Quantifying soluble isoforms of amyloid precursor protein in cerebrospinal fluid with a SRM-MS based assay ─ method development

Abstract: Alzheimer’s is a widespread neurodegenerative disease, growing larger and larger in the world. Once developed, the disease has no cure. To this day, there is only mitigating drugs. To be able to start this treatment rapidly, a method to distinguish healthy individuals from prospective Alzheimer’s diseased needs to be developed. Cerebrospinal fluid is thought to contribute to the development of such method, through its close substitution of fluids, molecules and proteins from the brain. It may provide a progressive marker of the disease, a substance differently expressed in the healthy and diseased; a biomarker. The aim of the present study was to develop and evaluate a stable method for degradation and analysis of peptides and proteins in the cerebrospinal fluid using mass spectrometry techniques, such as selective reaction monitoring. Mass spectrometry is often used after a first dimension separation with liquid chromatography. Three degradation methods were evaluated, resulting in a protocol with the detergent DOC being the most beneficial. Tryptic peptides occurred in a concentration of 10 % w/v due to the SDS-PAGE gels and database searches concomitantly. The elution pattern from the liquid chromatography enables a narrow selection in the sensitivity for each peptide. Chromatographic preferences such as column, hydrophobicity and time span was determined, and unwanted peptides filtered away. A specific protein, the amyloid precursor protein APP, is thought to play a significant part in the development of Alzheimer’s disease. The protein is located in the neurons, cleaved and processed to produce the neurodegenerating plaques found in the brain at the diseased. Three isoforms are found in the neurons, APP695, APP751 and APP770. When cleaved, a shorter soluble tryptic peptide is generated from all APP isoforms. This was the target for the current study, as a potential progressive biomarker. The method developed was able to separate and distinguish the soluble APP751 isoform, but not the APP695 or APP 770 isoforms, most probably due to glycosylations of the two resembling isoforms.