High sensitivity analysis of tau phospho, non-phospho, and MTBR peptides in Alzheimer's disease CSF and plasma using Orbitrap Astral

Introduction

Recently active treatments against Alzheimer"s disease (AD) have been made avaliable, creating an even greater need for biomarkers to diagnose and monitor the treatment of patients. Different phosphorylated forms of tau (pTau) have emerged as the most promising biomarkers for diagnosis and staging of AD. Their low plasma concentrations have so far required millilitres for mass spectrometry (MS) measurement, severely restricting the applicability of MS in clinical studies. In addition, there is an interest to measure the parts of tau that constitutes the core of the tangles that are formed in the brain, the microtubule binding region (MTBR). The tau fragments containing the MTBR are present at very low concentrations in cerebrospinal fluid (CSF) and plasma. Here, we evaluated the recently introduced Orbitrap Astral mass spectrometer for quantification of CSF and plasma tau in the context of Alzheimer"s disease.

Methods

Both paired patient CSF and plasma samples submitted to the routine lab at the Sahlgrenska University Hospital/Mölndal [CSF biomarker (total-tau, pTau181, amyloid β1-42) defined AD and non-AD controls] cohort of plasma samples were analysed on an Orbitrap Astral (Thermo Fisher Scientific). Analysis was performed using parallel reaction monitoring (PRM) to measure tryptic tau peptides. Six phospho and eight non-phospho peptides (of which six were isoform specific reflecting contributions from both the central and peripheral nervous systems) were monitored: 45-68 0N, 131-138 PNS, 275-291 PNS, 175-190-p181, 195-209, 195-209-p199, 195-209-p 202, 195-209-p 205, 212-221, 212-221-p217, 225-240-p231, 275-280 4R, 275-286 3R, 282-290 4R. Pooled samples were used for method validation (determination of minimal sample volume, LLOQ, precision).

Preliminary data

All 14 tau peptides in the assay were detectable both in CSF and plasma. Tau175-190-p181 could be measured down to 50 amol on-column, corresponding to 2 µL CSF and 212-221-p217 could be measured in the same CSF sample volume down to 10 amol on-column (CV=1.6%). In previous studies using Orbitrap Fusion Lumos, 250 µL CSF was used for analysis. For plasma, the analyses were performed using sample volumes of 250 µL, i.e., a quarter of the 1-mL-volume required previously.

The high detection sensitivity of the Orbitrap Astral enabled detection of pTau forms in significantly lower CSF and plasma sample volumes compared with previously published studies, and with low technical variation. Moreover, because of the high sampling rate, the degree of multiplexing could be increased without compromising the analytical quality.

Novel Aspect

Orbitrap Astral enabled detection by MS of pTau and MTBR biomarkers in significantly lower volumes than previously possible and allowed for a higher degree of multiplexing, with high technical precision.