Proteomics raw data of feline mammary tumors
Description
Distinct proteomic signatures with specific protein expression patterns data were identified in formalin-fixed, paraffin-embedded (FFPE) feline mammary carcinoma cribiform and tubulopapillary samples using mass-spectrometry-based analysis and compared with human breast cancer proteomic data obtained from Human Protein Atlas. We hypothesized that specific proteins are responsible for the mechanisms of disease progression in cribiform and tubulopapillary and that similar prognostic markers may be identifiable in both feline mammary carcinoma and human breast cancer tissues. The FFPE samples (10 X 10 µm) were deparaffinized and rehydrated in sequential, decreasing concentration ethanol baths, followed by protein extraction, quantification, in-gel tryptic digestion, and mass spectrometry to identify the protein. Four proteins each were specific for cribiform and tubulopapillary feline mammary carcinoma, while 2 proteins, indicative of poor prognosis, similar to human breast cancer were identified in both feline groups.
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Steps to reproduce
Twenty one feline mammary carcinoma samples were collected from adult female cats and seperated into 2 groups: cribiform (CRIB, n = 12), and tubulopapillary (TUB, n = 9), and subjected to tissue sample preparation. Tissue were sectioned and cut into 10 slices of 10 µm thickness each and placed into a tube for protein extraction, followed by deparaffinization which involved incubation in xylene baths twice, each for 20 minutes at room temperature. The samples were rehydrated using sequential ethanol baths (100%, 95%, 75% ethanol) for 10 minutes each, and protein extraction was done by adding RIPA buffer (150 mmol NaCl, 1% Triton X-100, 1% sodium deoxcholate, and 0.1% SDS in 50 mmol Tris-HCl at pH 7.5; Peach et al., 2012), with the addition of protease inhibitor (0.8 mmol EDTA, 1.0 μg/mL aprotinin, 1.0 μg/mL leupeptin, 35.0 μg/mL PMSF in 50 mmol Tris-HCl at pH 7.2; Carvalho et al., 2021). Following sample sonication in ice bath, centrifugtion at 10,000 × g, 4°C for 30 minutes (Baker et al., 2002; Souza et al., 2009) and tryptic digestion of supernatant was done. Protein quantification was performed using A280 method (NanoDrop™ 2000 spectrophotometer, ThermoFisher), and SDS-PAGE run was conducted until the samples reached the seperating gel, followed by in-gel trypsin digestion (Shevchenko et al., 2006). Mass spectrometry analysis was done using Orbitrap Velos with ECD (Thermo Fisher) coupled to Easy-nLC II system with chromatographic separation setting of 150 mm column, 300 nL/min flow, over 45 minutes with MS full scan run at m/z 300-1600, resolution 60,000. Data processing and bioinformatic analysis involved protein identification (MaxQuant v1.5.8) using UnipProt database (Felis catus). The data were normalized to exclude outliers and analyzed by a non-hierarchical clustering (MetaboAnalyst 5.0 software). Protein intensity was performed by fold change, t-test, and volcano plots was used to determine the different abundances of proteins between groups (FDR < 0.05). KEGG pathway was used to analyse the pathways and gene ontology to determine the biological process, cellular component and molecular function of abundant proteins at FDR settings of < 0.05 was done using ShinyGO 0.82. All proteins encountered were compared with human breast cancer data from The Human Protein Atlas.
Institutions
- Universidade Estadual de Londrina Departamento de Clinicas Veterinarias
- Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de Medicina Veterinaria e Zootecnia Campus de Botucatu