Interactomes of neural cell adhesion molecule 1 and the metal ion transporter ZIP6 in mouse NMuMG epithelial cells with transforming growth factor beta 1-induced fibroblastoid morphology
The prion protein (PrP) evolved from the subbranch of ZIP metal ion transporters comprising ZIPs 5, 6 and 10, raising the prospect that the study of these ZIPs may reveal insights relevant for understanding PrP function. PrP and ZIP6 are required for the execution of a cellular program known as epithelial-to-mesenchymal transition (EMT). Polysialylation of neural cell adhesion molecule 1 (NCAM1) during EMT is also controlled by PrP. Here we report the ZIP6 interactome and ZIP6-dependent NCAM1 interactome in a mouse cell EMT model.
Sample Processing Protocol
NMuMG wild-type and ZIP6 knockout clones produced using CRISPR-Cas9 as well as wild-type clones with NCAM1 knockdown by siRNA were cultured to full confluence. For each experiment, three biological replicates of each clone were maintained and immunoprecipitated in parallel. To induce EMT, all cell cultures were treated with 6.4 ng/ml of transforming growth factor beta 1 every 24 hours for 48 hours. Following treatment, cells were washed with ice cold PBS, then exposed to 2% formaldehyde in PBS for 15 minutes to crosslink their proteins. Crosslinking was stopped using 125 mM glycine. Cells were then washed with ice cold PBS before undergoing lysis in 1% NP-40 in 150 mM HEPES (pH 8.0) with protease inhibitors. Insoluble cellular debris was cleared by centrifugation. Immunoprecipitations with ZIP6 and NCAM1 specific antibodies were conducted with Protein A agarose and Protein G sepharose beads, respectively. Cell lysates were exposed to bead-immobilized antibodies overnight on a turning wheel at 4°C, the slurry was then washed with lysis buffer followed by 10 mM HEPES (pH 8.0). Elution was effected with 0.2% trifluoroacetic acid in 20% acetonitrile then the acid and organic solvent were removed by vacuum centrifugation. Immunoprecipitates were dissolved in 9 M urea, heated to 0°C in the presence of 5 mM tris(2-carboxyethyl) phosphine then alkylated with 10 mM 4-vinylpyiridine (4-VP). The urea concentration was reduced to 1.5 M by dilution with water prior to overnight digestion with trypsin 37°C. Digested immunoprecipitates generated with anti-ZIP6 were covalently modified with Tandem Mass Tags (Thermo Fisher Scientific) while those generated with anti-NCAM1 were covalently modified with iTRAQ reagents (Sciex). Digested and covalently modified immunoprecipitates were purified by C18 reversed-phase and strong cation exchange cartridges (Agilent Technologies) and the eluates were dried in a centrifugal concentrator then disolved in aqueous 0.1% formic acid. and applied to C18 nanocapillary columns (25 cm long Acclaim PepMap RSLC with 100 Å pore size, 2 µm particle size, 75 µm inner diameter) using an EASY-nLC 1100 system (Thermo Fisher Scientific). Peptide separation was performed at 300 nl/minute using a binary mobile phase gradient with mobile phases containing 0.1% (v/v) formic acid in water or acetonitrile. Over the gradient, the organic content of the mobile phase was increased from 0 to 30% over 180 minutes then to 100% by 240 minutes. The nano-HPLC system was coupled by online nanoscale electrospray ionization (ESI) to an Orbitrap Fusion Tribrid mass spectrometer. The MS data acquisition method included three scan types. Each data acquisition cycle began with a survey scan of the 400–2000 m/z range in the orbitrap at 120,000 resolution with automatic gain control set to 2e5 counts. Next, the most intense precursor ions carrying two to seven charges were separately isolated, subjected to collision-induced dissociation (CID) and their fragments detected in the linear ion trap. Finally MS3 with higher-energy collision-induced dissociation (HCD) was performed on the ten most intense fragments from each MS2 scan. The orbitrap was used to obtain MS3 scans at 60,000 resolution with an automatic gain control target of 1e5 counts at a maximum injection time of 120 ms. The combined cycle time for this method was 3 seconds during which as many precursors as possible were analyzed.
Data Processing Protocol
Peptide sequencing was performed using Mascot (Version 2.4; Matrix Science Ltd, London, UK) and Sequest HT search engines embedded in Proteome Discoverer software (Version 1.4; Thermo Fisher Scientific). The international protein index (IPI) mouse database (Version 3.87) was used as the protein sequence source in peptide-spectral matching. Peptide and protein level quantification was based on MS3 data.