Fig. 1: Experimental procedures for the quantitative measurement of Aβ42 oligomer populations during an ongoing amyloid fibril self-assembly reaction using 3H labelling or MS. | Nature Chemistry

Fig. 1: Experimental procedures for the quantitative measurement of Aβ42 oligomer populations during an ongoing amyloid fibril self-assembly reaction using 3H labelling or MS.

From: Dynamics of oligomer populations formed during the aggregation of Alzheimer’s Aβ42 peptide

Fig. 1

a, We incubated varying concentrations of Aβ42 or Aβ40 monomers and collected aliquots at the desired time points during the aggregation reaction. For each time point (Δt), we used centrifugation to remove the fibrils. We then isolated the oligomeric fraction, which encompassed species in the range of trimers to ~22-mers, through SEC. We used a Superdex 75 column with a void volume of ~7 ml and the monomer was eluted at 14–16 ml. b,c, After separation through SEC, we used liquid scintillation counting (b) or MS (c) to measure the oligomer concentrations. b, We used liquid scintillation counting to measure the absolute mass concentration of peptides that eluted between 7 and 13 ml in the case of 3H-labelled Aβ42. c, We used MS of natural abundance peptides, in which each fraction (1 ml) was lyophilized, redissolved in 20 μl of H2O, supplemented by a defined amount of 15N-Aβ42 (10 pmol) and AspN enzyme, digested overnight and analysed by matrix-assisted laser desorption/ionization–time of flight MS. The peptide concentration in each fraction was determined as the ratio r of the integrated area of the 14N peak at 1,906 m/z and the 15N peak at 1,928 m/z as c = r × 10 nM. The total oligomer concentration at each time point Δt was calculated as the sum over fractions 7–12. The relative Aβ concentration in each fraction was then calculated by dividing c by the summed concentrations over fractions 7–12. d, Observed concentration of oligomers versus aggregation time Δt. This procedure, which requires 10–16 min for oligomer isolation (Supplementary Section 1), provides a rapid and quantitative readout of the time evolution of oligomeric populations. a.u., arbitrary units.

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