New insights into the metal-induced oxidative degradation pathways of transthyretin

Abstract

The amyloidogenic mechanism of transthyretin is still debated but understanding it fully could lend insight into disease progression and potential therapeutics. Transthyretin was investigated revealing a metal-induced (Cr/Cu) oxidation pathway leading to N-terminal backbone fragmentation and oligomer formation; previously hidden details were revealed only by FT-IM-Orbitrap MS and surface-induced dissociation.

Figure 1.

Time evolution of TTR under ambient nano-ESI conditions showing the sequential addition of 64 Da corresponding to either one zinc or four oxidations. At t = 0 min, the peaks to the right of the apo peak correspond to Zn(II) binding; however, at later times, the mass shifts are attributed to Cys-10 oxidation. The red peaks correspond to the backbone fragmentation of TTR between Cys-10 and Pro-11 which occurs simultaneously with TTR oxidation.

Figure 2.

(A) ATDs for the [TTR + 14H⁺ + n*64 Da]¹⁴⁺ ions (where n = 0–4) directly after loading (black) and after 20 hours of continuous analysis by a theta emitter (red). ATDs are the average of 8 IM-MS runs. (B) Deconvoluted ATDs of the incubated sample showing two additional conformations populated by TTR after 20 hours.

Figure 3.

(A) Mass spectra of TTR tetramer as a function of time collected on a Waters Synapt G2 equipped with a custom SID cell. (B) The SID products of the [TTR + 15H⁺]¹⁵⁺ tetramer. Monomers, dimers, and b10/y117 fragments are observed. The b10 fragment is doubly oxidized. (C) A magnified look at the [M + 4H⁺]⁴⁺ monomer of TTR reveals a time dependent oxidation of TTR monomer as well as Zn(II) and 2-mercaptoethanol (ME) binding. [TTR + 15H⁺]¹⁵⁺ retains the native conformation (Figure S1) and is used for SID to retain higher ion abundances.

Figure 4.

The mass spectra of [TTR + 14 H⁺ + 4 NEM]¹⁴⁺ at 0 and 360 minutes. Equivalent backbone fragmentation occurs at 360 minutes compared to 64 minutes for TTR with Zn(II) and no NEM (Figure 1). The dotted line represents the m/z for apo-TTR.

Figure 5.

[TTR + 8H⁺]⁸⁺ monomer fragment obtained directly after the addition of equimolar Cr. This addition of Cr in solution immediately induces oxidation without the need for an applied potential.