Mass spectrometric measurement of six site‐specific tau phosphorylations in CSF and blood of Alzheimer's disease patients

Abstract

AbstractBackgroundHyperphosphorylation of tau is responsible for its loss of normal physiological function, gain of toxicity and its aggregation to form NFTs. Increased phosphorylated tau is detected in Alzheimer´s disease (AD) CSF and blood, and changes in the tau phosphorylation pattern may reflect different stages of the disease progression. A better understanding on how the levels of site‐specific phosphorylations are modulated across the AD continuum in these biofluids may help to provide with a more accurate diagnosis.MethodWe developed an LC‐MS method based on parallel reaction monitoring (PRM) using Orbitrap MS to measure tau phosphorylations at positions pT181, pS199, pT205, pT217 and pT231 in CSF and plasma, and additionally p396 in CSF. Tau enrichment prior to LC‐MS differed between biofluids. For CSF, 250 µl of sample were prepared by performing partial PCA protein precipitation, followed by SPE and tryptic digestion. For plasma, 1 ml samples were subjected to immunoprecipitation using a combination of several tau antibodies, followed by tryptic digestion and SPE.ResultThe performance of the method was first evaluated by analysis of CSF from neurochemically characterized AD patients and controls (n=40). All measured phosphorylations showed significant separation between groups (p<0.001) (Fig. 1). In addition, pT181, pT217 and pT231 showed good correlations with SIMOA immunoassays. In plasma, a set of AD patients and controls (n=26) were analysed. All phosphorylations showed an increase in AD, and most significantly pT205 (p=0.003) and pT231 (p=0.008) (Fig 2). Interestingly, these two phosphorylations were the ones that showed higher correlation with p‐tau CSF (Spearman r=0.52 and r=0.63, respectively). Plasma values were also compared to the respective SIMOA immunoassays. Finally, the method was used to measure samples from the Translational biomarker In Aging and Dementia (TRIAD) cohort (n=209), consisting of young individuals (<30 years old), cognitive unimpaired, mild cognitive impairment (MCI) and AD patients.ConclusionWith our LC‐MS method to quantify site‐specific p‐tau levels, we have established the tau phosphorylation pattern in AD biofluids. Our data indicates that phosphorylation at position T231 is the one that shows a higher fold change between AD and controls both in CSF and blood.