Florey Department of Neuroscience and Mental Health - Theses
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ItemApplying matrix assisted laser desorption-ionization mass spectrometry to identify and quantify isoforms of amyloid beta in human brain( 2017)Amyloid beta (Aβ) plays a central pathogenic role in Alzheimer's Disease (AD), however its great heterogeneity remains largely unexplored as most research focuses on only two specific species: Aβ1-40 and Aβ1-42. More than fifty truncated species of amyloid beta have been identified, as well as a multitude of additional post translational modifications including pyroglutamation, oxidation and glycosylation. This spectrum of isoforms is either cumbersome or impossible to resolve with prevailing methods such as ELISA and western blots. Matrix Assisted Laser Desorption-Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS) allows specific and simultaneous identification of these species. My thesis employed this method, with prior enrichment of donated brain samples with amyloid beta immunoprecipitation, to characterise these isoforms in different fractions of human brain: the cytosolic (TBS) fraction, the peripheral membrane/vesicular (Na2CO3) fraction, the integral lipid/membrane (urea/detergent) fraction, and the polymerised fibrillary (formic acid) fraction. At least fourteen different Aβ isoforms were resolved, most in the formic acid fraction and membrane fraction, with relatively less in the sodium carbonate fraction. Extending the methodology, isotopically labelled ‘heavy’ internal standards were utilised to allow for absolute quantification of some prominent truncations, including Aβ4-42 and Aβ1-42. This showed a mean of 704 pmol/g wet brain of Aβ4-42 in formic acid fractions of brains with Alzheimer’s pathology, and 438 pmol/g in the membrane fractions of brains with Alzheimer’s pathology. Aβ1-42 was present in the formic acid fractions and membrane fractions of those with Alzheimer’s Disease in mean values of 1.3 nmol/g and 232 pmol/g respectively. These values were greater than those in the control brains. It was also seen that Aβ4-42 ionised approximately 1.3 fold the extent of AβH1-42, and approximately 3 fold that of AβH1-40. Noting the difficulties inherent in ionising entire species of amyloid beta, enzymatic cleavage with Lys-N was undertaken to improve reliability and throughput of MALDI-TOF MS analysis. Lys-N is a metalloendopeptidase that preferentially cleaves N-terminals of lysine groups. These cleaved products of Aβ are more amenable to MALDI-TOF MS than products of C-terminal cleavage. Lys-N was first isolated from the fruiting bodies of pleurotus ostreatus, achieving a 3.94 fold purification. This was then applied to synthetic amyloid beta and MALDI-TOF MS analysis, preferentially resolving N terminal truncated isoforms of Aβ. With further refinement, this method would allow a specific and robust approach to characterising and quantifying multiple species of Aβ to picomole amounts.