Figure 1. Experimental procedures for the quantitative measurement of Aβ42 oligomer populations during an ongoing amyloid fibril self-assembly reaction using tritium labelling or mass spectrometry.
(a) We incubated varying concentrations of Aβ42 or Aβ40 monomers and collected aliquots at desired time points during the aggregation reaction. For each time point (), we used centrifugation to remove fibrils. We then isolated the oligomeric fraction, encompassing species in the range of trimers to ca. 22-mers, through size-exclusion chromatography (SEC). We used a Superdex 75 column for which the void volume is ca. 7 mL and the monomer elutes at 14-16 mL. After separation through SEC, we used (b) liquid scintillation counting or (c) mass spectrometry (MS) to measure oligomer concentrations. In (b), we used liquid scintillation counting to measure the absolute mass concentration of peptides eluting between 7 and 13 mL in the case of 3H-labelled Aβ42. In (c), we used MS of natural abundance peptides, in which case each fraction (1 mL) was lyophilised, redissolved in 20 μL H2O, supplemented by a defined amount of 15N-Aβ42 (10 pmol) and AspN enzyme, digested overnight, and analysed by MALDI-TOF MS. The peptide concentration in each fraction was determined as the ratio r of the integrated area of the 14N peak at 1906 m/z and the 15N peak at 1928 m/z as nM. The total oligomer concentration at each time point 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 . This procedure, which requires 10-16 minutes for oligomer isolation (Materials and Methods in Supplementary Sec. 1), provides a rapid and quantitative readout of the time evolution of oligomeric populations.