Identification of a Novel Phosphorylation Site on Alpha-Synuclein Within Erythrocyte Ghost Membranes Restricted; Files Only
Chiok, Joshua (Spring 2024)
Abstract
Background: Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder and is on the rise within the aging population of the United States. PD is typically detected during its advanced stages, when significant neuron degeneration has occurred. The challenge of diagnosing PD early is of the utmost importance, with significant implications for improving the overall quality of life of individuals living with the disease. A potential solution to achieving early PD diagnosis lies in the development of minimally invasive diagnostic biomarkers such as the detection of phosphorylated alpha-synuclein in RBCs utilizing mass spectrometry. The goal of this study is to discover novel phosphorylation sites in erythrocyte ghost membranes that can later be evaluated as potential biomarkers for diagnosing PD.
Methods: A pool of RBCs were obtained from The Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL). Erythrocyte ghost membranes were extracted from the AIBL pool of RBCs and then underwent phosphoenrichment via immobilized metal affinity chromatography using the Agilent AssayMap Bravo platform. After phosphoenrichment, samples were placed in a Bruker timsTOF fleX coupled to a Bruker nanoElute2 nanoHPLC. Data was acquired using a default data dependent acquisition (DDA) method on MS. Data was analyzed using Skyline and FragPipe software.
Results: 0.5% glycolic acid in the binding buffer of Fe-NTA IMAC phosphoenrichment was optimal. 10% acetonitrile in the elution buffer of Fe-NTA IMAC phosphoenrichment was optimal. Fe-NTA IMAC had a greater percentage of unique phosphopeptides and contained our target protein alpha-synuclein in comparison to our TiO2 OMAC phosphoenriched samples that did not contain alpha-synuclein. 100 µg of erythrocyte ghost membranes was the optimal sample load for MS analysis. Alpha-synuclein was found to be phosphorylated at Y125 and S129 as documented in previous studies. One novel phosphorylation site, S87, was discovered.
Conclusions and Relevance: Optimization of phosphoenrichment of digested frontal cortex brain samples using novel technology, such as the Agilent AssayMap Bravo platform, offers an important methodological advancement for future studies seeking to investigate phosphorylated aSyn biomarkers in PD. The discovery of pS87 in erythrocytes warrants further evaluation in future research as a potential biomarker for early PD diagnosis, which could improve health outcomes in affected patients.
Table of Contents
Abstract……………………………………………………………………………………………1
Introduction………………………………………………………………………………………..3
Figure 1. Previously described alpha-synuclein phosphorylation sites in the literature…..7
Methods…………………………………………………………………………………………..11
Figure 2. Schematic Workflow of TiO2 Phosphoenrichment…………………………….15
Results……………………………………………………………………………………………16
Figure 3. Glycolic Acid Optimization of Bravo Fe-NTA Binding Buffer……………….17
Figure 4.Acetonitrile Optimization of Bravo Fe-NTA Elution Buffer….……………….18
Figure 5. Fe-NTA vs TiO2 Phosphopeptide Comparison………………………………...20
Figure 6. Erythrocyte Ghost Membrane Sample Load Optimization for Phosphoenrichment……………………………………………………………………... 21
Figure 7. Y125, S87, and S129 Alpha-Synuclein Phosphorylation Sites in Erythrocyte Ghost Membranes ……………………………………………………………………….23
Discussion………………………………………………………………………………………..25
Future Directions………………………………………………………………………………...33
References………………………………………………………………………………………..35
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File download under embargo until 24 May 2025 | 2024-04-29 22:00:56 -0400 | File download under embargo until 24 May 2025 |
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