Protein Extraction From FFPE Kidney Tissue Samples: A Review of the Literature and Characterization of Techniques

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.

Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC–MS

“…As shown in Figure a, compared to 0.5% SDS, the presence of 3% SDS in the cocktail resulted in >45% increase in the recovery of signature peptides of brentuximab and CD30, a 44% increase in the median recovery of the 40 tissue markers, as well as a 24% increase in the total protein recovery (all p < 0.05, t -test). Nonetheless, higher concentrations of SDS appeared to be counterproductive, leading to the significantly lower recovery of target signature peptides but not total protein content, which may arise from lower efficiency in delinking reaction caused by a high concentration of the anionic detergent. , Therefore, SDS concentration at 3% was determined to be optimal.…”

“…Nonetheless, higher concentrations of SDS appeared to be counterproductive, leading to the significantly lower recovery of target signature peptides but not total protein content, which may arise from lower efficiency in delinking reaction caused by a high concentration of the anionic detergent. 28,29 Therefore, SDS concentration at 3% was determined to be optimal. Previously, we found that salt in SC buffer enhanced protein extraction efficiency from fresh tissues.…”

Disease-Specific α-Synuclein Seeding and Structural Properties in Lewy Body Disease and Multiple System Atrophy are Preserved in Formaldehyde-Fixed Paraffin-Embedded Human Brain

“…Extraction of sufficient amount of high-quality proteins from FFPE tissue has consistently been reported to be challenging, particularly in seeding studies where the detection of misfolded αSyn was relatively low (75-76% sensitivity) [28,29]. Many studies have investigated different deparaffinization methods and extraction buffers, including commercially available FFPE protein extraction kits, to achieve a higher protein yield from FFPE tissue [46][47][48][49]. Although commercially available protein extraction kits can be effective for proteomic analysis, the extraction leaves the proteins suspended in extraction buffers that are composed of proprietary reagents, which can alter the performance of SAAs.…”

Mass Spectrometric-Based Proteomics for Biomarker Discovery in Osteosarcoma: Current Status and Future Direction

“…In recent years, proteomics technology has advanced dramatically. It is now capable of highly comprehensive and quantitative peptide profiling of archived biological materials, including Formalin-Fixed Paraffin-Embedded (FFPE) cancer tissues [ 56 , 57 ].…”