Proteomic analysis of FFPE tissue samples from liver cancer
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Yang Zhao
PROVIDER: PXD060181 | iProX | Thu Mar 27 00:00:00 GMT 2025
REPOSITORIES: iProX
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Project description:Fixed-Tissue ChIP-seq for H3K27Ac profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in FFPE tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27Ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65°C in more stringent detergent conditions and a minimized sonication step, to produce robust genome-wide H3K27Ac maps from clinical samples. We demonstrate FiTAc-seq suitability to generate high quality enhancer landscapes and SE annotation in a number of archived FFPE samples from distinct tumor types. This approach will be of great interest both for basic and clinical researchers. The entire protocol from FFPE blocks to sequence ready library can be accomplished within 4 days.
2020-06-26 | GSE140806 | GEO
Project description:Fixed-Tissue ChIP-seq for H3K27Ac profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in FFPE tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27Ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65°C in more stringent detergent conditions and a minimized sonication step, to produce robust genome-wide H3K27Ac maps from clinical samples. We demonstrate FiTAc-seq suitability to generate high quality enhancer landscapes and SE annotation in a number of archived FFPE samples from distinct tumor types. This approach will be of great interest both for basic and clinical researchers. The entire protocol from FFPE blocks to sequence ready library can be accomplished within 4 days.
2020-06-26 | GSE140805 | GEO
Project description:Fixed-Tissue ChIP-seq for H3K27Ac profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in FFPE tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27Ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65°C in more stringent detergent conditions and a minimized sonication step, to produce robust genome wide H3K27Ac maps from clinical samples. We demonstrate FiTAc-seq suitability to generate high quality enhancer landscapes and SE annotation in a number of archived FFPE samples from distinct tumor types. This approach will be of great interest both for basic and clinical researchers. The entire protocol from FFPE blocks to sequence ready library can be accomplished within 4 days.
2020-06-26 | GSE147446 | GEO
Project description:In recent years, the use of formalin-fixed paraffin-embedded (FFPE) tissues has increased within cancer research and clinical proteomics. However, the use of FFPE tissues can be demanding due to paraffin embedding and cross-links introduced by the formaldehyde. Here, we developed a workflow implementing efficient paraffin removal and protein extraction from FFPE tissues, followed by optimized digestion using suspension trapping (S-trap), with different clinical proteomics strategies. To gain proteomic depth, the optimized sample preparation protocol was combined with isobaric labeling and applied to three lung adenocarcinoma patient samples. The peptides eluted from the S-trap were labeled with TMT without any desalting step in between. In total, 8,163 proteins were commonly quantified across all channels, and specific proteins related to clinical outcome and histopathological subtype were detected. In addition, we developed a protocol in the S-trap for studying endogenous lysine acetylation stoichiometry using FFPE tonsil tissue. Our method allows identification and localization of lysine acetylation and relative quantification comparison between samples. We could identify 1,839 acetylated peptides belonging to 1,339 protein groups. The results were compared to frozen tissue samples and no notable differences in acetylation pattern were observed. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that is easy to scale up using the S-trap 96-well plate. Our results demonstrate compatibility of the S-trap with TMT labeling. And, for the first time, we showed that it is biologically relevant to study endogenous lysine acetylation in FFPE tissues, contributing to a better use of the existing FFPE collections around the world.
2020-12-18 | PXD020157 | Pride
Project description:Well-characterized archival FFPE tissues are of much value for prospective biomarker discovery studies. Protocols that offer high-throughput and good reproducibility are essential in proteomics. Therefore, we developed a workflow implementing efficient paraffin removal and protein extraction from FFPE tissues, followed by digestion using suspension Trapping (S-Trap). The protocol was then combined with isobaric labeling/TMTpro 16plex and applied to lung adenocarcinoma patient samples. In total, 9,585 proteins were quantified, and proteins related to clinical outcome and histopathological subtype were detected. Since acetylation is known to play a major role in cancer development, an on-filter acetylation protocol was developed for studying endogenous lysine acetylation. Our method allows identification and localization of lysine acetylation and quantitative comparison between samples. We identified 1,839 acetylated peptides belonging to 1,339 protein groups and showed that the data obtained from FFPE tissues is in concordance with acetylation data from frozen tissues. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that resolves known proteomic-related challenges. We demonstrate compatibility of the S-Trap with TMTpro 16plex labeling. And, for the first time, we prove that it is feasible to study endogenous lysine acetylation stoichiometry in FFPE tissues, contributing to better utility of the existing global tissue archives.
