Antibody-free measurement of cerebrospinal fluid tau phosphorylation across the Alzheimer's disease continuum

Background: Alzheimer's disease is characterized by an abnormal increase of phosphorylated tau (pTau) species in the CSF. It has been suggested that emergence of different pTau forms may parallel disease progression. Therefore, targeting multiple specific pTau forms may allow for a deeper understanding of disease evolution and underlying pathophysiology. Current immunoassays measure pTau epitopes separately and may capture phosphorylated tau fragments of different length depending on the non-pTau antibody used in the assay sandwich pair, which bias the measurement. Methods: We developed the first antibody-free mass spectrometric method to simultaneously measure multiple phosphorylated epitopes in CSF tau: pT181, pS199, pS202, pT205, pT217, pT231, and pS396. The method was first evaluated in biochemically defined Alzheimer's disease and control CSF samples (n = 38). All seven pTau epitopes clearly separated Alzheimer's disease from non-AD (p < 0.001, AUC= 0.84-0.98). We proceeded with clinical validation of the method in the TRIAD (n = 165) and BioFINDER-2 cohorts (n = 563), consisting of patients across the full Alzhei-mer's disease continuum, including also young controls (< 40 years), as well as patients with frontotemporal dementia and other neurodegenerative disorders.Results: Increased levels of all phosphorylated epitopes were found in Alzheimer's disease dementia and A beta positron emission tomography-positive patients with mild cognitive impairment compared with A beta-negative con-trols. For Alzheimer's disease dementia compared with A beta-negative controls, the best biomarker performance was observed for pT231 (TRIAD: AUC = 98.73%, fold change = 7.64; BioFINDER-2: AUC = 91.89%, fold change = 10.65), pT217 (TRIAD: AUC = 99.71%, fold change = 6.33; BioFINDER-2: AUC = 98.12%, fold change = 8.83) and pT205 (TRIAD: AUC = 99.07%, fold change = 5.34; BioFINDER-2: AUC = 93.51%, fold change = 3.92). These phospho-epitopes also discriminated between A beta-positive and A beta-negative cognitively unimpaired individuals: pT217 (TRIAD: AUC= 83.26, fold change = 2.39; BioFINDER-2: AUC = 91.05%, fold change = 3.29), pT231 (TRIAD: AUC= 86.25, fold change = 3.80;BioFINDER-2: AUC = 78.69%, fold change = 3.65) and pT205 (TRIAD: AUC = 71.58, fold change = 1.51; BioFINDER-2: AUC = 71.11%, fold change = 1.70).Conclusions: While an increase was found for all pTau species examined, the highest fold change in Alzheimer's disease was found for pT231, pT217 and pT205. Simultaneous antibody-free measurement of pTau epitopes by mass spectrometry avoids possible bias caused by differences in antibody affinity for modified or processed forms of tau, provides insights into tau pathophysiology and may facilitate clinical trials on tau-based drug candidates.

Automated summary

This study presents the first antibody-free mass spectrometric method for simultaneously measuring multiple phosphorylated epitopes in CSF tau, providing insights into the pathophysiology of Alzheimer's disease and potentially facilitating clinical trials for tau-based drug candidates.