Dataset Information

Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories

ABSTRACT: In this study, we evaluated the feasibility of proteomics and phosphoproteomics on formalin-fixed paraffin-embedded (FFPE) lung tissue for protein extraction protocols, quantification methods, pre-analytics, and sample size. First, we compared three protocols for the extraction of proteins from FFPE tissues and used the best-performing protocol to acquire a deep proteome of lung adenocarcinoma and squamous cell carcinoma. We quantified >8,000 proteins and >14,000 phosphosites with a tandem mass tag (TMT11) approach and 6,700 proteins and 7,000 phosphosites with a microscaled TMT approach, enabling the analysis of FFPE needle biopsies with limited amounts of tissue. This is a considerable increase in coverage compared to label-free quantification techniques. We also evaluated pre-analytical variables such as the influence of fixation times and the duration of heat-assisted de-crosslinking on protein extraction efficiency and proteome coverage. Our improved workflows provide quantitative information on protein abundance and phosphosite regulation for most relevant oncogenes, tumor suppressors, and signaling pathways in lung cancer. We also present general guidelines to what proteomics and phosphoproteomics methods are best suited for different applications for retrospective cancer studies.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung

SUBMITTER: Corinna Friedrich

LAB HEAD: Philipp Mertins

PROVIDER: PXD024800 | Pride | 2021-05-05

REPOSITORIES: Pride

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Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories.

Friedrich Corinna C Schallenberg Simon S Kirchner Marieluise M Ziehm Matthias M Niquet Sylvia S Haji Mohamed M Beier Christin C Neudecker Jens J Klauschen Frederick F Mertins Philipp P

Nature communications 20210611 1

Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable resource for retrospective clinical studies. Here, we evaluate the feasibility of (phospho-)proteomics on FFPE lung tissue regarding protein extraction, quantification, pre-analytics, and sample size. After comparing protein extraction protocols, we use the best-performing protocol for the acquisition of deep (phospho-)proteomes from lung squamous cell and adenocarcinoma with >8,000 quantified proteins and >14,000 phosphosites with a ...[more]

PMID: 34117251

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Project description:Bilateral retinoblastoma has served as a critical model for studying genetic predisposition to cancer, however international incidence of unilateral retinoblastoma is variable across populations suggesting the involvement of environmental factors. DNA from Human Papilloma Virus (HPV) has been found in human retinoblastoma tissue through polymerase chain reaction based methods in several independent reports. To search for HPV oncoproteins and characterize transcriptional profiles in retinoblastoma, we analyzed tumor tissue obtained after enucleation and cultured for one week from 13 previously untreated Mexican children with retinoblastoma. We identified the presence of E7 HPV oncoproteins using western blot analysis in HPV positive tumors, and using non-supervised methods of classification on gene expression microarrays data, tumors clustered according to the presence or absence of HPV DNA identified by PCR. These methods were unable to discriminate retinoblastoma tissues by laterality. 2,404 genes were differentially expressed between HPV positive and HPV negative retinoblastoma, including a group of genes involved in a transcriptional response to viral infection. This molecular evidence supports the hypothesis that HPV is involved in the molecular pathogenesis of these tumors. The 13 children and tumors examined here, are incident cases participating in a larger case-control study at two Pediatric Hospitals of third level of medical attention in Mexico City, that form part of the public health system. Nine different tumors positive and four negative to HPV by PCR were included in the experimental design. From these, seven are unilateral and six are bilateral and are considered biological replicas. Two unilateral and two bilateral cases were chosen to make technical replicas. From the surgical specimens, two tumor portions were obtained, one was frozen, DNA was extracted from it, and HPV status was assigned using MY09 and MY11 primers by PCR. Another portion of tumor tissue was used to grow in culture, separating viable cells from non viable cells through a ficoll gradient. In the eye, retinoblastoma usually grows into the vitreous which has no cells, and thus the tissue obtained is predominantly formed by tumor cells and few endothelial cells from capillaries in the tumor. Retinoblastoma grow in culture as non adherent cells, and when adherent cells were also present, cell suspension was moved to a new dish to keep retinoblastoma cells as the main or only cellular type as possible. We used a two channel platform with in house spotted microarrays. Since retinoblastoma has known classes: uni and bilateral, we sought to verify these classes or discover new, we used a class discovery approach and a reference experimental design. As the reference sample we used RNA form exponentially growing Saccharomyces cerevisiae because major signaling pathways and basic cellular processes among Saccharomyces cerevisiae and mammalian cells including cell cycle control and DNA repair mechanisms have a high degree of conservation. Also because yeast is easy to grow, and thus a reliable and robust source of reference RNA.

Dataset Information

Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories

Publications

Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories.

Similar Datasets

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