Deciphering proteome alterations in neurodegeneration with mass spectrometry

Daniel Hornburg PhD, Vice-President Biomarkers & Precision Medicine, Bruker Daltonics, Stefan Foser, Vice President Global Pharma, Bruker Daltonics, and Torsten Müller, Business Development Manager, Proteomics, Bruker Daltonics, explore how MS-based proteomics can be used to dissect disease biology with spatial resolution. Impact of neurodegeneration for ageing societies Neurological conditions have become the leading cause of illness and disability worldwide, affecting more than 3 billion people, according to the Global Burden of Disease study supported by the World Health Organization.1 Among these, neurodegenerative diseases stand out for their progressive nature, lack of curative treatments, and growing prevalence in ageing populations. It is estimated that one-third of the population in industrialised countries will eventually develop a neurodegenerative disease like Alzheimer's.2 Beyond the profound toll on the individuals who experience progressive cognitive and motor decline, as well as their families and caregivers, these conditions also impose a rising and substantial socio-economic burden on healthcare systems. Lengthy disease courses and the need for long-term care amplify the issue. For Alzheimer’s in particular, its slow onset, progressive deterioration over decades, and lack of a cure underscore the critical need to deepen our understanding of the molecular cues that lead to neurodegeneration, with the goal of identifying effective therapeutic and preventative avenues. The central role of proteostasis in neurodegeneration To understand the molecular mechanisms underpinning neurodegenerative diseases, it is essential to examine protein homeostasis – the cellular processes responsible for maintaining protein balance. Protein homeostasis, or proteostasis, is the regulation and maintenance of the cellular functional protein environment (proteome), ensuring that proteins are correctly folded, modified, and translocated, as well as appropriately degraded when damaged or no longer needed. Disruption of proteostasis is a hallmark of neurodegenerative diseases leading to the accumulation of misfolded proteins or protein aggregates that impair cellular function and trigger neuronal death. For instance, Huntington's disease is characterised by the formulation of extended protein aggregates that cause widespread neuronal toxicity and severe clinical phenotypes. Similarly, ALS is associated with hundreds of genetic risk factors which converge on pathways that destabilise proteome integrity. A particularly aggressive form of ALS is associated with the genetic risk factor C9orf72, which has been reported to drive pathology through production of a di-peptide repeat that forms resilient aggregates, sequestering hundreds of different proteins and driving widespread proteostasis collapse.3,4 MS-based proteomics: a window into neurodegeneration Proteomics is a key technology for advancing our understanding of neurodegeneration because it allows for the comprehensive analysis of dynamics in protein abundance, modifications, interactions, and degradation pathways. A crucial aspect of this approach is the concept of proteoforms, which are the diverse structural and functional variants a single protein can take due to genetic variation, alternative splicing, and posttranslational modifications (PTMs). Investigating proteoforms provides a more nuanced understanding of protein function and dysfunction in neurodegenerative diseases. Mass spectrometry (MS) enables the sensitive and high-throughput identification and quantification of disease-relevant proteome alterations from model systems to human tissues. This level of resolution is crucial to provide critical insights into the molecular mechanisms underlying neurodegenerative diseases, from early-stage pathology to advanced disease. By capturing this holistic view of the proteome, including its diverse proteoforms, MS-based proteomics can facilitate the identification of potential therapeutic targets and biomarkers, while highlighting the biological pathways and complex biological processes that contribute to disease progression.5 Technological advances driving proteomic discovery in neurodegenerative research By enabling high-throughput, unbiased, and highly sensitive analysis of proteins, MS offers powerful insights into disease-relevant processes and biomarker development (see Figure 1). As MS technologies evolve in resolution, sensitivity, and throughput, they now span applications from single-cell analysis to population-scale biomarker discovery, and deep tissue profiling providing a systems-level understanding of neurodegenerative processes. Below, we highlight the key applications where MS-based proteomics is advancing our understanding of neuronal disorders and enabling novel therapeutic avenues: Probing Proteome Insolubility: Mass Spectrometry in the Analysis of Neurodegenerative Protein Aggregates Many neurodegenerative diseases are characterized by protein aggregation. MS-based proteomics can analyse the composition and structure of these aggregates, helping to understand their formation and impact on cellular function. Because these aggregates have been shown to be very resilient (hard to dissolve) and can harbor additional PTMs, targeted and epitope-based technologies struggle to dissect the complexity of these subcellular