2020-12-18 | PXD021964 | Pride
Project description:Well-characterized archival FFPE tissues are of much value for prospective biomarker discovery studies. Protocols that offer high-throughput and good reproducibility are essential in proteomics. Therefore, we developed a workflow implementing efficient paraffin removal and protein extraction from FFPE tissues, followed by digestion using suspension Trapping (S-Trap). The protocol was then combined with isobaric labeling/TMTpro 16plex and applied to lung adenocarcinoma patient samples. In total, 9,585 proteins were quantified, and proteins related to clinical outcome and histopathological subtype were detected. Since acetylation is known to play a major role in cancer development, an on-filter acetylation protocol was developed for studying endogenous lysine acetylation. Our method allows identification and localization of lysine acetylation and quantitative comparison between samples. We identified 1,839 acetylated peptides belonging to 1,339 protein groups and showed that the data obtained from FFPE tissues is in concordance with acetylation data from frozen tissues. In summary, we present a reproducible sample preparation workflow optimized for FFPE tissues that resolves known proteomic-related challenges. We demonstrate compatibility of the S-Trap with TMTpro 16plex labeling. And, for the first time, we prove that it is feasible to study endogenous lysine acetylation stoichiometry in FFPE tissues, contributing to better utility of the existing global tissue archives.
2020-12-18 | PXD021986 | Pride
Project description:The reliability of differential expression analysis on FFPE expression profiles from Affymetrix arrays is questionable, due to the wide range of percent-present values reported in studies which profiled FFPE samples on Affymetrix arrays. Moreover the validity of externally defined gene-modules in FFPE microarray expression profiles is unknown. Using eight breast cancer tumors with available frozen and FFPE samples, five sample-matched data sets were generated from different combination of Affymetrix arrays, amplification-and-labeling kit and sample preservation method. The reliability of differential expression analysis was investigated by developing de novo ER/HER2 pathway gene-modules from matched data sets and validating it on external data set using ROC analysis. Spearman's rank correlation coefficient of module scores between matched FFPE-frozen expression profiles was used to measure reliability of externally defined gene-modules in FFPE expression profiles. Independent of array/amplification-kit/sample preservation method used, de novo ER/HER2 gene-modules derived from all matching data sets showed similar prediction performance during independent validation (AUC range; ER: 0.92-0.95, HER2: 0.88-0.91), except for de novo HER2 gene-module derived from FFPE data set with 3'IVT kit (AUC: 0.67-0.72). Further not all gene-module based biological signals present in frozen expression profiles can be recovered from matching FFPE microarray expression profiles using the currently available FFPE specific sample preparation kits. The gene-module based biological signal extracted from FFPE RNA, using microarrays, may not be as reliable as that from their frozen counterpart, if the sample preparation protocol used with FFPE RNA failed to recover relevant genes involved in the biological signal.
2018-09-05 | GSE93337 | GEO
Project description: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.
2021-09-10 | PXD023891 | Pride
Project description:The reliability of differential expression analysis on FFPE expression profiles from Affymetrix arrays is questionable, due to the wide range of percent-present values reported in studies which profiled FFPE samples on Affymetrix arrays. Moreover the validity of externally defined gene-modules in FFPE microarray expression profiles is unknown. Using eight breast cancer tumors with available frozen and FFPE samples, five sample-matched data sets were generated from different combination of Affymetrix arrays, amplification-and-labeling kit and sample preservation method. The reliability of differential expression analysis was investigated by developing de novo ER/HER2 pathway gene-modules from matched data sets and validating it on external data set using ROC analysis. Spearman's rank correlation coefficient of module scores between matched FFPE-frozen expression profiles was used to measure reliability of externally defined gene-modules in FFPE expression profiles. Independent of array/amplification-kit/sample preservation method used, de novo ER/HER2 gene-modules derived from all matching data sets showed similar prediction performance during independent validation (AUC range; ER: 0.92-0.95, HER2: 0.88-0.91), except for de novo HER2 gene-module derived from FFPE data set with 3'IVT kit (AUC: 0.67-0.72). Further not all gene-module based biological signals present in frozen expression profiles can be recovered from matching FFPE microarray expression profiles using the currently available FFPE specific sample preparation kits. The gene-module based biological signal extracted from FFPE RNA, using microarrays, may not be as reliable as that from their frozen counterpart, if the sample preparation protocol used with FFPE RNA failed to recover relevant genes involved in the biological signal.
2018-09-05 | GSE93336 | GEO