Isobaric Colocalisation analysis (iCOLA) of A375P and A375M2 melanoma cells

ABSTRACT: To analyse protein-protein interactions on a global scale, we devised a methodology termed COLA, which uses subcellular fractionation combined with quantitative proteomics to generate multivariate subcellular localisation signatures for identified proteins. Bootstrapped clustering is then used to match proteins with significant similarity in their localisation signatures. A variarion of this method optimised for rapid analysis of interactome dynamics using isobaric labelling (iCOLA) was developed and used to compare interactomes of A375P melanoma cells which are weakly metastatic, against a highly metastatic derivative named A375M2. Cells were fractionated using two parallel subcellular fractionation procedures, resulting in a total of 9 fractions. The first fractionation procedure uses a serial solubilisation approach, resulting in five subcellular fractions (cytosol, total membrane, nuclear lumen,chromatin-bound nuclear, and cytoskeleton). The second procedure uses centrifugation coupled with aqueous biphasic extraction resulting in four fractions (total nuclear, intracellular membranes, plasma-membrane, and cytosol + microsomes). For quantifications, fractions were TMT labelled using a TMT 10plex labelling kit (Thermo). A total cell lysate control was also included as the 10th channel, with protein intensities in each subcellular fraction being normalised to the total lysate control intensities. Three sets of samples were analysed: two biological replicates of A375P (sample sets A and B), and one A375M2 sample(sample set C). We compared A and B to assess iCOLA reproducibility, and B and C to assess interactome dynamics between A375P and A375M2 cells.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Melanoma

SUBMITTER: Faraz Mardakheh  

LAB HEAD: Faraz Mardakheh

PROVIDER: PXD005277 | Pride | 2016-12-21

REPOSITORIES: Pride