Immunoprecipitation of Disease-Associated Proteins for Alzheimer’s Research

Background

Alzheimer’s disease is the most common neuropsychiatric disorder in the aging population and is characterized by extracellular deposits of aggregated amyloid-β (Aβ) peptides, intracellular tangles, and loss of synapses. The last several decades have seen an explosion in studies of the role of Aβ in the progression of Alzheimer’s disease which has been translated into promising novel therapies with putative disease-modifying effects. Up until recently, no biomarkers reflecting the third hallmark, progressive dysfunction and degeneration of synapses, have been available.

Methods

The target proteins and peptides are immunoprecipitated from human cerebrospinal fluid, plasma or brain tissue extract using magnetic Dynabeads. The beads are coated with antibodies having epitopes directed towards the protein of interest. Depending on the application, KingFisher mL or KingFisher Flex are used for automatic washing, enrichment and elution of target proteins and peptides. The immunoprecipitated samples are analysed by high resolution mass spectrometry (Q-Exactive), MALDI-TOF/TOF and/or on a TSQ Vantage mass spectrometer for MRM quantification.

Results

Immunoprecipitation (IP) in combination with mass spectrometry (MS) is a powerful tool for studying disease-associated proteins. Using this technique, researchers have shown that the metabolism of Aβ is far more complicated than previously known. To date, more than 40 different endogenous APP and Aβ peptides, including post-translational modifications and peptides ranging from Aβ1-13 and up to Aβ1-42 together with N-terminal truncated peptides, have been identified. As a consequence of these findings, several pharmaceutical companies have applied IP-MS in clinical trials to monitor target engagement. With the developed method we have shown that the postsynaptic protein Neurogranin (Ng) is metabolized into a series of endogenous C-terminally peptide species and that the endogenous peptide Ng48-76, which is the dominating endogenous peptide in human brain tissue, is significantly increased in Alzheimer’s disease compared to controls. This endogenous Ng peptide is not generated in the CSF, but is most probably a result of synaptic degeneration occurring in the central nervous system during the development of the disease.

Conclusions

The IP-MS methodology has been applied in translational clinical studies from cell and animal studies to human studies. This research has led to a completely new understanding of the complexity of the metabolism of Aβ, with consequences for both the understanding of disease mechanisms and drug development for Alzheimer’s disease.

Speaker

Erik Portelius, Associate Professor, Neurochemistry, The Sahlgrenska Academy at the University of Gothenburg

Erik Portelius received his PhD in 2009 from the University of Gothenburg, Sweden. Dr. Portelius is Associate Professor at the Clinical Neurochemistry Lab at Sahlgrenska University Hospital in Gothenburg and leads the proteomic section.

His major research interests include method development for studies on cerebrospinal fluid and plasma biochemical markers for clinical diagnosis of Alzheimer’s disease and monitoring treatment effects in clinical trials. The main focus of this research is in the discovery of novel biomarkers for early Alzheimer’s disease diagnosis and APP/Aβ metabolism and aggregation as well as neuronal and synaptic degeneration.

He has about 100 original research papers which have > 2,500 citations, with an H–index of 32.

Portelius has received a number of scientific awards, such as the Astrup Prize in 2016 (The Nordic Society for Clinical Chemistry) and European Grand Prix for Young Researcher – SCOR in 2017. 

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