Extraction of Proteins from Formalin‐Fixed, Paraffin‐Embedded Tissue Using the Qproteome Extraction Technique and Preparation of Tryptic Peptides for Liquid Chromatography/Mass Spectrometry Analysis

Géoui, Thibault; Urlaub, Henning; Plessmann, Uwe; Porschewski, Peter

doi:10.1002/0471142727.mb1027s90

Cited by 14 publications

(16 citation statements)

References 10 publications

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“…Therefore increasing numbers of studies have examined the potential of using more widely available FFPE tissue for proteomics studies. Encouragingly, it has been shown in multiple studies that similar proteomics data can be generated from FFPE and snap-frozen tissue 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 . Overall these studies found that the use of snap-frozen tissue resulted in slightly greater protein detection than FFPE, but that the majority of proteins detected in FFPE tissue were similarly detected in frozen tissue.…”

Section: Discussionmentioning

confidence: 99%

Proteomic analysis of neurons microdissected from formalin-fixed, paraffin-embedded Alzheimer’s disease brain tissue

Drummond

Nayak

Ueberheide

et al. 2015

Sci Rep

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The vast majority of human tissue specimens are formalin-fixed, paraffin embedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer’s disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer’s disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue.

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Section: Discussionmentioning

confidence: 99%

Proteomic analysis of neurons microdissected from formalin-fixed, paraffin-embedded Alzheimer’s disease brain tissue

Drummond

Nayak

Ueberheide

et al. 2015

Sci Rep

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“…Multiple comparative proteomic reports showed that the retrieved molecular information varied by extraction method, tissue type, and instrument, and made comparison of results between studies difficult (Hood et al, ; Geoui et al, ; Kojima et al, ; Mason, ). Because the lysine side chains are mostly involved in the reaction with formaldehyde, some studies reported the lysine (K) to arginine (R) terminal peptide ratio (K/R), as a way to evaluate and classify chemical variations found in FFPE tissues.…”

Section: Methods To Preserve and Store Tissuesmentioning

confidence: 99%

“…The impact of formaldehyde-induced protein modifications and the potential benefit of FFPE tissues as a surrogate for the FF tissues in the last decade has led to numerous studies that examined equivalence of FF-and FFPE-preserved tissues. Most of them used paired FFPE and FF tissues (Geoui et al, 2010;Bell et al, 2011;Gámez-Pozo et al, 2012;Kojima et al, 2012;Y Zhang, Muller, et al, 2015) to evaluate similarities in retrieved proteins; to a lesser extent, paired FFPE-and OCT-embedded FF tissues were also reported in proteomic studies (Scicchitano et al, 2009;Nirmalan et al, 2011;Holfeld et al, 2018). Measurement of the overlap of so-called diagnostically relevant proteins in paired FF and FFPE nonalcoholic steatohepatitis (NASH) 10 µm thick human liver tissues revealed that archived specimens could potentially be used for biomarker discovery of NASH (Bell et al, 2011).…”

Section: A Ffpe Tissues-formalin-fixed Paraffin-embeddedmentioning

confidence: 99%

Proteome analysis of tissues by mass spectrometry

Đapić

Baljeu‐Neuman

Uwugiaren

et al. 2019

Mass Spectrometry Reviews

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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, 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 mm2 obtained with laser capture microdissection to much larger amounts that weight several milligrams.

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“…In search for standardization commercially available kits are more and more used for archieval FFPE tissue [ 30 – 33 ], although considerable limitations concerning selective loss of extracted proteins have been reported [ 34 ]. The latter is particularly relevant for membrane proteins, which are difficult to extract and detect but are highly relevant in deciphering the pathogenesis of cancer and for the development of novel targeted therapies [ 35 ].…”

Section: Introductionmentioning

confidence: 99%

Systematic evaluation and optimization of protein extraction parameters in diagnostic FFPE specimens

et al. 2022

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Objectives Formalin-fixed paraffin-embedded (FFPE) tissue is the standard material for diagnostic pathology but poses relevant hurdles to accurate protein extraction due to cross-linking and chemical alterations. While numerous extraction protocols and chemicals have been described, systematic comparative analyses are limited. Various parameters were thus investigated in their qualitative and quantitative effects on protein extraction (PE) efficacy. Special emphasis was put on preservation of membrane proteins (MP) as key subgroup of functionally relevant proteins. Methods Using the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was measured by protein concentration and western blot analysis of cellular compartment markers as well as liquid chromatography-coupled mass spectrometry (LC–MS). Results Commercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval. Conclusions Preanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.

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Extraction of Proteins from Formalin‐Fixed, Paraffin‐Embedded Tissue Using the Qproteome Extraction Technique and Preparation of Tryptic Peptides for Liquid Chromatography/Mass Spectrometry Analysis

Cited by 14 publications

References 10 publications

Proteomic analysis of neurons microdissected from formalin-fixed, paraffin-embedded Alzheimer’s disease brain tissue

Proteomic analysis of neurons microdissected from formalin-fixed, paraffin-embedded Alzheimer’s disease brain tissue

Proteome analysis of tissues by mass spectrometry

Systematic evaluation and optimization of protein extraction parameters in diagnostic FFPE specimens

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