proteome agglomerates. MS does not rely on the detection of certain epitopes and can detect hundreds of thousands of individual peptides, including PTMs, even after dissolving resilient protein deposits under harsh denaturing conditions. Multiple studies investigated composition of protein aggregates associated with neurodegeneration, identifying hundreds of proteins sequestered into these insoluble cellular deposits.4,6,7 Besides the compositional analysis of these aggregates in relation to their neurotoxicity in the mechanism of action (MOA) studies, chemoproteomics geared to targeting problematic proteins for degradation may elucidate powerful therapeutic avenues dealing with toxic protein variants (discussed in “Targeting the Problem”). Mapping the Diseased Interactome: Protein-Protein Interaction Networks in Neurodegeneration Protein aggregates are a specific case of (mostly aberrant) protein-protein interactions. Since most proteins engage in stable or transient interactions, understanding not only protein aggregates but also the broader landscape of protein complexes is essential for elucidating physiological and pathological processes. MS-based proteomics can help to map protein interaction networks, revealing how disruptions in these networks contribute to neurodegenerative diseases. With the advent of high throughput proteomics, large-scale interactions studies have become feasible,8 achieving high enough sensitivity to detect even trace protein interactions in clinical isolates. Importantly, changes in interaction networks driven by PTMs or disease-associated mutations cannot be elucidated through genetic or targeted affinity approaches. In contrast, MS-based methods have proven particularly powerful in these contexts, such as in the study of hyperphosphorylated Tau, offering critical insights into disease-associated interactome remodeling.9 Similar approaches hold great promise for uncovering pathological interaction networks in other neurodegenerative diseases. Decoding Post-Translational Modifications: Molecular Fine Print of Neurodegenerative Pathology Neurodegenerative diseases often involve abnormal PTMs, such as phosphorylation, ubiquitination, and glycosylation. A prominent example is the above-mentioned hyperphosphorylated Tau in Parkinson’s Disease or the downstream processing of peptides in Alzheimer’s that leads to a variety of toxic peptide products.10 MS-based proteomics can detect and characterise these PTMs in contrast to targeted affinity-based methods from a variety of tissues and model systems, driving mechanistic understanding and biomarker discovery as well as shedding light on potential therapeutic targets. Developments in software and data acquisition methods (GlycoScape and GlycoPASEF Bruker, Billerica MA) could further reveal the role of less explored PTMs such as glycosylation in the spectrum of neurodegenerative disorders though large-scale bulk analysis in neuronal cell culture or tissue biopsies. Zooming into the Neuron: Single-Cell and Subcellular Proteomics for Cellular Heterogeneity Recent advancements in MS sensitivity allow for the analysis of proteomes at sub single-cell level, enabling comprehensive proteomics insights for the smallest cells, down to single bacteria and subcellular proteomes.11,12 This sensitivity is particularly useful for studying heterogeneous cell populations in the brain and understanding cell-specific contributions to neurodegenerative diseases, in particular from organoids or working with limited material. While isolating single neurons can be challenging given their complex and fragile morphology, the ability to detect thousands of proteins and tens of thousands of peptides from only a few picogram (less than a single HeLa cell) opens the opportunity to work with highly limited amounts of material e.g., from axonal cell regions. Spatial Proteomics in Context: Resolving Regional and Temporal Dynamics of Neurodegeneration Deep visual proteomics (DVP) approaches combine MS with imaging techniques to study the spatial distribution of proteins within tissues. DVP could drive understanding of the regional specificity of protein changes in the brain resolving distinct brain regions and cell types for their role in disease pathology. Here, laser capture microdissection (LCM) technologies along with unbiased, high sensitivity MS highlight new opportunities to study diseases at a sub tissue resolution.13 These technologies hold tremendous potential to resolve spatial and temporal disease phenotypes and progression in neurodegeneration from human tissues or organoids. Many neurodegenerative disorders affect specific areas of the brain, with prion-like spreading towards neighboring regions14 and intricate neuroinflammatory processes15 being proposed as key drivers of disease progression. Resolving spatial and temporal progress in model systems or post-mortem human tissue may reveal better insights into the pathological cascade. Immunopeptidomics and Neuroinflammation: Unraveling Immune Surveillance in the Degenerating Brain Immunopeptidomics, the study of peptides presented by major histocompatibility complex (MHC) molecules, has significant links to neurodegeneration through various mechanisms like immunogenicity. MS-based proteomics can directly survey cell surface decoration of immunopeptides and even monitor MHC shed into circulation.17 Moreover, recent innovation for tailored