Optimization of protein extraction for proteomic analysis of formalin-fixed and paraffin-embedding (FFPE) biopsy specimens from males with breast cancer.

Abstract

e13119 Background: Male breast cancer corresponds to 1% of breast cancer diagnoses. Given this low incidence, the tumors detected have a worse prognosis and lethality. Formalin fixation and paraffin-embedding (FFPE) methods are the most common to maintain biopsied samples. It is a low-cost procedure and is a valuable resource for the study of pathological molecular mechanisms and research of potential biomarkers and therapeutic target molecules. However, in this technique cross-links are formed compromising protein extraction, resulting in low yield and quality for mass spectrometry analysis. Objective: To present an optimized method of protein extraction from male breast FFPE samples for proteomic analysis. Methods: Four protein extraction protocols were optimized and tested in female breast cancer FFPE samples from Haroldo Juaçaba Hospital/Cancer Institute of Ceará to preserve the male sample. Each protocol was performed using a total of eight 5uM tumor slides. Different deparaffinization processes were tested followed by distinct extraction buffers and variable incubation time. The characteristics of each of the protocols and optimizations are presented in the table. The quantification of the protein profile was carried out in nanodrop 2000. The protocol with the highest yield was used in the male breast cancer sample FFPE. Analysis of the protein profile in polyacrylamide gel (SDS PAGE) was performed. Results: Protocol IV presented the highest yield of protein (16.9 mg/mL) and protocols I, II, and III presented, 0.46 mg/mL and 0.28 mg/ml, and 2.3 mg/mL, respectively. Protocol IV was selected to be used in the male breast cancer sample and the yield was 14.26 mg/mL, similar to the female sample. Protein bands from male and female samples using protocol IV were detected on the SDS-PAGE gel. Conclusions: Protocol IV performed a satisfactory protein yield in breast cancer samples FFPE. [Table: see text]