Dataset Information

Human osteoblasts quantitative proteomic analysis after cocultivation with endothelial cells

ABSTRACT: We previously described that the coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves the differentiation of both cell types, leading to the formation of functional blood vessels and enhanced bone regeneration. The objective of this study was to further delineate the multifaceted interactions between both cell types. To investigate the proteome of hOBs after cocultivation with HUVECs we used stable isotope labeling by amino acids in cell culture

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Osteoblast, Cell Culture

SUBMITTER: Oscar Winninger

LAB HEAD: Günter Finkenzeller

PROVIDER: PXD011844 | Pride | 2018-12-03

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20150731_VD_CFP859_sample1_01.raw	Raw
	20150731_VD_CFP859_sample1_02.raw	Raw
	20150731_VD_CFP859_sample1_03.raw	Raw
	20150731_VD_CFP859_sample1_04.raw	Raw
	20150731_VD_CFP859_sample1_05.raw	Raw

Items per page:

1 - 5 of 23

Publications

Increased differentiation and production of extracellular matrix components of primary human osteoblasts after cocultivation with endothelial cells: A quantitative proteomics approach.

Simunovic F F Winninger O O Strassburg S S Koch H G HG Finkenzeller G G Stark G B GB Lampert F M FM

Journal of cellular biochemistry 20180820 1

Coculturing of bone-forming and blood vessel-forming cells is a strategy aimed at increasing vascularity of implanted bone constructs in tissue-engineering applications. We previously described that the coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves the differentiation of both cell types, leading to the formation of functional blood vessels and enhanced bone regeneration. The objective of this study was to further delineate the multifac ...[more]

PMID: 30126049

Similar Datasets

Project description:Metastasizing tumor cells exit the vascular system through dynamic interactions with endothelial cells that line the internal surface of vessels. While extravasation is a key event within the metastatic cascade, the signals regulating tumor cell adhesion to the endothelium and their subsequent transendothelial migration are poorly understood. Here, we combined Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and phosphoproteomic analysis to identify cell-specific signaling pathways regulated between interacting breast cancer cells and endothelial cells (see PRIDE repository PXD001558). Further co-culture experiments were performed alongside the phosphoproteomic analysis in order to control for the protein quantity. These are presented here. Using SILAC, cell-specific labels were introduced into MDA-MB-231-LM2 cells (Human Breast cancer) and HUVECs (Human endothelial cells) to ensure each cell type had a distinct and traceable phosphoproteome when tumor and endothelial cells were co-cultured. To probe regulatory signaling events triggered in cancer cells following contact with endothelial cells, we labeled LM2 cells with medium or heavy isotopomers of arginine and lysine (Arg+6 Da, Lys+4 Da and Arg+10 Da, Lys+8 Da respectively). Heavy-labeled LM2 cells were collected by enzyme-free cell dissociation buffer, thereby preserving membrane proteins and adhesion receptors, and seeded onto a monolayer of light-labeled (Arg+0 Da, Lys+0 Da) HUVECs. Following 15 min of co-culture, non-adherent LM2 cells were gently removed and cancer cells that had attached to the endothelial layer were lysed together with the HUVECs. In parallel, medium-labeled LM2 cells were collected under the same conditions and maintained as suspension cells in monoculture to represent circulating tumor cells prior to any contact with the endothelium. These were then added to the harvested LM2-HUVEC co-culture in a 1:1 ratio of heavy:medium-labeled cells to provide a point of reference. Based on the SILAC labeling of the different cell populations, light-labeled peptides were assigned to HUVECs, medium-labeled peptides to monocultured LM2 cells in suspension, and heavy-labeled peptides to LM2 cells that had made contact with HUVECs. As such, the heavy/medium ratio for each peptide was used to quantify phosphorylation-dependent signaling changes occurring specifically in the LM2 cells upon contact with HUVECs. Conversely, to elucidate signaling events in HUVECs that were initiated by contacting cancer cells, light-labeled LM2 cells were seeded on top of a confluent monolayer of heavy-labeled HUVECs, while medium-labeled HUVECs were maintained in monoculture. Following 15 min of co-culture, the unattached LM2 cells were removed. Cells were lysed, mixed in a 1:1 ratio of heavy:medium HUVECs, followed by membrane fractionation and phosphoproteomic analysis as described above. A variation in phospho-peptide quantity could be due to the experimental difficulties in mixing heavy and medium labels in a 1:1 ratio or a quick regulation of the protein quantity through modification of its expression/degradation balance. Thus, we performed relative quantification of non-phosphorylated peptides in parallel with the phosphoproteomic analysis to account for changes in total protein abundance (data presented here) or correct for experimental bias. Cytoplasmic fractions were analyzed by LC-MS/MS before TiO2 enrichment and their median log2(H/M) were used for normalization of the phosphoproteomic dataset. In order to increase the number of proteins quantified in the LM2 cells, we performed a supplementary co-culture experiment and fractionated the peptides by SDS-PAGE. In order to get a confident relative quantification of Ephrin type-A receptor 2 (EPHA2), we performed 2 independent experiments followed by EPHA2 IP and LC-MSMS.