Fig 1: Workflow of the study, quality of the data and comparison of ECM in patients with TAV and BAV. A Workflow for the study. Aortic valve tissues from AVD patients with BAV and TAV were collected during open-heart surgery and stored in a biobank. The 88 samples were simultaneously processed using a compartment protein extraction kit. The ECM fractions were fully solubilized in 3.5–4% SDS, digested using S-Trap™ columns, protein abundances were quantified using label-free liquid chromatography tandem mass-spectrometry (LC–MS/MS), and the results were validated using independent methods. B Distribution of intensities for 651 quantified proteins (minimum 2 peptides per protein) with no missing values in the 88 samples. C Proteins with low CVs from different intensity ranges. D Enrichment analysis of the Gene Ontology (GO) cellular compartments among the quantified proteins. E Differential abundances of the 651 proteins in the TAV versus BAV group. Proteins that passed p-value p < 0.01 and fold change FC > 1.5 criteria are shown in red. F Several markers of BAV and TAV AVD ECM. The horizontal line in the box plots represents the median, 25th and 75th percentiles, and the whiskers represent measurements to the 5th and 95th percentiles. In figure, GO Gene Ontology; AVD aortic valve disease; BAV bicuspid aortic valve; TAV tricuspid aortic valve; ECM extracellular matrix; CV coefficient of variation; TNFRSF11B tumour necrosis factor receptor superfamily member 11B; PLTP phospholipid transfer protein; TNC Tenascin C; APOA4 Apolipoprotein A-IV; CTSD Cathepsin D; ANXA3 Annexin A3