Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry

Abstract

The key to understanding amyloid disease is the characterization of oligomeric species formed during the early stages of fibril assembly. Here we have used electrospray ionisation-ion mobility spectrometry-mass spectrometry to identify and structurally characterize the oligomers formed during amyloid assembly from beta(2)-microglobulin (beta(2)m). Beta(2)m oligomers are shown to have collision cross-sections consistent with monomeric units arranged in elongated assemblies prior to fibril formation. Direct observation, separation, and quantification of transient oligomeric species reveals that monomers to tetramers are populated through the lag phase with no evidence for the significant population of larger oligomeric species under the conditions employed. The dynamics of each oligomeric species were monitored directly within the ensemble at concentrations commensurate with amyloid formation by observing the subunit exchange of (14)N- and (15)N-labeled oligomers. Analysis of the data revealed a decrease in oligomer dynamics concomitant with increasing oligomer size and the copopulation of dynamic dimeric and trimeric species with more stable trimeric and tetrameric species. The results presented map the events occurring during the lag phase of fibril formation and give a clear insight into the structural characteristics and dynamic nature of the beta(2)m oligomers, demonstrating the existence of elongated assemblies arising from an intact amyloidogenic protein during fibril formation.

Fig. 1.

ESI-IMS-MS data for β₂m (pH 2.5) one minute into the lag time of fibril assembly. M-monomer, D-dimer, T-trimer, and Q-tetramer; the number adjacent to each letter refers to the charge state of those ions. The scaling is adjusted to “square root” or “log” as appropriate to display the weaker ions clearly. The summed m/z spectrum is shown on the right.

Fig. 2.

Average collision cross-sections (Ω) over all charge states measured by ESI-IMS-MS for β₂m monomer and oligomers, and a range of protein standards. Error bars are one standard deviation in Ω between charge states of a protein. Black circles represent the average Ω for each β₂m species at pH 2.5: β₂m-N is compact β₂m monomer, β₂m-P partially folded, and β₂m-A acid-unfolded; three conformers copopulated at pH 2.5. β₂m-red is acid-unfolded, reduced β₂m at pH 2.5. D = dimer, T = trimer and Q = tetramer. Open triangles represent the Ωs of proteins analyzed at pH 6.5 (Table S1). Proteins of note: β₂m(S55C)-dimer is a covalent dimer of two natively-folded β₂m monomers bridged by a mutationally introduced intermolecular disulphide bond; α-synuclein is a natively-unfolded protein; transthyretin tetramer (TTR) and human leukocyte antigen complex I (HLA-I) are globular proteins of similar mass to the β₂m tetramer; I27₅ is a linear polymer of five I27 domains; ((I27-pL^WT)₃I27) is a linear polymer of four I27 domains and three protein-L domains arranged alternately. Ribbon structures of native β₂m at pH 6.5, HLA-I (3HLA), TTR (1ICT), and I27₅ (1TIT modified) are shown. Expected Ωs were calculated for a range molecular weights (MW) assuming a perfect sphere of density 0.44 Da/ų (—) (27).

Fig. 3.

(A) ¹⁴N- and ¹⁵N-sub-unit exchange of the +10 trimer and +11 tetramer ions. Samples of ¹⁴N- and ¹⁵N-labeled preformed β₂m oligomers at 20 °C were mixed together in solution in a 1∶1 ratio before ESI-MS analysis. m/z spectra at t = 40 sec and t = 320 min after mixing are shown. Black dashed lines show the ¹⁴N- or ¹⁵N-only β₂m oligomers, red dashed lines show the ¹⁴N/¹⁵N mixed β₂m oligomers. (B) Oligomer exchange dynamics. Individual peak areas were calculated and normalized to total signal at each time point. The data were summed so that the peak area for the reactants (¹⁴N- and ¹⁵N-only β₂m oligomers; black), and the products (¹⁴N/¹⁵N mixed β₂m oligomers; red), could be compared. The resulting datasets were fitted to a double exponential decay (solid line). (Fig. S5 for further details).

Fig. 4.

Subunit exchange investigated in oligomers formed early in fibrillogenesis initiated by urea exchange. ¹⁴N- and ¹⁵N-labeled β₂m were each unfolded in 8 M urea and pH 2.5 fibrillogenesis initiated by urea exchange. The solutions were incubated for 1, 30, and 60 min prior to mixing (1∶1 v/v) and immediate ESI-MS analysis. 100% ¹⁴N-β₂m and 100% ¹⁵N-β₂m are shown in red and blue, respectively. The mixed ¹⁴N/¹⁵N signals are shown in black. Both sets of ions undergo rapid subunit exchange at the earliest time point, resulting in oligomers with mixed labels. After 30 min, the +11 tetramer and +8 trimer ions are less dynamic, as depicted by the lower intensity of oligomers with mixed labels and the higher intensity of all ¹⁴N- and all ¹⁵N-labeled β₂m peaks.

Fig. 5.

Model of the events occurring during the onset of β₂m fibril formation under acidic conditions (pH 2.5, 100 mM ammonium formate). Monomeric β₂m copopulates three distinct states at pH 2.5: native-like compact, partially folded, and acid-unfolded ( and 15); oligomer formation occurs in < 2 min and results in the detection of monomers to tetramers in the lag phase. The dimer (red) is a highly dynamic species with Ω only 5% larger than that of the acid-unfolded monomer. Further association proceeds by stacking monomeric subunits with Ωs consistent with an elongated structure. Initial association of the monomer to the dimer results in a dynamic trimer that undergoes structural change giving rise to a longer-lived, less dynamic species (green). The tetramer (blue) evolves in a similar manner, first forming with a dynamic structure that subsequently becomes more stable as revealed by biphasic oligomer exchange kinetics.