Enrichment of Intact Presenilin 1 by Sequential Membrane Solubilization and GELFrEE Electrophoresis for LC-MRM MS

摘要

Alzheimer's disease (AD) affects more than 5.3 million Americans and costs in the neighborhood of dollar172 billion in healthcare and lost wages annually (Alzheimer's Association, http://alz.org). Presenilin-1 (PS1) is the enzyme in the gamma secretase complex that generates both normal and amyloidogenic A-beta (A-beta40 and 42, respectively) from the parent protein amyloid precursor protein (APP); while both peptides are produced in normal subjects, A-beta42 levels are generally lower than A-beta40 (reviewed by Hardy and Selkoe, 2002). A-beta42 is called amyloidogenic because it is the peptide that comprises the amyloid plaques that are hallmark of AD brain. While 150 different point mutations in PS1 have been mapped (DeStrooper, 2007), which constitutes nearly 1/3 of the residues in PS1, the vast majority of AD patients do not have mutations in PS1, yet all have serum and CSF A-beta42/A-beta40 ratios that are higher than in normal subjects, indicative of aberrant PS1 function. We hypothesize that structural non-genomic modifications in PS1 such as oxidative modifications cause altered function of the enzyme causing abnormal A-beta42/A-beta40 ratios which may precede neurodegenerative disease. The objective of this study was to develop purification approaches to enrich for mammalian PS1 from tissues for mass spectrometry studies of structural alterations and how they correlate with activity.