Dataset Information

Protein extraction method improvement for FFPE kidney tissue samples

ABSTRACT: The vast majority of tissue biopsies from patients in hospital environments are included in paraffin (FFPE- formalin-fixed and paraffin-embedded) 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 techniques, since these modifications do not suppose a drawback for this technique; however, crosslinks makes the protein extraction process a challenging one. 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. This work aims to establish a deparaffinization and protein extraction protocol from paraffin-embedded kidney samples, which finally allows us to obtain high quantity and quality protein, suitable for the application of proteomic techniques. To achieve this; we have designed different protocols using a range of buffers with diverse compositions and characteristics. Our results showed that xylene can eliminate the formalin fixation from the kidney tissue. Moreover, strong detergents (SDS-sodium dodecil sulfate- or analogues) and reducing agents (e.g. DTT- Dithiothreitol-) in the extraction buffer appear to be more important to obtain a good protein extraction yields and a good number of proteins identifications by LC-MSMS.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

SUBMITTER: Susana Bravo

LAB HEAD: Susana Belen Bravo Lopez

PROVIDER: PXD023891 | Pride | 2021-09-10

REPOSITORIES: Pride

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Publications

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

García-Vence Maria M Chantada-Vazquez Maria Del Pilar MDP Sosa-Fajardo Ana A Agra Rebeca R Barcia de la Iglesia Ana A Otero-Glez Alfonso A García-González Miguel M Cameselle-Teijeiro José M JM Nuñez Cristina C Bravo Juan J JJ Bravo Susana B SB

Frontiers in medicine 20210513

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 protei ...[more]

PMID: 34055835

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Project description:Formaldehyde fixation is widely used for long-term maintenance of tissue. However, due to formaldehyde-induced cross-links, fixated tissue proteins are difficult to extract hampering the performance of mass spectrometry (MS)-proteomic analysis on formaldehyde-fixated tissue. Recent years saw the use of different combinations of high-temperature and solubilizing agents (usually derived from antigen retrieval techniques) to unravel formaldehyde-fixated paraffin-embedded tissue proteomes. However, in order to achieve protein extraction yields similar to fresh-frozen tissue high-temperature heating is necessary. Such harsh extraction conditions may affect, labile post-translational modifications such as phosphorylations resulting in the loss of important protein information. The objective of the present work is to assess cleavable fixative reagents that allow tissue preservation as well as efficient protein extraction from fixated tissue for MS-proteomics under mild conditions. To this regard, we investigated disuccinimidyl tartrate (DST) and dithiobis[succinimidylpropionate] (DSP) as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups leading to amide bond formation. Linkers can be cleaved with sodium metaperiodate (cis-diols) or reducing agents (disulfide bonds), respectively. Our results show that reversible protein crosslinking allows tissue fixation with morphology preservation comparable to formalin. In addition, cleavage of DSP improves protein recovery from fixated tissue by a factor of 18 and increases the number of identified proteins by approximately 20% under mild extraction conditions avoiding the need for sample boiling, which could affect labile post-translational modifications. A major advantage of DSP over formaldehyde is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by periodic cleavage, while effective, resulted in side reactions that prevented effective protein extraction and subsequent protein identification. Protein crosslinkers, which can be cleaved under mild conditions, are thus viable alternatives to formaldehyde as tissue fixatives facilitating protein analysis from paraffin embedded, fixated tissue.

Dataset Information

Protein extraction method improvement for FFPE kidney tissue samples

Publications

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

Similar Datasets

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