Proteomic Aggregation Quantification in Plasma

Proteostat Protein Aggregation Assay was purchased from Enzo Life Sciences (ENZ-51035, Farmingdale, NY, USA). Plasma proteins were incubated with the commercial reagent sensitive to protein aggregation at a ratio of 2 μL reagent to 98 μL of diluted plasma samples. Positive and negative controls were included, as indicated. Proteostat Protein Aggregation standards (ENZ-51039, Enzo Life Sciences, Farmingdale, NY, USA), containing stressed and unstressed IgGs, were used to establish a standard curve and calculate the percentage of aggregated protein in test samples. Fluorescence was measured using a Tecan Infinite M200 microplate reader (TECAN, Männedorf, Switzerland), with excitation set at 550 nm and an emission filter of 600 nm. For Thioflavin T (ThT) (211760050, Thermo Fischer Scientific, Waltham, MA, USA) staining, 5 µL of human plasma was loaded onto wells of a 96-well microplate, in duplicate. Samples were then mixed with 10 µM ThT in 100 mM sodium phosphate buffer, pH 7.4, to a final volume of 250 µL per well [29 (link)]. Fluorescence readings were acquired using the Tecan Infinite^® M200 PRO microplate reader at an excitation of 444 nm and emission of 485 nm.