Driving Therapeutic Innovation in Neurodegenerative Disease with Hydrogen Deuterium eXchange Mass Spectrometry

Global Statistics

• Dementia: An estimated 50 million people globally have dementia, with projections to reach 139 million by 2050.
• Parkinson’s Disease: Affects 7-8 million people worldwide.
• Multiple Sclerosis: Believed to affect over 1.8 million individuals globally.
• Motor Neuron Disease (ALS): Global estimates range from 1.9 to 6 cases per 100,000 people.
• Overall Neurological Conditions: The World Health Organization reports that over 1 in 3 people are affected by neurological conditions.
• UK: Over 1 million people in the UK live with neurodegenerative diseases.
• Leading Cause of Disability: Neurological conditions are the leading cause of illness and disability worldwide. 

The most prevalent and well-known human neurodegenerative diseases include Alzheimer’s Disease, Parkinson’s Diseases, Amyotrophic Lateral Sclerosis, Multiple Sclerosis and Huntington’s Disease. Many of these diseases are characterised by dysregulation at the protein level, including the development of toxic protein aggregates which can detrimentally impact brain physiology.

Proteomics is the study of proteins in biological systems and therefore lends itself perfectly to the study of protein dysregulation and degradation. To truly understand the mechanisms of neurodegenerative disease, structural and regulatory processes of molecular proteins must be comprehensively recognised. Proteomics has seen recent advances in technology which has led to the development of novel drugs for personalised medicine administration. Additionally, proteomics has assisted in Biomarker discovery, whereby protein species can be identified and subsequently tracked leading to earlier diagnosis and evaluation of treatment response.

Several structural proteins have been isolated as the key attribute with regards to neurodegenerative disease. Alpha-synuclein in Parkinson’s, tau in Alzheimer’s, and TAR DNA-binding protein 43 (TDP-43) in ALS. These proteins are involved in a diverse range of cellular functions including the regulation of neurotransmitter release and RNA splicing; and as a result, are susceptible to aggregation cascades of protein misfolding, organisation and accumulation. It is currently under debate as to whether there is a direct causal link between protein aggregates and neuronal dysfunction. However, limiting protein misfolding is considered to be a key goal in the development of novel therapeutic interventions across all neurogenerative disease.

Over the last few decades, Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) has emerged as an exceptionally powerful tool in understanding the dynamic processes that contribute to pathological changes in neuropathy. This technique has an inherent ability to measure protein structural disorder whilst simultaneously capturing transient structural conformations; something a comparable technique such as X-ray Crystallography is unable to encapsulate. HDX-MS is based on the concept that backbone amide hydrogens exchange with deuterons when exposed to deuterium oxide (D₂O). The additional mass, as a result of this process, can subsequently be measured in a Mass Spectrometer.  This isotopic exchange between protons and deuterons can be influenced by a number of factors; including pH, temperature, ionic strength and the physical structure of the protein itself.

A standard HDX-MS work flow can be divided into several steps:

1. Proteins are exposed to D₂O over a set period of time to facilitate hydrogen-deuterium exchange.
2. Exchange reaction is quenched by rapidly reducing pH to ~2.5 and temperature to 0°C. This prevents the back-exchange of deuterium to hydrogen.
3. Denaturing agents such as guanidinium and reducing agents such as TCEP (Tris(2-carboxyethyl)phosphine) may be used to reduce disulphide bridges and facilitate the protein unfolding.
4. Proteins are digested with an immobilised acid-stable protease such as pepsin, generating overlapping peptides and providing resolution of HDX behaviours.
5. Peptides are then separated via liquid chromatography (LC) and identified and analysed by tandem MS.

Deuterium uptake data can then be extracted and mapped onto the protein sequence. Subsequent analysis involves comparing uptake patterns under different conditions in order to deduce structural and dynamic changes. As demonstrated in the above diagram, HDX-MS can be divided into two distinct processes depending on the desired interests. Continuous labelling, whereby proteins are exposed to deuterated solvents for varying incubation periods. This permits the progressive exchange of amide hydrogens and are useful for studying overall protein dynamics, as well as the effect of mutations on its flexibility and binding. Pulsed Labelling is ideal for studying dynamic processes such as protein folding, aggregation or stressor-induced conformational shifts. In this instance, protein samples are exposed to denaturants, temperature or aggregation-inducing factors and finally a brief exposure to a deuterated solvent. This results in the ability to capture transient intermediates or rapid conformational changes, typically associated with alpha-synuclein or amyloid aggregation.

