For the past two decades, HX-MS has been widely applied to characterize protein conformation. Its ability to directly study disordered and uncrystallizable aggregates (Nettleton et al., 2000
; Tobler and Fernandez, 2002
) makes it an appealing approach for analyzing Aβ oligomeric species. When studied in isolation, protofibrils, and fibrils of Aβ have all previously shown HX patterns distinct from monomer (Chen et al., 1997
; Jablonowska et al., 2004
; Kheterpal et al., 2000
; Kraus et al., 2003
; Wang et al., 2003
). However, purified fractions of each species were used for those studies, and mixtures were not investigated. Here, we have implemented a HX-MS technique that allows the simultaneous estimation of the solvent accessibility of each species, as well as the distribution of various structural species in an Aβ mixture. By making the HX-MS measurement directly on mixed, aged samples without pre-purification or use of stabilizing agents, there is greater assurance that Aβ structures and distributions obtained are representative of those present during in vitro toxicity studies. SEC has been be used to characterize soluble oligomer size distributions but do not provide structural information (Murphy, 2007
; Pallitto and Murphy, 2001
). Chemical modification methods such as PICUP (Bitan et al., 2003
) are an alternative, but chemical modification comes with the possibility of introducing a change in Aβ structure during the measurement. The HX-MS approach developed here can be applied to mixtures without purification or introduction of chemical agents.
It should be acknowledged that as implemented here, HX-MS alone does not provide detailed information about the state of folding (e.g., secondary or tertiary structure) or degree of oligomerization of intermediates. Further, it is not possible to distinguish such species with high resolution (e.g., dimers from trimers, etc.). However, we have shown that combined with complementary analysis, the identity of resolved components in distributions can be made, and a quantitative analysis of the results is possible. This is particularly important for the intermediates which are challenging to study by any method—both those observed in the 4 and 10 h samples, as well as in the more rapidly dissociating intermediates observed in freshly diluted Aβ samples.
For the freshly prepared samples of Aβ, the ratio of monomer to low molecular weight oligomer is approximately 1:1. This indicates that a fair amount of structured oligomer was formed promptly once diluted into PBS buffer. The HX-MS measurement itself does not indicate the size of the oligomer, but it must be sufficiently strongly associated both structurally and kinetically to exclude solvent (Paterson et al., 1990
). AFM image of fresh Aβ sample does not show many oligomers of significant size (AFM ), suggesting that if the protected species is an oligomer, it is small. Monomer, dimer and trimer, formed by Aβ(10–30) fragments in ammonium acetate pH 7.0, were reported to have similar, highly exposed (>90%) hydrogen exchange patterns after only 6 s (Jablonowska et al., 2004
). This longer time constant for protection in our case is likely due to additional stability provided by the additional residues. In particular, residues 31–36 are proposed to be important components of β-sheet structure of fibril by both proline mutagenesis (Williams et al., 2004
) and solid-state NMR (Petkova et al., 2004
). Therefore, the species formed immediately upon dissolution of Aβ(1–40) into PBS is likely a distinct oligomeric species from the low molecular weight oligomer formed in ammonium acetate by Aβ(10–30).
The hydrogen exchange kinetic data for the fresh Aβ samples like that shown in were analyzed using a model for the EX1 exchange kinetics regime, assuming dissociation of a dimer to monomer (see Supplementary Information
). While we have not established the protected species in is a dimer, this modeling approach provides a starting point for quantitative interpretation and comparison of the fresh sample HX-MS data with other studies (Fogle et al., 2006
). Triplicates collected for a range of labeling times were fitted well by the model (see Supplementary Information
). From the fit, a dissociation rate constant of 0.43 ± 0.08 (10−3
) was obtained. This value is of the same order as the value of 0.92 ± 0.13 (10−3
), obtained using for a similar model applied to surface plasmon resonance data for Aβ(1–40) under similar, although not identical conditions (Hu et al., 2006
). This dissociation time scale is also consistent with the ability to separate a similar low molecular weight oligomer from monomer in longer than 30 min by SEC (Pallitto and Murphy, 2001
). Moreover, in those studies, a comparable ratio of low molecular weight oligomer to monomer was observed in SEC chromatograms as we observe in the 10 s labeling time HX mass spectrum ().
