Comparison of two FFPE preparation methods using label-free shotgun proteomics: Application to tissues of diverticulitis patients

Quesada-Calvo, Florence; Bertrand, Virginie; Longuespée, Rémi; Delga, Agnès; Mazzucchelli, Gabriel; Smargiasso, Nicolas; Baiwir, Dominique; Delvenne, Philippe; Malaise, Michel; Pauw‐Gillet, Marie‐Claire De; Pauw, Edwin De; Louis, Édouard; Meuwis, Marie‐Alice

doi:10.1016/j.jprot.2014.08.013

Cited by 18 publications

(13 citation statements)

References 33 publications

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“…We previously demonstrated the ability to obtain an efficient and repeatable protein extraction using limited quantities of FFPE material before data-independent label-free analysis [24]. We performed macrodissection to favor the selection of cells of interest, removing abundant stromal tissue surrounding the tumor and decreasing the impact of response factors originated from neighboring tissues.…”

Section: Discussionmentioning

confidence: 99%

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OLFM4, KNG1 and Sec24C identified by proteomics and immunohistochemistry as potential markers of early colorectal cancer stages

Quesada-Calvo¹,

Massot²,

Bertrand

et al. 2017

Clin Proteom

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BackgroundDespite recent advances in colorectal cancer (CRC) diagnosis and population screening programs, the identification of patients with preneoplastic lesions or with early CRC stages remains challenging and is important for reducing CRC incidence and increasing patient’s survival. MethodsWe analysed 76 colorectal tissue samples originated from early CRC stages, normal or inflamed mucosa by label-free proteomics. The characterisation of three selected biomarker candidates was performed by immunohistochemistry on an independent set of precancerous and cancerous lesions harbouring increasing CRC stages.ResultsOut of 5258 proteins identified, we obtained 561 proteins with a significant differential distribution among groups of patients and controls. KNG1, OLFM4 and Sec24C distributions were validated in tissues and showed different expression levels especially in the two early CRC stages compared to normal and preneoplastic tissues.ConclusionWe highlighted three proteins that require further investigations to better characterise their role in early CRC carcinogenesis and their potential as early CRC markers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-017-9143-3) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

“…FFPE sections for a total of 300 mm 2 of macrodissected tissue per patient were treated as previously described [11]. Deparaffinization was performed by incubating the samples twice in xylene and followed by ethanol (100%) incubation (5 min each).…”

Section: Methodsmentioning

confidence: 99%

OLFM4, KNG1 and Sec24C identified by proteomics and immunohistochemistry as potential markers of early colorectal cancer stages

Quesada-Calvo¹,

Massot²,

Bertrand

et al. 2017

Clin Proteom

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“…Through multiple techniques they identified 193 proteins (by two or more peptides). Quesada-Calvo et al obtained 101.5 g/mm 3 from human colorectal cancer using FFPE-FASP and 84.2 g/mm 3 using On Slice AR [21]. Three FFPE-FASP replicates identified 884 proteins, and three On Slice AR replicates identified 622 proteins.…”

Section: Discussionmentioning

confidence: 99%

Two methods for proteomic analysis of formalin-fixed, paraffin embedded tissue result in differential protein identification, data quality, and cost

2015

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Formalin-fixed paraffin-embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC-MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in-solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre-analytical variations and analyzed with three technical replicates by LC-MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre-analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained.

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“…Methods have been reported for protein extraction and mass spectrometry from formalin-fixed and paraffinembedded (FFPE) tissue; however, a limited number of reports exist on mass spectrometry proteomic analysis of OCT embedded tissue samples (Azimzadeh et al, 2010;Tian et al, 2011;Quesada-Calvo et al, 2015;Zhang et al, 2015). These published reports are mainly discovery proteomics data, which focuses on qualitative appearance of peptide signals rather than absolute peptide quantitation.…”

Section: Discussionmentioning

confidence: 99%

An Optimized Method for Protein Extraction from OCT-Embedded Human Kidney Tissue for Protein Quantification by LC-MS/MS Proteomics

Vrana

Goodling

Afkarian

et al. 2016

Drug Metabolism and Disposition

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The existing biobanks of remnant tissue from clinically indicated kidney biopsies are attractive potential reservoirs for quantification of clinically relevant human tissue proteins by quantitative proteomics. However, a significant caveat of this strategy is that the tissues are often preserved in optimal cutting temperature (OCT) medium. Although OCT is an effective method of preserving the morphologic and immunohistological characteristics of tissues for later study, it significantly impacts efforts to quantify protein expression by liquid chromatography-tandem mass spectrometry methods. We report here a simple, reproducible, and cost-effective procedure to extract proteins from OCT-embedded tissue samples. Briefly, the excess frozen OCT medium was scraped before thawing from the tissue specimens stored at -80°C for ∼3 months. The tissue samples were homogenized and diethyl ether/methanol extraction was performed to remove the remaining OCT medium. The recovered protein was denatured, reduced, and alkylated. The second step of protein extraction and desalting was performed by chloroform/methanol/ water extraction of denatured proteins. The resultant protein pellet was trypsin-digested and the marker proteins of various kidney cellular compartments were quantified by targeted selective reaction monitoring proteomics. Upon comparison of peptide signals from OCT-embedded tissue and flash-frozen tissue from the same donors, both individual protein quantities, and their interindividual variabilities, were similar. Therefore, the approach reported here can be applied to clinical reservoirs of OCT-preserved kidney tissue to be used for quantitative proteomics studies of clinically relevant proteins expressed in different parts of the kidney (including drug transporters and metabolizing enzymes).

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Comparison of two FFPE preparation methods using label-free shotgun proteomics: Application to tissues of diverticulitis patients

Cited by 18 publications

References 33 publications

OLFM4, KNG1 and Sec24C identified by proteomics and immunohistochemistry as potential markers of early colorectal cancer stages

OLFM4, KNG1 and Sec24C identified by proteomics and immunohistochemistry as potential markers of early colorectal cancer stages

Two methods for proteomic analysis of formalin-fixed, paraffin embedded tissue result in differential protein identification, data quality, and cost

An Optimized Method for Protein Extraction from OCT-Embedded Human Kidney Tissue for Protein Quantification by LC-MS/MS Proteomics

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