Characterizing Early Aggregates Formed by an Amyloidogenic Peptide by Mass Spectrometry

Abstract

Self-assembly of peptides and proteins into the ordered fibrillar aggregates known as amyloid fibrils has potential for achieving control over molecular-level architecture and macroscopic properties. Polypeptide fibrils have been suggested as templates for ordering inorganic components in nanoassemblies, [1] or as the basis for biomaterials. [2][3][4] The ordered self-assembly of proteins and polypeptides is also of interest in the context of the protein misfolding disorders, including Alzheimers disease and type 2 diabetes. The vast majority of systems that form amyloid-like fibrils assemble by a nucleation and growth mechanism [5] whereby the formation of a multimeric nucleus of polypeptide molecules drives selfassembly. The considerable experimental difficulties associated with observing a small and potentially rare aggregate or aggregates in a soup of molecules have impeded characterization of these triggers to growth.Herein we probe by time-course mass spectrometry (MS) and ion-mobility mass spectrometry (IM-MS) the early aggregation states of an amyloidogenic endecapeptide (sequence YTIAALLSPYS) derived from amino acid residues 105-115 of the human plasma protein transthyretin (TTR 105-115). The mobility K of an ion (and its corresponding reduced mobility K 0 ) is obtained from the arrivaltime distribution (ATD) of a given ion after it has passed through a drift cell filled with a buffer gas under the influence of a weak electric field. K is inversely related to the orientationally averaged collision cross-section W by a modified version of Equation (1):where z is ion charge, e is electron charge, N 0 the buffer gas number density, m the reduced mass of the buffer gas and ion, k B the Boltzmann constant, T the effective temperature, and W is the momentum transfer collision integral.The use of MS methods, and in particular IM-MS, can provide molecular detail on the early stages of aggregation of amyloidogenic peptides and proteins. A large body of work assessing the configurations of species formed during polypeptide aggregation has utilized MS techniques: for example, Bowers and co-workers have examined the self-assembly of Ab, [6] a-synuclein, [7] IAPP, [8] and most recently a prion protein fragment.[9] The unique benefit of this approach is illustrated by a study of early aggregate species formed by the Alzheimers-related peptide Ab1-42 and the non-amyloidogenic variant Ab1-42 F19P (by Bernstein and co-workers [10] ). Resolved species in an ATD following nESI-MS of Ab1-42 were assigned to dimer, tetramer, hexamer, and dodecamer (n = 1, 2, 3, and 6 respectively) whereas the F19P mutant contained only dimer and tetramer. Furthermore, the absence of aggregates intermediate between putative hexamers and dodecamers led the authors to speculate that the hexamer was the building block of protofibril formation, a finding corroborated by cross-linking studies. [11][12][13] Recent work by Ashcroft and co-workers [14] describes the conformation of b2m oligomers up to the tetramer, finding compact dimers and elongate...

Traveling‐wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross‐sections of human insulin oligomers

“…[27][28][29][30][31][32] For example, data on the structural features of early oligomeric intermediates associated with amyloid fibril formation have been very difficult to obtain using traditional structural biology techniques, [33] but IM-MS has been very successful in revealing details about the critical early stages of amyloid-b and other fibril-forming peptides. [34][35][36][37][38][39] Two different IM methods are commonly used. [23] Electrostatic drift-tubes have a constant electric field along the axis of ion transmission, and the absolute Ω can be determined directly based on the measured drift-time, gas pressure, temperature, and other known instrumental and physical parameters using the Mason-Schamp equation.…”

Traveling-wave ion mobility-mass spectrometry reveals additional mechanistic details in the stabilization of protein complex ions through tuned salt additives

“…It is now well established that MS can yield insights into the composition, stoichiometry and connectivity of heterogeneous multiprotein assemblies at relatively low concentrations (Aquilina et al 2003; Heck and van den Heuvel 2004; Hernandez et al 2006; Sharon and Robinson 2007; Zhou et al 2008; Hernandez et al 2009). When combined with IM, it becomes possible to separate species not only according to their mass-to-charge ratio (m/z) but also according to their ability to traverse an ion guide containing inert gas under the influence of a weak electric field, thus yielding ion size and shape information (Clemmer and Jarrold 1997; Wyttenbach and Bowers 2007; Kaddis et al 2007; Bohrer et al 2008; Scarff et al 2009; Ekeowa et al 2010; Uetrecht et al 2010; Knapman et al 2010; Smith et al 2010; Cole et al 2010). Traveling wave IM separations specifically, that utilize a series of low-voltage ‘waves’ to propel ions for such size-dependant separations, have enabled most of the modern applications of IM-MS to structural biology (Giles et al 2004; Scarff et al 2008; Zhong et al 2011; Giles et al 2011).…”

Adding a new separation dimension to MS and LC–MS: What is the utility of ion mobility spectrometry?

“…Oligomerization of IAPP, being considered as a pathogenic process in type II diabetes, has also been extensively investigated by IM-MS [213,214]. Time-course MS and IM-MS were reported to probe the early aggregation states of an amyloidogenic endecapeptide derived from amino acid residues 105-115 of the human plasma protein transthyretin [215]. Early Aβ oligomers, which are thought to be responsible for the neurodegeneration of AD patients, have also been analyzed using IM-MS [31].…”