Parkinson’s Disease

Parkinson’s Disease (PD) is a largely sporadic and multifactorial neurodegenerative disease resulting in problems with both motor and non-motor function. Impaired motor function relates to the archetypical tremor, muscle rigidity and bradykinesia (slow movement). As the disease advances, symptoms involving non motor function becomes more apparent. Typically this can include worsening of cognition, behavioural changes and psychosis. Although the underlying pathomechanisms of Parkinson’s is still poorly understood a consistent abnormality of intracellular assembly of alpha-synuclein protein and it’s aggregation within neurons ultimately leads to neuronal degeneration. Alpha-synuclein is a monomeric, negatively charged intrinsically disordered protein (IDP) of 140 amino acids in length, whose precise molecular function is still unclear. Several studies have used HDX-MS to monitor the self-assembly, oligomerisation and misfolding of alpha-synuclein monomers into higher-order neurotoxic soluble oligomers and their intermediates. This structural transition underlies a toxic gain-of-function mechanism, a better understanding of which could provide an attractive target for therapeutic intervention in Parkinson’s Disease. A secondary target that has garnered significant attention in the development of novel neurotherapeutics is the large, multidomain Roco protein Leucine-Rich Repeat Kinase 2 (LRRK2). One of the strongest genetic risk factors involves mutations in the LRRK2 gene, resulting in increased susceptibility to both familial and sporadic Parkinson’s Disease. Mutations likely drive PD pathogenesis via enhanced kinase activity and hyperphosphorylation of substrate proteins. LRRK2 is primarily located in the cytoplasm but can also be found on the mitochondrial outer membrane. As such, the protein has been implicated in neurodegeneration through a multitude of pathways and extensive effort has been put into developing inhibitors of LRRK2 kinase activity. Thirdly, Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a member of the ubiquitin–proteasome system and is estimated that it comprises up to 5% of total soluble brain protein. The genetic association between UCH-L1 and neurodegeneration is subject to much debate, with one study identifying a point mutation in the UCHL1 gene as a contributing factor to Parkinson’s Disease, while conversely a polymorphism at S18Y confers a reduced susceptibility to the condition. Furthermore, UCHL1 mutations have also been associated with ALS. Regardless, UCHL1 remains a prime target for therapeutic development in PD and ALS. Finally, DJ1, also known as Parkinson disease protein 7, is a deglycase which has been proposed to be neuroprotective by sensing and allowing cells to respond to Reactive Oxygen Species, and is essential for the maintenance of healthy mitochondria. The protein can inhibit the aggregation of alpha-synuclein, and mutations result in an increased susceptibility to Parkinson’s Disease. A recent study utilized HDX-MS to decipher the mechanistics of enhanced enzyme activity through the interaction of dimeric DJ1 with two separate activating peptides. The dynamic HDX-MS information revealed unique peptide binding mechanisms, which facilitated improved substrate binding and enhanced enzyme activity.

Alzeimer’s Disease

Alzheimer’s Disease is the most common form of dementia, which refers to a family of conditions which display a progressive decline in memory, cognition and language ability. Pathogenesis is complex and caused by the concerted action of several factors including age, lifestyle and environment. The disease also has a genetic component. Alzheimer’s manifests sporadically, usually affecting those over the age of 65. Alzheimer’s is principally characterised by the accumulation and deposition of toxic versions of two proteins. The aggregation and deposition of these two proteins disrupt normal synaptic functioning, ultimately resulting in neuronal degeneration and dementia. Amyloid-beta is the principal component of the amyloid plaques that are found in the brain tissue of Alzeimer’s patients. It has long been considered one of the key drivers in the progression of the disease. Of the two amyloid-beta species, it is unclear which represents its true pathological form, and as such, no fully effective therapies targeting the protein currently exist. Amyloid-beta can assemble and dissemble, and form into large, insoluble amyloid fibrils, which have inherent neurotoxicity and form the basis of amyloid plaques. It has been extremely difficult to generate meaningful structural data by traditional methods such X-ray crystallography. As HDX-MS is not limited by crystallisation trials, it may represent an excellent alternative to assess the structural and conformational dynamics of the protein. Microtubule-associated protein tau is another key player in Alzheimer’s Disease. The protein provides stability and support to microtubules, aiding in their assembly. One of the primary hallmarks of AD and similar tauopathies is the pathophysiological deposition of hyperphosphorylated, aggregated tau in the brain, resulting in aberrant synaptic communication and neuronal loss.

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), which is also commonly referred to as motor neuron disease is a relatively rare, progressive and fatal disorder which leads to neurodegeneration of upper and lower motor neurons which oversee voluntary muscle contraction, eventually leading to muscle weakness and wasting. As of 2025, there are three drugs available to treat ALS, riluzole, radicava, and tofersen, with only marginal or conflicting benefits observed. The primary disease-associated protein in ALS is TDP-43, aggregation of which can be prevented by RNA binding. HDX-MS was therefore recently employed to provide a detailed picture of the structural interplay between RNA and TDP-43 complexes.

Spinocerebellar Ataxia Type 1

Spinocerebellar ataxia type 1 (SCA1) is a rare neurodegenerative disorder, primarily a result of an extended repeat sequence in a specific gene, which encodes the protein ataxin-1. The disease is an inherited, autosomal dominant condition. Which leads to a progressive decline in motor function, gait and balance. No disease-modifying treatments are currently available, but several therapies are in development. HDX-MS was elegantly employed to assess the structural interaction of ataxin-1 with adaptor proteins, which indirectly contribute to ataxin-1 stability. HDX-MS pinpointed a region of ataxin-1 which likely interacts or causes a conformation rearrangement or shielding effect, which ultimately prevents complex formation.

Full Article

Driving Therapeutic Innovation in Neurodegenerative Disease with Hydrogen Deuterium eXchange Mass Spectrometry

Andrea Pierangelini, Benedikt M. Kessler, Darragh P. O’Brien

Molecular & Cellular Proteomics | Articles in Press | 101017 | June 2025