PRIDE Assigned Tags:Biomedical Dataset
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Immunohistochemical markers distinguishing cholangiocellular carcinoma from pancreatic ductal adenocarcinoma discovered by proteomic analysis of microdissected cells
Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types which arise from epithelial cells of the pancreatobiliary system. Due to their histologic and morphologic similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. Biomarkers with high specificity and sensitivity for the differentiation of these tumour types would therefore be a valuable tool, but so far none are available. Here, we address this problem by comparing microdissected CCC and PDAC tumour cells in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of primary and secondary tumour tissue.
Sample Processing Protocol
Tumour tissue was collected during surgery, frozen in liquid nitrogen and stored at -80°C until further processing. For laser capture microdissection (LCM) the tissue was cryosectioned and stained with cresyl violet on PEN membrane-covered slides. LCM was performed on a Palm MicroBeam (Carl Zeiss Microscopy). Cells were lysed by sonication and tryptic protein digestion was carried out in solution using RapiGest surfactant as recommended by the manufacturer. LC-MS/MS analyses were performed on an Ultimate 3000 RSLCnano system (Dionex, Idstein, Germany) online coupled to an LTQ Orbitrap Elite (Thermo Scientific, Bremen, Germany). MS/MS spectra were acquired in a data-dependent mode.
Data Processing Protocol
Ion intensity-based label-free quantification was performed using Progenesis QI for proteomics (ver. 2.0.5387.52102, Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK). LC-MS runs were aligned to account for retention time shifts. Detected ions were filtered for those charged positively 2-, 3- or 4-fold and exhibiting more than 2 isotopes. Further details about peptide quantification have been described previously (Megger et al., 2013). Protein identification was performed using Proteome Discoverer (ver. 1.4) (Thermo Scientific, Bremen, Germany) searching the UniProt database (Release 2014_05; 545.388 entries) via Mascot (ver. 22.214.171.124) (Matrix Sciences Ltd., London, UK). Information about the search parameters has been published (Padden et al., 2014). Proteins quantified with only one unique peptide were removed from the study. A significant regulation was defined as a fold change > 2 and an FDR-corrected ANOVA p-value < 0.05.
Padden J, Ahrens M, Kälsch J, Bertram S, Megger DA, Bracht T, Eisenacher M, Kocabayoglu P, Meyer HE, Sipos B, Baba HA, Sitek B. Immunohistochemical Markers Distinguishing Cholangiocellular Carcinoma from Pancreatic Ductal Adenocarcinoma Discovered by Proteomic Analysis of Microdissected Cells. Mol Cell Proteomics. 2015 Dec 7. pii: mcp.M115.054585 PubMed: 26644413
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||46610||CCC vs. PDAC||1771||7536||5413||31815||6350||
|2||46611||CCC vs. PDAC||3493||22416||16259||30564||15579||
|3||46612||CCC vs. PDAC||2708||16438||11961||31950||11903||
|4||46613||CCC vs. PDAC||1862||9822||7126||32784||7969||
|5||46614||CCC vs. PDAC||1392||6864||5033||31721||5747||
|6||46582||CCC vs. PDAC||2078||9142||6497||33991||7762||
|7||46615||CCC vs. PDAC||2230||9378||6724||34275||8015||
|8||46581||CCC vs. PDAC||1702||8594||6294||33960||7052||
|9||46616||CCC vs. PDAC||2108||8457||6201||33722||7300||
|10||46617||CCC vs. PDAC||2000||10273||7608||31012||8410||