Antibody-free quantification of seven tau peptides in human CSF using targeted mass spectrometry.

Pauline Bros,Audrey Gabelle,Sylvain Lehmann,Vincent Delatour,Christophe Hirtz,Jérôme Vialaret,Nicolas Barthelemy

doi:10.3389/fnins.2015.00302

Abstract

Tau protein concentration in cerebrospinal fluid (CSF) is currently used as a sensitive and specific biomarker for Alzheimer's disease. Its detection currently relies on ELISA but the perspective of using mass spectrometry (MS) to detect its different proteoforms represents an interesting alternative. This is however an analytical challenge because of its low concentration in the CSF, a biological fluid collected in small volume by lumbar puncture, and with a high structural heterogeneity. To overcome these issues, instead of using immunocapture as previously done, we rather relied on an original two steps pre-fractionation technique of CSF: perchloric acid (PCA) followed by micro solid phase extraction (μSPE). We could then measure seven tau trypsic peptides by Multiple Reaction Monitoring (MRM) on a triple quadrupole mass spectrometer. Quantification was performed using isotopically labeled 15N- recombinant tau protein as internal standard and validated using CSF pools with low, medium, or high tau concentrations (HTCs). Repeatability, intermediate precision, linearity, limit of quantification (LOQ), and recovery were calculated for the different peptides. This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.

Highlights

Alzheimer’s disease (AD) is the most common form of dementia, a general term encompassing loss of memory and cognitive functions interfering with activities of daily living
Development, Linearity, and Recovery Starting from 22 validated peptides of cerebrospinal fluid (CSF) tau protein obtained with high resolution mass spectrometry (MS) in PRM mode (Barthelemy, under review), 7 peptides (GAAPPGQK, SGYSSPGSPGTPGSR, TPSLPTPPTREPK, TPSLPTPPTR, LQTPVPMPDLK, IGSTENLK, SPVVSGDTSPR) were validated using triple quadrupole in Multiple Reaction Monitoring (MRM) mode (Table 1)
We presented for the first time an MRM based multiplex assay for tau in the CSF that did not necessitate any immuno-capture

Summary

Introduction

Alzheimer’s disease (AD) is the most common form of dementia, a general term encompassing loss of memory and cognitive functions interfering with activities of daily living. Tau protein is synthesized by a single microtubule-associated protein tau (MAPT) gene (chromosome 17q21) in humans (James et al, 2015) mainly expressed in neurons (Perrin et al, 2009). Tau protein is known to bind neuronal microtubules, promote their assembly, and stabilize them (Drechsel et al, 1992). The hyperphosphorylation occurring in case of AD caused its release from microtubules, destabilizing the axons, and triggering neuronal death (Sergeant et al, 2005). The full quantification of tau in CSF remained an analytical challenge. This protein has 6 different isoforms (ranging from 352 to 441 amino acids), and is subject to many different post-translational modifications like phosphorylation, glycosylation, and oxidation (Hernandez and Avila, 2007). Tau is present at very low concentration (Sjögren et al, 2001; Blennow and Vanmechelen, 2003) in CSF which is a highly complex matrix

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Results

Conclusion