic diseases such as diabetes, hypertension, obesity, or chronic kidney disease have been studied for many years through physiological, biochemical, and genetic approaches, contributing to the understanding of the pathophysiology of these diseases [1,2,3]
formalin-fixed and paraffin-embedded (FFPE) kidney tissue samples were deparaffinated with xylene at 65◦C or mineral oil at 95◦C, and the buffer selected for the extraction step was buffer 6 (RIPA LB)
Visible bands were observed in the 1D Sodium dodecyl sulfate (SDS)-PAGE gels with the three protocols; only well-defined bands were observed after using the protocol 3 with both deparaffinization solvents
Summary
Chronic diseases such as diabetes, hypertension, obesity, or chronic kidney disease have been studied for many years through physiological, biochemical, and genetic approaches, contributing to the understanding of the pathophysiology of these diseases [1,2,3]. [5,6,7,8,9] These samples represent an almost endless biorepository for DNA, RNA and protein analyses. One of the main problems is trying to extract proteins from FFPE specimens, due to the nature of the cross-links formed after the treatment with formalin [10,11,12] (see Figure 1). These molecular crosslinks difficult protein extraction and more aggressive extraction techniques than those used for fresh or frozen tissues are necessary [7, 11, 12]
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