Optimization of a Protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications: Development of a Standard Operating Procedure

Data de publicação: 01/09/2022 Data Ahead of Print: 01/09/2022

Autores da FMUP

• Francisca Almeida Saraiva

  Autor

• Carla Margarida Castanheira Sousa

  Autor

• Cristina Maria Rodrigues Pinheiro Gavina

  Autor

• Joaquim Adelino Correia Ferreira Leite Moreira

  Autor

• Inês Maria Falcão Sousa Pires Marques

  Autor

Participantes de fora da FMUP

• Trindade, F
• Ferreira, AF
• Martins, D
• Mendes, VM
• Manadas, B
• Vitorino, R

Unidades de investigação

• Cirurgia e Fisiologia

• Laboratório Associado RISE- Rede de Investigação em Saúde

  

  Investigador

  Fernando Carlos De Landér Schmitt

• Medicina

• RISE-Health

  

  Investigador

  Fernando Carlos De Landér Schmitt

Abstract

The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcification. The reproducibility of proteome studies is key in clinical translation of the findings. Thus, we aimed to optimize a protocol for aortic valve homogenization and protein extraction and to develop a standard operating procedure (SOP), which researchers can use to maximize protein yield while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenization with a Potter apparatus with a more advanced bead-beating system. Once we confirmed the superiority of the latter, we further optimized it by testing the effect of beads size, the number of homogenization cycles, tube capacity, lysis buffer/tissue mass ratio, and two different lysis buffers. Optimal protein extraction was achieved with 2.8 mm zirconium dioxide beads, in two homogenization cycles, in the presence of 20 mu L RIPA buffer/mg tissue, using 2 mL O-ring cryotubes. As a proof of concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterized, resulting in the quantification of proteins across six orders of magnitude and uncovering some putative proteins dysregulated by sex.

Keywords

• aortic valve; calcification; homogenization; protein extraction; zirconium dioxide beads; proteomics; SOP