A phosphopeptide enrichment strategy for investigating phosphorylation in Alzheimer’s disease and related tauopathies Open Access
Kearney, Masin (Spring 2025)
Abstract
Alzheimer’s disease (AD) is a tauopathy characterized by cognitive decline and neuropathological changes driven by the aggregation of extracellular amyloid-beta plaques and intracellular tau-containing neurofibrillary tangles (NFTs). Tau is hyperphosphorylated prior to NFT formation, suggesting that abnormal phosphorylation contributes to neurotoxicity by altering protein structure and function. In addition to tau hyperphosphorylation, global phosphorylation-dependent signaling cascades are implicated in the pathogenesis of tauopathies. Other tauopathies that share tau pathology with AD include frontotemporal dementia with parkinsonism (FTDP) and progressive supranuclear palsy (PSP). However, overlapping clinical and neuropathological features among these diseases complicate differential diagnosis, and the absence of effective treatments underscores the need for therapeutic development. Phosphoproteomics enables the enrichment and identification of proteins within these signaling pathways, providing insight into potential biomarkers and therapeutic targets. However, studying phosphoproteins is challenging due to their low abundance and the presence of non-phosphorylated isoforms. To address this, immobilized metal affinity chromatography (IMAC) followed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to investigate the phosphoproteome. Brain lysates from AD, FTDP, PSP, and non-demented control frontal cortex brain tissue samples were digested and subjected to phosphopeptide enrichment using Fe-NTA magnetic beads, followed by data-dependent acquisition LC-MS/MS analysis. Raw MS data were processed with FragPipe, missing values were imputed, and statistical tests were performed to identify differentially enriched proteins and gene ontology categories. Consistent with previous studies, microtubule-associated protein tau (MAPT) was more highly phosphorylated in AD brain tissue than in FTDP, PSP, or control samples. Future work will scale and automate phosphopeptide enrichment from brain tissue and additional biofluids, such as cerebrospinal fluid (CSF) and plasma, to further explore AD biomarkers and underlying mechanisms. A more comprehensive understanding of biomarkers across brain, CSF, and plasma may enable non-invasive diagnostics and uncover novel therapeutic avenues for tauopathies.
Table of Contents
Chapter 1: Introduction ............................................................................................... 2
1.1 Phosphorylation ........................................................................................................... 2
1.2 Phosphorylation in Disease .......................................................................................... 3
Cancer ................................................................................................................................................... 3
Neurodegeneration ................................................................................................................................ 4
1.3 Tauopathies ................................................................................................................. 4
Tau ......................................................................................................................................................... 4
Tau in Disease ........................................................................................................................................ 5
Primary Age-Related Tauopathy (PART) .................................................................................................. 6
1.4 Alzheimer’s Disease (AD) .............................................................................................. 7
Overview ................................................................................................................................................ 7
Clinical Symptoms ................................................................................................................................. 7
Neuropathology ..................................................................................................................................... 8
Diagnosis ............................................................................................................................................... 9
1.5 Frontotemporal Dementia with Parkinsonism Linked to Chromosome-17 (FTDP-17) .... 10
Overview .............................................................................................................................................. 10
Clinical Symptoms ............................................................................................................................... 11
Neuropathology ................................................................................................................................... 12
Diagnosis ............................................................................................................................................. 12
1.6 Progressive Supranuclear Palsy (PSP) ......................................................................... 12
Overview .............................................................................................................................................. 12
Clinical Symptoms ............................................................................................................................... 13
Neuropathology ................................................................................................................................... 13
Diagnosis ............................................................................................................................................. 14
1.7 Why study phosphorylation?....................................................................................... 14
Diagnosis ............................................................................................................................................. 15
Treatment ............................................................................................................................................ 16
1.8 How to study phosphorylation?................................................................................... 17
Immunoassays ..................................................................................................................................... 17
Proteomics........................................................................................................................................... 18
1.9 Hypothesis and Specific Aims ..................................................................................... 21
Hypothesis ........................................................................................................................................... 21
Aim 1: Establishing Viability of Fe-IMAC Phosphopeptide Enrichment for Brain Tissue Analysis ........... 23
Aim 2: Enhancing Fe-IMAC Sensitivity and Reproducibility for Phosphoproteomics.............................. 23
Aim 3: Comparative Phosphoproteomic Analysis of AD and Related Tauopathies ................................ 24
Chapter 2: Aim 1: Development of Fe-IMAC Phosphopeptide Enrichment for Brain ... 25
2.1 Methods ..................................................................................................................... 25
2.1.1 Brain Tissue Homogenization ...................................................................................................... 25
2.1.2 Protease Digestion ...................................................................................................................... 26
2.1.3 Fe-NTA Magnetic Bead Preparation ............................................................................................. 26
2.1.4 Phosphopeptide Enrichment ....................................................................................................... 27
2.1.5 Liquid Chromatography – Tandem Mass Spectrometry (LC-MS/MS) ............................................ 28
2.1.6 Database Search and Quantification ........................................................................................... 28
2.1.7 Differential Abundance, Ontological Enrichment, and Data Visualization .................................... 29
2.2 Results ....................................................................................................................... 30
Chapter 3: Aim 2: Enhancing Fe-IMAC Sensitivity and Reproducibility for Phosphoproteomics ................................................................................................. 33
3.1 Methods ..................................................................................................................... 33
3.1.1 Brain tissue homogenization........................................................................................................ 33
3.1.2 Protease Digestion ...................................................................................................................... 33
3.1.3 Fe-NTA Magnetic Bead Preparation ............................................................................................. 35
3.1.4 Phosphopeptide Enrichment ....................................................................................................... 36
3.1.5 Liquid Chromatography – Tandem Mass Spectrometry (LC-MS/MS) ............................................ 36
3.1.6 Database Search and Quantification ........................................................................................... 36
3.1.7 Differential Abundance, Ontological Enrichment, and Data Visualization .................................... 36
2.2 Results ....................................................................................................................... 37
Chapter 4: Aim 3: Comparative Phosphoproteomic Analysis of AD and Related Tauopathies .............................................................................................................. 40
4.1 Methods ..................................................................................................................... 40
4.1.1 Brain Tissue Homogenization ...................................................................................................... 40
4.1.2 Protease Digestion ...................................................................................................................... 41
4.1.3 Fe-NTA Magnetic Bead Preparation ............................................................................................. 41
4.1.4 Phosphopeptide Enrichment ....................................................................................................... 41
4.1.5 Liquid Chromatography – Tandem Mass Spectrometry (LC-MS/MS) ............................................ 42
4.1.6 Database Search and Quantification ........................................................................................... 42
4.1.7 Differential Abundance and Ontological Enrichment ................................................................... 42
4.2 Results ....................................................................................................................... 43
Chapter 5: Discussion ............................................................................................... 48
References ............................................................................................................... 59
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