Pulsed hydrogen/deuterium exchange mass spectrometry for time‐resolved membrane protein folding studies

Rempel

Proc. Natl. Acad. Sci. U.S.A.

et al. 2013

112

179

Probing the conformational changes of amyloid beta (Aβ) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aβ 42 peptide, the proposed causative agent in Alzheimer's disease. The approach incorporates pulsed hydrogen-deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen-deuterium exchange does not require modified Aβ 42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aβ 42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu 2+ ions. This MS approach has sufficient structural resolution to allow interrogation of Aβ aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.soluble Aβ oligomers | amyloid beta peptide | copper | electrospray ionization | Finke-Watsky mode P rotein aggregation is one of the immediate causes of Alzheimer's, Parkinson, and Huntington diseases, motivating biophysical studies of the responsible proteins. More than 20 small proteins undergo amyloidosis in humans. In Alzheimer's disease (AD), the aggregation of the 40-or 42-aa-long amyloid beta (Aβ) peptide, generally called Aβ 40 or Aβ 42 , respectively, is proposed to be involved in the onset of the disease (1, 2). Aβ 42 is more amyloidogenic and more neurotoxic than Aβ 40 . Although the amyloid-cascade hypothesis suggests that the Aβ-containing amyloid plaques are responsible for neurodegeneration (3-7), other studies suggest that soluble aggregates of Aβ 42 are more neurotoxic than the amyloid plaques (8-13).The amyloid plaques in AD-affected brains contain high levels of copper, zinc, and iron (14-20). Among these, Cu has drawn the most attention because the Aβ precursor protein is likely a Cuchaperone protein (21). Several studies of Cu 2+ -Aβ 40 interactions show that Cu 2+ can promote Aβ 40 aggregation (14,18,19).The structure of Aβ 42 and its aggregates, although studied extensively, remains of high interest. Studies of amyloid fibrils invoke X-ray crystallography (22-24), EM (19,25,26), and thioflavin T fluorescence (19, 27), revealing the polypeptide's global behavior, whereas NMR studies provide residue-level information for the fibrils (28-30). Nevertheless, we know little about soluble Aβ aggregates owing to their intrinsically high heterogeneity.MS should offer an opportunity for investigating soluble aggregates of Aβ 42 . Thus far, there are no MS-based, timedependent studies of the formation of soluble aggregates. Moreover, there are no ot...

mentioning

confidence: 99%

Pulsed hydrogen–deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aβ) peptide aggregation

Rempel

Proc. Natl. Acad. Sci. U.S.A.

et al. 2013

112

179

“…Mass spectrometry (MS) has sparked a resurgence, and in some cases a re‐learning, of the HX method. A growing number of studies incorporate HX‐MS into biophysical analyses of protein folding, protein‐ligand interactions, membrane protein dynamics and therapeutic antibody characterization . MS should open many new frontiers.…”

Section: Introductionmentioning

confidence: 99%

HX‐MS2 for high performance conformational analysis of complex protein states

et al. 2015

Water-mediated hydrogen exchange (HX) processes involving the protein main chain are sensitive to structural dynamics and molecular interactions. Measuring deuterium uptake in amide bonds provides information on conformational states, structural transitions and binding events. Increasingly, deuterium levels are measured by mass spectrometry (MS) from proteolytically generated peptide fragments of large molecular systems. However, this bottom-up method has limited spectral capacity and requires a burdensome manual validation exercise, both of which restrict analysis of protein systems to generally less than 150 kDa. In this study, we present a bottom-up HX-MS 2 method that improves peptide identification rates, localizes high-quality HX data and simplifies validation. The method combines a new peptide scoring algorithm (WUF, weighted unique fragment) with data-independent acquisition of peptide fragmentation data. Scoring incorporates the validation process and emphasizes identification accuracy. The HX-MS 2 method is illustrated using data from a conformational analysis of microtubules treated with dimeric kinesin MCAK. When compared to a conventional Mascot-driven HX-MS method, HX-MS 2 produces two-fold higher a/b-tubulin sequence depth at a peptide utilization rate of 74%. A Mascot approach delivers a utilization rate of 44%. The WUF score can be constrained by false utilization rate (FUR) calculations to return utilization values exceeding 90% without serious data loss, indicating that automated validation should be possible. The HX-MS 2 data confirm that N-terminal MCAK domains anchor kinesin force generation in kinesin-mediated depolymerization, while the C-terminal tails regulate MCAK-tubulin interactions.

Biochemical Society Transactions

“…Continuous labelling experiments provide information on the dynamics of the protein studied. A variety of other approaches can also be taken, including pulsed HDX experiments, which primarily are used to measure shortlived folding intermediates [35,37,45,46]. The most common application of HDX-MS has been in the determination of differential exchange rates of proteins upon varying stimuli ( Figure 1B).…”

Section: Hydrogen Deuterium Exchange Mass Spectrometry: Practicementioning

confidence: 98%

“…Of critical note these studies all used HDX-MS in combination with a variety of biophysical methods including X-ray crystallography, Cryo-EM and chemical cross-linking to fully define the molecular mechanism of regulation. A variety of novel strategies have been developed to study integral membrane systems using HDX-MS, including the use of protein membrane nanodiscs [21,91,92] and lipid bicelles [46,93]. The complementarity of HDX-MS with other structural techniques allows for the interrogation of molecular determinants regulating very larges protein complexes, including membrane-spanning and membraneinteracting assemblies.…”

Section: Studying Membrane Proteins By Hdx-msmentioning

confidence: 99%

Probing the dynamic regulation of peripheral membrane proteins using hydrogen deuterium exchange–MS (HDX–MS)

Vadas

Burke

2015

Many cellular signalling events are controlled by the selective recruitment of protein complexes to membranes. Determining the molecular basis for how lipid signalling complexes are recruited, assembled and regulated on specific membrane compartments has remained challenging due to the difficulty of working in conditions mimicking native biological membrane environments. Enzyme recruitment to membranes is controlled by a variety of regulatory mechanisms, including binding to specific lipid species, protein-protein interactions, membrane curvature, as well as post-translational modifications. A powerful tool to study the regulation of membrane signalling enzymes and complexes is hydrogen deuterium exchange-MS (HDX-MS), a technique that allows for the interrogation of protein dynamics upon membrane binding and recruitment. This review will highlight the theory and development of HDX-MS and its application to examine the molecular basis of lipid signalling enzymes, specifically the regulation and activation of phosphoinositide 3-kinases (PI3Ks).