Evaluation of an SP3 protocol for the mass spectrometry-based quantification of t-tau and tau proteoforms in artificial CSF and brain.

RATIONALE: Diagnosis and differentiation of Alzheimer’s disease (AD) from other tauopathies is challenging, especially at the early stages. In this aim, analytical methods are essential to identify and to confirm new biomarkers. Antibody-dependent approaches may not capture all the diversity of proteoforms when dealing with complex proteins such as tau. METHODS: We adapted and evaluated the single-pot solid-phase-enhanced sample-preparation (SP3) protocol for antibody-free extraction of tau-protein in diluted human serum or human brain, following perchloric acid precipitation. A total of 13 non-modified peptides were quantified by high-resolution mass spectrometry (HRMS) after digestion of tau by trypsin. RESULTS: We significantly improved the basic SP3 protocol by carefully optimizing the organic solvents and incubation time for tau binding, as well as the digestion step for the release directly from the beads of the 13 tau peptides. These optimizations proved to be primarily beneficial for the most hydrophilic tau peptides, increasing the sequence coverage. Mean recovery of the 13 non-modified peptides quantified by LC-HRMS was of 53%, with LODs ranging from 0.75 to 10ng/mL. CONCLUSIONS: Finally, we tested the optimized SP3 protocol on pathological tau extracted from the soluble fraction from an AD brain sample (middle frontal gyrus). We could successfully identify and quantify several biologically relevant tau peptides, including representative peptides of two isoforms and two phospho-peptides (p-Tau217 and p-Tau181).