Unraveling the Structure and Dynamics of Ac-PHF6-NH2 Tau Segment Oligomers

The aggregation of the proteins tau and amyloid-β is a salient feature of Alzheimer’s disease, the most common form of neurodegenerative disorders. Upon aggregation, proteins transition from their soluble, monomeric, and functional state into insoluble, fibrillar deposits through a complex process involving a variety of intermediate species of different morphologies, including monomers, toxic oligomers, and insoluble fibrils. To control and direct peptide aggregation, a complete characterization of all species present and an understanding of the molecular processes along the aggregation pathway are essential. However, this is extremely challenging due to the transient nature of oligomers and the complexity of the reaction networks. Therefore, we have employed a combined approach that allows us to probe the structure and kinetics of oligomeric species, following them over time as they form fibrillar structures. Targeting the tau protein peptide segment Ac-PHF6-NH2, which is crucial for the aggregation of the full protein, soft nano-electrospray ionization combined with ion mobility mass spectrometry has been employed to study the kinetics of heparin-induced intact oligomer formation. The oligomers are identified and characterized using high-resolution ion mobility mass spectrometry, demonstrating that the addition of heparin does not alter the structure of the oligomeric species. The kinetics of fibril formation is monitored through a Thioflavin T fluorescence assay. Global fitting of the kinetic data indicates that secondary nucleation plays a key role in the aggregation of the Ac-PHF6-NH2 tau segment, while the primary nucleation rate is greatly accelerated by heparin.


Figure
Figure S3.CD spectrum of Ac-PHF6-NH2 pep�de 100 μM in 10 mM AA with 1.5 μM heparin and 20 μM ThT taken a�er incuba�on at room temperature a�er 33 days.

Figure
Figure S4.(A)Averaged mass spectra without heparin measured immediately a�er the sample prepara�on (blue) and a�er 24 hours (pink).(B) Averaged mass spectra with addi�on of heparin (final concentra�on 1.5 µM) measured immediately a�er addi�on of heparin (blue) and a�er 24 hours (pink).The main peak present is the singly charged monomer (m/z 790.5) denoted as 1 1+ (n z+ ), where n is number of monomers and z is the charge state.The asterisk corresponds to the TuningMix calibrant of m/z 922.

Figure
Figure S5.(A) Quadrupole-selected total ion mobility spectrum of m/z 1580 of 20 µM of Ac-PHF6-NH2 pep�de in 10 mM AA with 1.5 µM heparin measured at 24 hours a�er heparin addi�on (shown in dark red, top).The oligomers are labeled as [2n] nz+ , where n is the number of monomers and z is the charge state.Quadrupole-selected total ion mobility spectrum of m/z 1580 of 50 µM of Ac-PHF6-NH2 pep�de in 10 mM AA, measured by us previously 1 (shown in black, botom).The dashed gray lines show the posi�on of the 10 5+ and 12 6+ oligomers previously assigned.The two le� peaks from the upper spectrum overlap with these lines.(B) Extracted mass spectra from the ion mobility peaks corresponding to the oligomers showing the isotopic patern.The spacing between the peaks indicates the triply-charged species 6 3+ and quadruply-charged oligomer 8 4+ , respec�vely.

Figure S6 .
Figure S6.Collision cross sec�on (CCS) values versus oligomer number n (shown in black), where n is the number of monomer units in the oligomer, for the Ac-PHF6-NH2 pep�de in 10 mM AA with 1.5 µM heparin.The green line corresponds to the isotropic growth 2 , where the CCS values scale as σ1•n 2/3 (n is the number of monomers in the oligomer and σ1 is the CCS value of the monomer).The red line corresponds to the stacked fibrillar growth 2 in one direc�on, where the CCS values has the following linear dependence on n: σ1 = 120•n + 275.Here, the slope a = 120 is the difference of CCS values between dimer (2 1+ ) and monomer (1 1+ ), and the intercept k = 275 is the CCS value of monomer (1 1+ ).

Figure S7 .
Figure S7.Normalized intensi�es of fibrils (green) and oligomers (pink) of 20 µM of Ac-PHF6-NH2 pep�de in 10 mM AA with 1.5 µM heparin over �me.The pink asterisk indicates the highest oligomer intensity.The solid green line shows the con�nuing growth of fibrillar species.

Table S1 .
Comparison of the CCS values of Ac-PHF6-NH2 pep�de measured on TIMS-Qq-ToF.
*The CCS values in blue indicate the oligomers, which were assigned to two conformers in the previous work.In the current work, the signal was too noisy to dis�nguish between conformers, therefore the closest CCS value from the previous work is compared to the current CCS value.

Table S2 .
Instrumental parameters on TIMS for IM-MS measurements.