Quantification of Tau in Cerebrospinal Fluid by Immunoaffinity Enrichment and Tandem Mass Spectrometry

Abstract

BACKGROUND Cerebrospinal fluid (CSF) tau is a common biomarker for Alzheimer disease (AD). Measurements of tau have historically been performed using immunoassays. Given the molecular diversity of tau in CSF, the selectivity of these immunoassays has often been questioned. Therefore, we aimed to develop an analytically sensitive and selective immunoaffinity liquid chromatography–tandem mass spectrometry (LC-MS/MS) (IA-MS) assay. METHODS IA-MS sample analysis involved the addition of an internal standard, immunoaffinity purification of tau using a tau monoclonal antibody coupled to magnetic beads, trypsin digestion, and quantification of a surrogate tau peptide by LC-MS/MS using a Waters Trizaic nanoTile ultraperformance LC microfluidic device. Further characterization of tau peptides was performed by full-scan MS using a Thermo Orbitrap LC-MS. CSF samples from a cohort of age-matched controls and patients with AD were analyzed by the IA-MS method as well as a commercially available immunoassay. RESULTS The IA-MS assay had intra- and interassay imprecision values of 3.2% to 8.1% CV and 7.8% to 18.9% C, respectively, a mean recovery of 106%, and a limit of quantification of 0.25 pmol/L and was able to quantify tau concentrations in all human specimens tested. The IA-MS assay showed a correlation of R2 = 0.950 against a total-tau immunoassay. In patients with AD, tau was increased approximately 2-fold. CONCLUSIONS Combining immunoaffinity enrichment with microflow LC-MS/MS analysis is an effective approach for the development of a highly selective assay to measure total tau and, potentially, other posttranslationally modified forms of tau in CSF.

Differential Mass Spectrometry Profiles of Tau Protein in the Cerebrospinal Fluid of Patients with Alzheimer’s Disease, Progressive Supranuclear Palsy, and Dementia with Lewy Bodies

“…However, our results are coherent with the study of Meredith et al [30] which described N-and C-terminal fragments associated with a 17 kDa central core fragment. Differences in output observed between the present study and those by Portelius et al [28] and McAvoy et al [27] can be accounted for in various ways different origins. First, the MS methods used were different, and the fact that different preanalytical methods were used in particular (precipitation versus immunocapture) may have yielded different results.…”

“…Here, thanks to an adaptation of the "protein standard for absolute quantification" (PSAQ) approach [16], an original purification protocol not relying on immunocapture, and the latest generation of MS analyzers, we could monitor in parallel 18 peptides encompassing the entire Tau protein sequence and isoform diversity. Previous MS attempts to monitor Tau in the CSF of patients were in fact limited to a few peptides and/or relied on immunoprecipitation procedures [27,28]. Importantly, preanalytical procedures are a major issue in the field of proteomics research, as Greco et al have pointed out in connection with the CSF in particular [29].…”

Pre-analytical and analytical factors influencing Alzheimer's disease cerebrospinal fluid biomarker variability

“…Production of candidate CRMs for Aβ 42 is an ongoing work with the efforts from the IFCC working group on CSF proteins [49] and from the BIOMARKAPAD consortium. Thereby, two candidate RMPs for quantification of Aβ 42 [50,51] and one for quantification of t-Tau [52] were recently reported.…”

Alzheimer's disease — Recent biomarker developments in relation to updated diagnostic criteria

“…Thus, we are in great need of improved and the recent publication of a selected reaction monitoring-based mass spectrometry method for t-tau [77]. In conclusion, great progress has been made in terms of standardization, of the preanalytical and analytical steps, and interpretation of results.…”

“…An alternative approach for future tau fragment assays may be the development of selected reaction monitoring (SRM) method as previously described [77]. The identification and quantification of a novel tau fragment may lead to increased specificity and improved differential diagnosis in many neurodegenerative diseases as well as to an improved understanding of the pathological role of tau in AD…”