acquisitions, enable highly effective coverage of singly and multiply charged immunopeptides16,18, and de novo sequencing and PTM search strategies19 significantly enhance the capabilities of MS-based immunopeptidomics to study immune homeostasis. The proteasome which degrades intracellular proteins plays a key role in in both decoration of MHC as well as in dealing with aberrantly folded proteins, hence providing an intriguing yet underexplored connection between immunopeptidomics and neurodegeneration.20 In addition, neuroinflammation plays a key role in the progression of neurodegeneration. Peptides derived from aggregated or misfolded proteins could elicit adaptive immune responses, contributing to neuroinflammation. Protein-aggregates themselves may also be linked to inflammation mediated by brain-resident cells including microglia and astrocytes, leading to neuronal damage and brain atrophy. By studying the peptides presented by MHC, immunopeptidomics can provide valuable insights into the immunogenicity and inflammatory processes involved in neurodegenerative diseases, helping to identify potential therapeutic targets and improve our understanding of disease mechanisms. From Biomarkers to Risk Trajectories: Proteomics-Driven Discovery in Body Fluids Identifying biomarkers for early diagnosis and monitoring disease progression is crucial. MS-based proteomics can discover and validate biomarkers in body fluids like cerebrospinal fluid (CSF) and blood, facilitating early detection and supporting personalised treatment strategies. In addition, longitudinal studies from minimally invasive liquid biopsies can be exploited to track proteome changes over time in disease models or patient samples, providing insights into disease progression and the effects of therapeutic interventions.21 Many neurodegenerative diseases develop for years if not decades at the molecular level before the onset of any symptoms. It could therefore be assumed that early risk trajectories are key for interventions. With increased throughout enabled by speed and robustness in MS-based proteomics for example with the introduction of trapped ion mobility spectrometry combined with a time-of-flight mass spectrometer (timsTOF),22,23 in conjunction with deep workflows using particle technologies,24,25,26, MS is ideally suited to deliver high-quality population scale access and discovery of novel circulating biomarkers. Targeting the Problem: Chemoproteomics and Protein Degraders in Neurodegenerative Therapeutics Chemoproteomics and targeted protein degraders offer promising avenues for understanding and treating neurodegenerative diseases 27. Benefiting from robust, quantitative, and deep insights into a cell’s protein repertoire across various model systems, MS has enabled unbiased chemoproteomics at scale.28,29 Chemoproteomics involves the use of chemical probes to study protein interactions and functions within the cellular environment, providing insights into the molecular mechanisms underlying neurodegeneration. In addition, those modifications could change the physicochemical properties of proteins sufficiently to potentially affect their aggregation/ disaggregation propensity, which may offer a novel therapeutic avenue. Targeted protein degraders, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, leverage the ubiquitin-proteasome system to selectively degrade disease-associated proteins. This approach is particularly valuable in neurodegenerative diseases, where protein aggregates and misfolded proteins play a central role in pathology. By facilitating the removal of these harmful proteins, targeted protein degraders could mitigate neuronal damage and improve cellular function.30 The integration of these technologies holds great potential for advancing basic research in neurodegeneration and developing innovative therapeutic strategies. By leveraging these approaches, MS-based proteomics can significantly enhance our understanding of the molecular mechanisms underlying neurodegenerative diseases and aid in the development of effective therapies. Figure 1: A schematic highlighting key proteomics-driven strategies to dissect the molecular underpinnings of neurodegenerative diseases. Conclusion In conclusion, neurodegenerative diseases pose a significant challenge to an ageing society, with disrupted proteome homeostasis playing a central role in their pathology. MS-based proteomics offers a powerful suite of tools to investigate these complex disorders. By enabling the identification and quantification of proteins, characterisation of PTMs, analysis of protein aggregates, and mapping of protein-protein interactions, MS-based proteomics provides critical insights into the molecular mechanisms driving neurodegeneration. As we continue to integrate MS-based proteomics into the study of neurodegenerative diseases, we move closer to uncovering the fundamental proteomic changes that underpin these debilitating conditions, ultimately contributing to the development of novel diagnostic and therapeutic strategies. References 1 GBD 2021 Nervous System Disorders Collaborators. Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021. Lancet Neurol. 2024;23(4):344–381. doi:10.1016/S1474-4422(24)00038-3. 2 World Health Organization. Over 1 in 3 people affected by neurological conditions, the leading cause of illness and disability worldwide. 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