Proteomic Analysis of Abdominal Aortic Tissue

Preparation of non-aneurysmal abdominal aortic tissue for mass spectrometry was performed virtually as previously described [33 (link)]. Briefly, formalin-fixed tissue was homogenized in a lysis buffer, denatured, alkylated, and subjected to overnight digestion with trypsin. Tryptic peptide samples were purified and tagged with 16-plex tandem mass tags (TMT, Thermo Scientific, Waltham, MA, USA) using mass tag 126 as the internal control (pool of all samples). Proteome data were protein abundances relative to the internal control. Mixed peptide samples were fractionated by high-pH chromatography followed by nano-LC–MS/MS analysis, virtually, as previously described [34 (link)] with the following modifications: Samples were analyzed using FAIMS Pro interface (Thermo Fischer Scientific, Slangerup, Denmark) and mass spectra were acquired by switching between CVs of −50 V and −70 V with a 2 s cycle time. All Eclipse raw data files were processed and quantified using Proteome Discoverer version 2.4 (Thermo Scientific, Waltham, MA, USA), also as previously described [34 (link)].