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P1‐019: Characterisation of tau and phospho‐tau populations within CSF: The relevance to Alzheimer's biomarker development
Author(s) -
Schofield Emma,
Russell Claire,
Lößner Christopher,
Prinz Thorsten,
Ward Malcolm
Publication year - 2012
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2012.05.294
Subject(s) - immunoprecipitation , biomarker , tau protein , phosphorylation , epitope , proteome , microbiology and biotechnology , chemistry , gene isoform , alzheimer's disease , biology , antibody , medicine , pathology , biochemistry , disease , immunology , gene
Background: Cerebral spinal fluid (CSF) measurement of Ab1-42, T-Tau and P-Tau181 are established Alzheimer’s disease (AD) biomarkers. However, previous LC-MS/MS analysis of PHF-Tau from post mortemAD brain revealed 12 novel phosphorylation sites (Hanger et al, 2007) and additionally phosphorylation of Tyr394 (Derkinderen et al, 2005). We have previously reported (ICAD 2011) the development and preclinical application of multiplexed quantitative mass spectrometry (MS) assays (Phospho-Tau SRM) for Tau phosphorylation screening and we now endeavour to extend thesemeasurements into CSF.Methods: Pooled hospital remnant CSF (Seralab Laboratories International Ltd.), depleted of abundant proteins (MARS-6 depletion column, Agilent), was prepared for 2-DE as described by Hanisch et al., 2010, with modifications. Tau and P-Tau specific antibodies (DAKO Tau, BT2, HT7, pT181, pS202, pT231 and pS396) were used to detect spots containing different epitopes. For MS, hospital remnant CSF (1mL) was precipitated with 2.5% perchloric acid prior to immunoprecipitation (IP) with Tau and P-Tau antibodies as described by Portelius et al., 2008, with modifications. Immunoprecipitated proteins were analysed using GeLC-MS/MS (Orbitrap Velos, ThermoFisher Scientific) and the PhosphoTau SRM assay (Proteome Sciences plc). Results: Here we describe the characterisation of numerous Tau and P-Tau moieties present within CSF. 2-DE highlights multiple features with distinct iso-electric points and molecular weights. Low molecular weight species are detected at w14kDa and at least one of these moieties is found to originate from the central region of Tau. A C-terminal fragment of Tau encompassing the full DAKO epitope is also observed. Preliminary LC-MS/MS analysis of Tau species enriched from hospital remnant CSF (500uL) via IP (BT2) identified the Tau peptide TPSLPTPPTR. Analysis via LC-SRM (1mL) identified SGYSSPGSPGTPGSR, STPTAEDVTAPLVDEGAPGK, IGSTENLK, SPVVSGDTSPR and STPTAEAEEAGIGDTPSLEDEAAGHVTQAR (1N Tau isoforms). Experiments with additional IP antibodies and phosphopeptide enrichment techniques will provide further insights. Conclusions: Phospho-Tau SRM assays provide the most comprehensive phospho-site specific array for Tau. With appropriate sample preparation to enrich Tau molecules from CSF, the Phospho-Tau SRM methodology can be extended from preclinical models of AD into clinical materials (CSF). The application of Phospho-Tau SRM assays to support clinical trials will add significant value to AD drug development activities providing further diagnostic utility.