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P1‐127: MASS SPECTROMETRIC QUANTIFICATION OF TAU PROTEIN ISOFORMS IN HUMAN CEREBROSPINAL FLUID
Author(s) -
Hioki Yusaku,
Kaneko Naoki,
Yoda Ritsuko,
Sekiya Sadanori,
Iwamoto Shinichi,
Tanaka Koichi
Publication year - 2018
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.2018.06.130
Subject(s) - chemistry , gene isoform , triple quadrupole mass spectrometer , chromatography , selected reaction monitoring , electrospray ionization , mass spectrometry , tau protein , immunoprecipitation , peptide , protein isoform , tandem mass spectrometry , biochemistry , alzheimer's disease , medicine , disease , pathology , gene , alternative splicing
Background: In recent years, accumulated tau proteins have been considered important as targets for early detection, diagnosis and treatment against Alzheimer’s disease (AD). Six tau isoforms are expressed in the human central nervous system, and imbalances in tau isoform ratios are associated with AD pathology. To determine tau isoform ratios in cerebrospinal fluid (CSF), we developed a mass spectrometric quantification method. Here we report a sensitive analysis of tau tryptic peptides in CSF by immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM).Methods: Tau protein was immunoprecipitated from human CSF using magnetic beads coupled with anti-tau monoclonal antibody. Then this immunoprecipitate was reduced, alkylated, and digested by trypsin in solution. The MRM data were acquired using a triple quadrupole mass spectrometer (LCMS-8060, Shimadzu Corporation, JAPAN) coupled with liquid chromatography system (Nexera, Shimadzu Corporation, JAPAN). Dimethyl sulfoxide (DMSO) and isopropyl alcohol (IPA) were used as post-column additives to improve ionization efficiency and/or charge state coalescence. Results:Our immunoprecipitation method for tau achieved recovery rate of equal to or more than 50% from artificial CSF. The digestion efficiency of tau was enhanced by an optimized condition. The post-column additions of DMSO and IPA greatly increased electrospray ionization (ESI) response of the peptides. We evaluated the signals of tryptic peptides of tau and selected target peptides in MRM from the perspective of isoform quantification. MRM transitions for the four target peptides were optimized: three peptides were specific to 1N/2N isoform (amino acids 45-67), 2N isoform (68-87) and 4R isoform (282290), and the other was common peptide to all isoforms (195209). To evaluate the sensitivity for the practical use, we tried to detect the peptides from human CSF of 1 mL or less. As a result, we clearly and reproducibly detected peptides from 4R isoformspecific site and common sequence. We will present the analytical evaluation for quantitation of tau in CSF samples using stable isotopic label. Conclusions:We successfully quantified peptide for 3R/ 4R isoform distinction from human CSF of 1 mL or less. Our method provides the means for the analysis of 4R isoform as well as total tau.