Optimisation of a protocol for Protein Extraction from Calcified Aortic Valves for Proteomics Applications

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 homogenisation and protein extraction are difficulted by tissue calcification. Reproducibility of proteome studies is key when minding clinical translation of the findings. Thus, we aimed to optimise a protocol for aortic valve homogenisation and protein extraction, and to develop a standard operating procedure (SOP), which can be used by researchers to maximise protein yield, while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenisation with a Potter apparatus, with a more advanced bead-beating system. Once confirmed the superiority of the latter, we further optimised it by testing the effect of beads size, the number of homogenisation 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 homogenisation cycles, in the presence of 20 µL RIPA buffer/mg tissue, using 2-mL O-ring tubes. As a proof-of-concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterised, resulting in the quantification of proteins across 6 orders of magnitude, and uncovering some putative proteins dysregulated by sex.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Tissue, Aortic Valve

DISEASE(S): Aortic Valve Stenosis

SUBMITTER: Fábio Trindade  

LAB HEAD: Fábio Trindade

PROVIDER: PXD034386 | Pride | 2022-10-13

REPOSITORIES: Pride