Figure 1.
SDS PAGE of Aβ oligomeric forms obtained at different times of incubation with H2O2 and horseradish peroxidase and visualized by (A) Coomassie staining, (B) anti-Aβ antibodies. (C) Gel stained with anti-di-Tyr antibody (left panel) at different reaction times shows the expected signal corresponding to the mass of a dimer. A gel developed with an anti-Aβ monoclonal antibody 6E10 (right panel). Arrow indicates the position of the BSA containing Di-Tyr cross-link (as a control). See also Figure S1.
Figure 2.
Di-tyrosine fluorescence spectra.
Fluorescence spectra collected at different times of incubation of Aβ with H2O2 and horseradish peroxidase, upon excitation at 315 nm. The two bands at 400 nm and 425 nm correspond to di-Tyr and tri/tetra-Tyr, respectively. Arrows indicate time of reaction in minutes.
Figure 3.
Spectra for covalently stabilized DIM9+.
Fragments of mass spectra collected before (right panel) and after 90 min of reaction (left panel). Shown is the fragment of spectra in which a new signal, absent before reaction, was observed. This signal corresponded to 9+ charged dimer at 963 m/z.
Table 1.
Experimental values of molecular masses (MB, MA), IMS drift times (tD) and collisional cross-section (Ω) for dimer and trimer.
Figure 4.
Fragments of IMS-MS spectra for different charged dimer.
Selected regions of ion mobility separated mass spectra (IMS-MS) covering dimeric signals in 2D rendition (colored panels, drift time at horizontal axis and m/z at vertical axis). Lower panels show the corresponding ion mobility drift time profiles, i.e., either projections of the signal group on the drift time axis or cross-sections at a different m/z value, as described below. Panels showing isotopic envelopes are cross-sections at indicated (blue arrows) drift time values correspond to a given oligomeric form, for instance DIM8+ denoting a non-covalently stabilized dimer charged 8+, whereas cDIM8+ is a covalently stabilized dimer charged 8+, etc. Spectra are shown before reaction with H2O2/HRP (A,C,E,G) and at 90 min of reaction (B,D,F,H). (A,B) 1082–1085 m/z region with overlapping MON4+ and DIM8+ signals present. The two drift time profiles are collected at m/z values indicated by black arrows, showing a new isotopic envelope after reaction (B), corresponding to cDIM8+, characterized by a smaller m/z and drift time (7.17 ms) longer than DIM8+/MON4+ envelope at 6.28 ms. (C,D) 1236–1240 m/z region, showing signals of the two structurally alternate (at 7.61 and 8.16 ms) DIM7+ forms before reaction (C), after incubation (D) accompanied by a strong new signal of cDIM7+ at smaller m/z and shorter drift time (6.95 ms). (E,F) 1442–1446 m/z region with MON3+ and DIM6+ signals present. The two drift time profiles are collected at m/z values indicated by black arrows. (G,H) 1731–1735 m/z region before reaction (G) showing signals of at least four structurally alternate DIM5+ forms, with no new forms after incubation (H) but with equilibrium shifted towards the most compact of the four forms present before reaction. Experiment was repeated at least three times. See also Figure S2.
Figure 5.
Fragments of IMS-MS spectra for different charged trimer.
Selected regions of ion mobility separated mass spectra (IMS-MS) covering trimeric signals in 2D rendition (colored panels, drift time at horizontal axis and m/z at vertical axis). Lower panels show the corresponding ion mobility drift time profiles, i.e. projections of the signal group on the drift time axis. Panels showing isotopic envelopes are cross-sections at indicated (blue arrows) drift time values which correspond to a given oligomeric form, for instance TRI8+ denoting a non-covalently stabilized trimer charged 8+, whereas cTRI8+ a covalently stabilized trimer charged 8+, etc. Spectra are shown before reaction (A,C) and at 90 min of reaction (B,D). (A,B) 1854–1858 m/z region showing signals of the two structurally alternate (at 10.25 and 12.2 ms) TRI7+ forms before reaction (A), after incubation (B) accompanied by a strong new signals of cTRI7+ at smaller m/z and shorter drift time (8.36 and 7.6 ms), i.e., corresponding to covalently stabilized more compact trimeric form. (C,D) 1624–1626 m/z region with two structurally alternate non-covalently stabilized forms of 8+ charged trimer (at 10.25 and 11.03 ms) before reaction, dominated after incubation by new trimeric forms covalently stabilized by a di-Tyr (at 9.04 and 9.7 ms) and a tri-Tyr (at 7.6 ms) bond. Experiment was repeated at least three times. See also Figure S2.
Figure 6.
Collisional cross section (Ω) for dimer and trimer.
Dependence of the collisional cross-section (Ω) of major dimeric (A) and trimeric (B) forms of Aβ obtained from the analysis of drift time profiles in IMS-MS spectra on the charge of the molecule before reaction (open triangles) and after di−/tri-Tyr crosslinking (solid triangles).
Figure 7.
Molecular models of cross-link Aβ oligomer.
Molecular models of the compact form of Aβ hexamer cross-linked by di- (A) or tri-Tyr (B) bond.
Figure 8.
Oligomer evolution scheme showing an on-pathway scenario, leading to fibrils, and off-pathway scenario.