Computational protocol: The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics

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[…] Cells were grown on sterilised coverslips, fixed in 4% formaldehyde, permeabilised with 0.2% Triton X‐100 and blocked with 1% BSA in PBS before incubation with the relevant primary antibody and, subsequently, with AlexaFluor®488/568/647‐conjugated secondary antibodies (Molecular probes). DNA was visualised with Hoechst, biotin with AlexaFluor®568‐conjugated streptavidin (Molecular probes) and actin with AlexaFluor®488/568/647‐conjugated phalloidin (Molecular probes). For structured illumination microscopy experiments, cells were grown on acid‐washed, No. 1.5, 18 mm square coverslips (high performance 170 ± 5 μm, Schott, Germany). For antibody uptake experiments, cells were starved overnight, incubated for 15 min at 4°C with HA antibody before uptake at 37°C in the presence of 10 μM LPA for the indicated time. Images were obtained using a 63× objective on a Zeiss LSM710 confocal microscope, a Zeiss AxioImager upright widefield epifluorescence microscope equipped with an ORCA Flash 4 v2 camera or, for SIM, on a Zeiss Elyra PS1 super‐resolution microscope. To measure colocalisation, images were taken from randomly selected fields of view, background subtracted and cells manually segmented using ImageJ. The Pearson's correlation coefficient was then calculated using the ImageJ plugin coloc2. Statistical analysis was performed in GraphPad Prism as indicated. [...] Samples were resuspended in MS solvent (3% acetonitrile, 0.1% TFA) for analysis on a Q Exactive (Thermo Scientific) coupled to an RSLC3000nano UPLC (Thermo Scientific). Peptides were resolved using a 50 cm C18 PepMap EASYspray column with a gradient rising from 97% solvent A (0.1% formic acid), 3% solvent B (80% acetonitrile, 0.1% formic acid) to 40% solvent B over 40 min. Data were acquired in a top 10 data‐dependent acquisition fashion with MS spectra acquired between m/z 400 and 1,400 at 70,000 fwhm. MS‐MS spectra were acquired at 17,500 fwhm and excluded from further fragmentation for 30 s.Raw files were processed as a single batch using the MaxQuant proteomics software package version 1.5.0.0 . Spectra were searched using the built‐in Andromeda search engine and the UniProt reference database for human proteins. Cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation and N‐terminal acetylation were selected as variable modifications. Both peptide and protein false discovery rates (FDRs) were set to 0.01, the minimum peptide length was set at seven amino acids, and up to two missed cleavages were tolerated. Some bioinformatics analysis was performed in the Perseus package bundled with MaxQuant . Data were filtered by removing matches to the reverse database, proteins only identified with modified peptides, and common contaminants and intensity values were log10 transformed. For label‐free experiments, data were uploaded to the CRAPome.org online analysis tool , . The default settings were used for all analysis in CRAPome (FC‐A, user, default, average; FC‐B, all, stringent, geometric; SAINT‐express, user, average, virtual controls 10, all replicates). Scores were downloaded and exported to ProHits‐Viz to make dot plots or to Cytoscape for network diagrams , .For SILAC experiments, heavy/light ratios were log2 transformed and outliers were identified using the significance A function (Benjamini–Hochberg procedure) in Perseus defining a threshold of 0.05. The significance A function was run on each triplicate ΔWWY, ΔRRL and ΔPIP2 experiment and gene names and SILAC ratios were compiled and carried forward for subsequent analysis. Principal component analysis (PCA) was performed in R using the mean heavy/light ratios of each significant protein and the “prcomp” and “biplot” functions.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD008686. […]

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