Development of quantitative methods for measuring Abeta peptides in Alzheimer’s disease brain

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master_Nyberg_Elisabeth_2017.pdf (45.31 MB)

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Kemian tekniikan korkeakoulu | Master's thesis
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Date

2017-12-11

Department

Major/Subject

Biosystems and Biomaterials Engineering

Mcode

CHEM3028

Degree programme

Master’s Programme in Life Science Technologies

Language

en

Pages

52 + 7

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Abstract

Abeta peptides aggregate into amyloid plaques in the brain, a pathological hallmark of the neurodegenerative Alzheimer’s disease. The formation of the plaques can be studied using isotopic labels. In this thesis a pipeline is established, in which soluble Abeta peptides are extracted from mouse brain homogenate and enriched using immunoprecipitation with monoclonal antibodies immobilized on magnetic beads. The sample is eluted with 5% formic acid and digested with trypsin. The tryptic fragment Abeta17-28 is utilized as a surrogate for the full-length peptide in the quantitation with on-line liquid chromatography and mass spectrometry (LC-MS), reaching a level of detection of 50 amol. The pipeline offers a sensitive technique for differentiating aggregation-prone peptides from their isotopic isoforms in a quantitative manner, paving the way for further studies on the propagation behavior of Abeta peptides.

Abeta-peptider anhopas till amyloida placker i hjärnan, ett patologiskt kännetecken för den neurodegenerativa sjukdomen Alzheimers sjukdom. Plackbildningen kan studeras med hjälp av isotopmärkning. I detta diplomarbete utvecklas en flerstegsprocedur, där lösliga Abeta-peptider extraheras ur homogeniserad mushjärna och anrikas med immunoprecipitering med monoklonala antikroppar bundna till magnetiska kulor. Provet elueras med 5% myrsyra och klyvs med trypsin. Det tryptiska fragmentet Abeta17-28 används som surrogat för Abeta1-40 i kvantfieringen med direktansluten vätskekromatografi och masspektrometri (LC-MS), med en detektionsnivå på 50 amol. Proceduren erbjuder en känslig metod som särskiljer aggregationsbenägna peptider från deras isotopiska isoformer på ett kvantitativt sätt, och på så sätt bereder en väg för vidare forskning om utbredningsmönstret av Abeta-peptider.

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Supervisor

Frey, Alexander

Thesis advisor

Lalowski, Maciej

Keywords

Alzheimer's disease, Abeta peptides, mass spectrometry, UPLC-MS, quantitation

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https://urn.fi/URN:NBN:fi:aalto-201712187965

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[dipl] Kemian tekniikan korkeakoulu / CHEM