Phospho-Proteomic analysis of tumour-endothelial bidirectional contact-initiated signalling
AIM:
The aim of the project is to identify regulated phosphosites in breast cancer cells (MDA-MB-231-4175-TGL, referred to as LM2 or MDA depending on the document) and endothelial cells (HUVEC) when they interact.
EXPERIMENTAL OUTLINE:
In 15 cm petry dishes, monolayers of HUVEC were grown to confluency. After 1h30 of HUVEC starvation, twice the number of MDA-MB-231 (starved overnight and detached with enzyme-free cell dissociation buffer) were plated onto the HUVECs. Co-culture was conduced for 15 min at 37 degrees in a small volume of serum free medium (3ml per 15cm dish). Then, non-attached LM2 were removed with the medium and counted to assess the number of cells still attached. SILAC labelling was used to differentiate cancer cells from endothelial cells and perform the quantification. Heavy label was systematically used for cells in co- culture; medium label was used for cells in mono-culture. Non-labelled cells were the other cell type (not taken into account in the analysis). Heavy-labelled and medium-labelled cells were mixed in a 1:1 cell ratio. The experiment was performed 5 independent times with labelled LM2 and 4 independent times with labelled HUVECs. Cell lysates were subjected to a rough membrane fragmentation, then membrane- enriched and cytoplasmic fractions were prepared in parallel (phospho- enrichment with TiO2, lys-C / trypsin digestion) before MS/MS. Raw data were searched against SwissProt database using PD1.3 / Mascot. Tables were extracted and for each identified peptide the position of the first amino-acid in the protein was used using the same database as the one utilised for the search.
DATA:
- "Samples.csv" contains the sample descriptions (experiment number, labelled cell type, fraction, sample name and raw file name).
- "SILAC1018TotV5_FAAposition.csv" is the raw table containing all the peptides identified in the experiment and the position of the first amino-acid in the protein sequence.
- “HUVsignV5spF.csv", "MDAsignV5spF.csv" and "MDAsignV5spFLMNA.csv" contain the spectra corresponding to the list of regulated targets after manual inspection.
- "HUVChecked.csv" and "MDAChecked.csv" are the equivalent tables after manual inspection of a broader range of phosphosites of interest.
- "DA-MDA-HUVEC" is the markdown document associated with the data analysis.
- “MDARegProt.csv” is the list of uniprot entry names associated to the proteins in the “Pathway” figures (manually assessed).
- “HUVRegProt.csv” is the equivalent for HUVEC regulated proteins.
- “TableRegPsites” and related document describe the construction of the tables with median of regulation fold per experiment.
In the folder "Proteomics-20151015" are all the documents regarding proteomics analysis: MS data from the Proteome Discoverer search are in the folders "Analysis_2" and "EPHA2-IP". More precised description is provided in the "TotProtAnalysis_Final" files. All the tables and figures from the analysis are in the folders "OutputTab" and "OutputFig".
DESCRIPTION:
The table "Samples.csv" contains the description of all samples analysed for the project.
- experiment: each experiment is labelled with "SILAC" and a number (from 010 to 019 - with no SILAC018).
- sample: each sample is labelled with "ps" of "psMLP" and a number. For each experiment, there are at least two samples prepared (membrane-enriched fraction and cytoplasmic fraction). There can be several sample preparations / technical replicates, injected different days (thus several "SpectrumFile").