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Formalin-fixed Human Ovarian Cancer Tryptic Peptide LC-MS/MS

ABSTRACT: LC-MS/MS (LTQ-Orbitrap, CID) data sets of 31 human ovarian cancer samples (FFPE). Sample material obtained by laser microdissection of cores from tissue microarray. Preparation matching MALDI imaging protocol (as described in Gustafsson, J Proteomics 2012).

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Stephan Meding

PROVIDER: PXD000079 | Pride | 2013-02-28

REPOSITORIES: Pride

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			Action	DRS
	ORB120820-04_JG-014-10_SH.RAW	Raw
	ORB120820-06_JG-014-09_SH.RAW	Raw
	ORB120820-08_JG-014-08_SH.RAW	Raw
	ORB120820-10_JG-014-07_SH.RAW	Raw
	ORB120820-12_JG-014-06_SH.RAW	Raw

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Publications

Tryptic peptide reference data sets for MALDI imaging mass spectrometry on formalin-fixed ovarian cancer tissues.

Meding Stephan S Martin Karina K Gustafsson Ove J R OJ Eddes James S JS Hack Sandra S Oehler Martin K MK Hoffmann Peter P

Journal of proteome research 20121213 1

MALDI imaging mass spectrometry is a powerful tool for morphology-based proteomic tissue analysis. However, peptide identification is still a major challenge due to low S/N ratios, low mass accuracy and difficulties in correlating observed m/z species with peptide identities. To address this, we have analyzed tryptic digests of formalin-fixed paraffin-embedded tissue microarray cores, from 31 ovarian cancer patients, by LC-MS/MS. The sample preparation closely resembled the MALDI imaging workflo ...[more]

PMID: 23214983

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Project description:The use of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues for high-throughput molecular techniques, such as microarray gene expression profiling has become widespread in molecular research area. However, working with FFPE tissues is challenging because of degradation, cross-linking with proteins, and RNA chemical modifications. Also, there is no generally accepted procedure for RNA extraction to microarray analysis. Thus, there is a need for a standardized workflow for FFPE samples to study microarray transcriptome profiling. Therefore, the main purpose of this study was to conduct a standardized process from deparaffinization to RNA extraction and microarray gene expression analysis. Firstly, deparaffinization procedure was optimized for FFPE samples and then Trizol, PicoPure RNA isolation kit, and Qiagen RNeasy FFPE kit performances were compared in terms of yield and purity. Finally, two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were also evaluated to determine which combination gives the best percentage of present call. Our optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol and U133_X3P array combination gave a significantly higher percentage of present calls than the 3’ IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms. These results present a workflow for microarray gene expression profiling of FFPE tissues. The findings also indicate that sufficient quality gene expression data can be obtained from FFPE-derived RNA.

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Novel protocol for spatial proteome analysis of microdissected formalin-fixed paraffin-embedded tissues

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2024-06-03 | PXD051706 | Pride

A non-hazardous deparaffinization protocol enables quantita-tive proteomics of core needle biopsy-sized formalin-fixed and paraffin embedded (FFPE) tissue specimens.

Project description:Most human tumor tissues that are obtained for pathology and diagnostic purposes are forma-lin-fixed and paraffin embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to prote-olytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a ‘green’ xylene-free protocol for accel-erated sample preparation of FFPE tissues based on paraffin-removal with hot water. Com-bined with tissue homogenization using disposable micropestles and a modified protein aggre-gation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label free quantitation of FFPE cores from human ductal breast carcinoma in-situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2=0.992) with a median %CV of 16.9%. Im-portantly, this small volume is amenable to tissue micro array (TMA) cores and core needle bi-opsies, while our results and the easy-of-use indicate that a further downsizing is feasible. Fi-nally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

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2021-09-09 | PXD022994 | Pride

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