P1–174: Quantitative mass spectrometry (SRM/MRM) for amyloid peptides, tau protein and apolipoprotein E in human cerebrospinal fluid for Alzheimer's disease diagnosis

Abstract

Recent improvements in mass spectrometry (MS) allow this technology to quantify with clinical grade analytical sensitivity and specificity, peptides and proteins in biological fluids. We believe that in some cases MS will represent a valuable alternative to traditional methods of immunochemical quantification of proteins. We followed this path for the quantitation of biomarkers in Alzheimer disease (AD) which represents major cause of dementia. AD is associated with specific apolipoprotein E (ApoE) isoforms, and with alteration of cerebrospinal fluid (CSF) biomarkers. As a matter of fact, the decrease of amyloid peptides (Aß) and the increase of Tau proteins in CSF are currently use for AD diagnosis. Many isoforms of these molecules exist and MS represent an interesting tool to quantify the diversity of the isoforms, and therefore, to improve AD diagnosis and follow-up. For this purpose, quantitative targeted mass spectrometry (SRM/MRM) was developed using a triple quadripole. Sample prefractionation (Solid Phase Extraction), trypsic digestion and sample clean-up were realised using an automated liquid handling robot. Quantotypic peptides (Aß1–40, Aß1–42, tau, ApoE̤) were synthetized in light and heavy (13C and 15N) versions and used in calibration curve to evaluate the Limit Of Detection (LOD) and Quantification (LOQ). Experiments were run on series of biological samples from control and AD patients. Optimal Multiple Reaction Monitoring methods for the different analytes were developed. Detection of specific Apo E peptides resulted in a rapid method for e2/e3/e4 phenotyping. Different isoforms of Aß and Tau proteins were detected with sensibility compatible with pathophysiological variations. Correlation with immuno-detection methods and validation of the clinical relevance of the results are on-going. The mass spectrometry detection of several isoforms of amyloid peptides, Tau protein, and apolipoprotein E in human cerebrospinal fluid represents an important achievement that opens new avenue for quantitative Clinical Chemistry Proteomics (qCCP). The perspective is to exploit these results to improve phenotyping, diagnosis and follow-up of dementia.