Proteomic analysis of 3D cultures of fibroblasts and Ras-transformed HaCaT keratinocytes
HaCaT, and its three retrovirally c-Ha-ras-transformed subclones, A5, II-4 and RT3 (abbreviated as ras-HaCaTs in the text) that exhibit benign, invasive and malignant phenotypes, respectively, were cultured as 3D monocultures and co-cultures with fibroblasts. After hypotonic lysis of the cells, the insoluble proteins were analysed by LC-MS/MS.
Sample Processing Protocol
The proteins from 3D culture were dissolved in 50 µl of 8 M Urea, 100 mM ammonium bicarbonate. The cysteines were reduced in 10 mM dithiotreitol at 37°C for 1 h and alkylated in 40 mM iodoacetamide at room temperature for 1 h. The proteins were digested with LysC/Trypsin mixture (Promega), at 650 rpm at 37°C for 4 h, after dilution of the digestion mixture to 0.8 M urea with 100 mM ammonium bicarbonate, the digestion was continued for additional 16 h, and subsequently filtrated by a Microcon ultrafiltration device with 10 kDa cutoff (Merck-Millipore, Billerica, MA, USA). The peptides were desalted by StageTips (Rappsilber J, Ishihama Y, Mann M. Anal Chem. 2003 Feb 1;75(3):663-70) and vacuum dried before the LC-MS/MS run. The peptides were dissolved in 1 % formic acid, and loaded on a nanoflow HPLC system (Easy-nLC1000, Thermo Fisher Scientific) coupled to the Q Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ionization source. The peptides were first loaded on a trapping column and subsequently separated inline on a 15 cm C18 column (75 µm x 15 cm, ReproSil-Pur 5 µ;m 200 Å C18-AQ, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany). The mobile phase consisted of 0.1% formic acid (solvent A) and acetonitrile/water (95:5 (v/v)) with 0.1% formic acid (solvent B). The peptides were separated with a 46 min gradient from 7 to 25 % of solvent B followed by 4 min gradient from 25 to 35 % of solvent B. Before the end of the run, the percentage of solvent B was raised to 100 % in 5 min and kept there for 5 min. Full MS scan over the mass-to-charge (m/z) range of 300-1750 with a resolution of 140,000 followed by data dependent acquisition of with an isolation window of 2.0 m/z and a dynamic exclusion time of 30 s was performed. The top 10 ions were fragmented by higher energy collisional dissociation (HCD) with a normalized collision energy of 27 and scanned over the m/z range of 200-2000 with a resolution of 17,500. After the MS2 scan for each of the top 10 ions had been obtained, a new full mass spectrum scan was acquired and the process repeated until the end of the 60-min run.
Data Processing Protocol
Tandem mass spectra were searched using the MaxQuant software (version 188.8.131.52) against a database containing reviewed (SwissProt) human sequences, of UniProtKB release 2017_06. Peptide-spectrum-match- and protein-level false discovery rate thresholds were set to 0.01. Carbamidomethyl (C), as a fixed modification, and oxidation (MKP) as dynamic modifications were included. A maximum of two missed cleavages by trypsin (also before P) was allowed. The LC-MS profiles were aligned (within 20 min), and the identifications were transferred to non-sequenced or non-identified MS features in other LC-MS runs (within 0.7 min).