Monomeric Aβ made up a much smaller fraction of the initial labeling distribution for the 4 and 10 h aged samples ( and ) compared to the freshly diluted Aβ samples. The relative abundance of the fully labeled Aβ peak increased only modestly with longer labeling times. This indicates a slow dissociation rate of the intermediate species to solvent exposed monomers with a time constant longer than our longest labeling time (1 h). For the 72 h aged sample (), similar behavior was observed. Our observation of no detectable fibril dissociation on 1 h time scale is consistent with previous reports of fibril dissociation occurring over much longer time scales (Cannon et al., 2004
; Hasegawa et al., 2002
). Similar behavior was also demonstrated for SH3 fibril (Carulla et al., 2005
For fibril formation, we have insufficient kinetic data to propose a detailed kinetic model. However, a highly protected species corresponding to washed fibrils () is clearly observed in the HX-MS spectra of 72 h aged samples () and that species can be isolated from lower the intermediates by centrifugation (). More importantly, as shown in both the HX-MS ( vs. 6) and AFM ( vs. C), the predominant subpopulation changed from intermediates (4 and 10 h) to fibril (72 h). This indicates that these are at least temporal precursors of fibril. Recently, it has been proposed that oligomers are off-pathway intermediates (Necula et al., 2007
). If so, under these conditions at least, they make up a significant fraction of material. Further, if they are to dissociate to more solvent exposed species before incorporation into fibrils, they do so with time constants much longer than our 1 h labeling times.
The proportion of intermediate present measured here is correlated with Aβ neurotoxicity reported in a previous study under the same aggregation conditions (Patel and Good, 2007
). In that study, SY5Y cells treated with 100 µM Aβ pre-incubated for 4 and 8 h showed much lower relative viability than cell cultures exposed to fresh and 72 h aged Aβ. The mass spectrometry determinations of oligomer subpopulations made here () showed that the intermediates are most abundant at 4 h, when toxicity was greatest. The AFM results further strengthened this correlation. In 4 h aged sample, the spherical species about 30–50 nm in diameter predominates (). These intermediate aggregates only account for a small fraction of total Aβ in both fresh and long time aged samples (72 h). A similar correlation between the concentration of intermediates and toxicity has also been found by other groups, though the intermediates exhibit different morphologies and are given different names, including low molecular weight oligomers, ADDLs, spherical aggregates, and protofibrils (Bucciantini et al., 2002
; Dahlgren et al., 2002
; Demuro et al., 2005
; Gong et al., 2003
; Kayed et al., 2003
; Kirkitadze et al., 2002
; Lambert et al., 1998
; Lee et al., 2007
; Lesne et al., 2006
; Walsh et al., 2002
In summary, for the first time, hydrogen exchange detected by mass spectrometry has been used to estimate distributions of aggregates in Aβ samples. Complementary measurements with AFM allowed assignment of distinct contributions in the distribution to monomer, very low molecular weight aggregates, larger intermediates, and fibrils. Freshly dissolved Aβ contains two components, monomer and low molecular weight oligomers in roughly equal amounts. This oligomeric species dissociates on the time scale of minutes. Aged samples were mixtures of monomer, intermediates and fibrils, the proportions of which varied with aging time. The intermediates formed in 10 h were larger (30–50 nm) and dissociated much more slowly than the very low molecular weight oligomers that formed immediately in freshly dissolved Aβ samples. These aggregation intermediates also had a measurably lower degree of solvent exposure than fibrils. The proportion of these intermediates in Aβ samples correlated with the neurotoxicity of the sample, suggesting that these intermediates are one of the most toxic Aβ species. Further residue level characterization of these toxic intermediates may aid in the molecular design of agents that prevent Aβ toxicity associated with Alzheimer’s disease. The HX-MS methods developed here may also be useful for characterizing the behavior of other complex aggregating systems such as designed self-assembling peptides and proteins with intermediate folded states prone to aggregation.