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

person Maria Júlia Barbosa Bezerra ICC, Fortaleza, Brazil info_outline Maria Júlia Barbosa Bezerra, Jackeline Almeida Silva, Rosane Oliveira Sant'Ana, Clarissa Gondim Picanço-Albuquerque, Maria Claudia dos Santos Luciano, Jose Fernando Bastos, Francisca Fernanda Barbosa Oliveira, Italo Aguiar Freire, Flávio da Silveira Bitencourt, Carlos Gustavo Hirth, Paulo Goberlanio de Barros Silva, Isabelle Joyce de Lima Silva-Fernandes

Authors person Maria Júlia Barbosa Bezerra ICC, Fortaleza, Brazil info_outline Maria Júlia Barbosa Bezerra, Jackeline Almeida Silva, Rosane Oliveira Sant'Ana, Clarissa Gondim Picanço-Albuquerque, Maria Claudia dos Santos Luciano, Jose Fernando Bastos, Francisca Fernanda Barbosa Oliveira, Italo Aguiar Freire, Flávio da Silveira Bitencourt, Carlos Gustavo Hirth, Paulo Goberlanio de Barros Silva, Isabelle Joyce de Lima Silva-Fernandes Organizations ICC, Fortaleza, Brazil, Instituto do Câncer do Ceará, Fortaleza, Brazil, Cancer Insititute of Ceará, Fortaleza, CE, Brazil, Hospital Haroldo Juaçaba, Ceará Cancer Institute, Fortaleza, Brazil Abstract Disclosures Research Funding No funding received None. 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. Protocol I Protocol II Protocol III Protocol IV Deparaffinization Xylene for 5 min and in ethanol for 5 min and dry for 30 min at room temperature X X Two incubations in xylene for 5 min X X X Rehydration 95% ethanol for 5 min followed by 70% ethanol for 5 min followed by distilled water for 10 min (Mohanty et al, 2021) X X X Lysis buffer Ureia 8M, NaCl 75mM e Tris 70 mM X X Tris 20 mM pH 8,8 and SDS 2% (Kuras et al., 2021) X TEAB 50 mM, DTT 100 mM and SDS 4% (Mohanty et al, 2021) X Incubation For 1h at 99°C (Kuras et al., 2020) X For 2h at 80°C (Shi et al, 2026) X