Tau protein profiling across brain of different tauopathies using high‐resolution mass spectrometry: quantification of isoforms and phosphorylations

AbstractBackgroundThe abnormal accumulation of the microtubule‐associated protein tau in the brain is the main contributing factor of a group of diseases called tauopathies, including Alzheimer’s disease (AD). Due to similarities, it is currently problematic to chemically characterize the tau pathology in these diseases. Our aim was to develop a multiplex assay to detect and quantify isoform‐specific peptides and phospho‐tau species in brain of different tauopathies, possibly holding disease‐specificity.MethodFrontal cortices from AD (n = 10), progressive supranuclear palsy (PSP, n = 11), Pick’s disease (PiD, n = 10), corticobasal degeneration (CBD, n = 10) and controls (n = 10) were processed to obtain a soluble (tris‐buffered saline, TBS) and a sarkosyl‐insoluble (SI) fraction. Fractions were immunoprecipitated using antibodies targeting all four tau regions. Tryptic peptides corresponding to isoforms and peptides carrying one, two or three phosphorylations were monitored by liquid chromatography/high‐resolution mass spectrometry, using isotope‐labelled protein and phospho‐peptide standards for quantification.ResultIn all samples, the 0N and 1N tau isoforms were most abundant. In the AD group we observed an increase of the 0N isoform in the SI fraction. The SI fraction also showed the 3R/4R isoform predominance characteristic of the different tauopathies, with the 3R being more abundant in PiD, 4R in PSP and CBD, while in AD and controls they had similar abundance. Further, in the SI fraction, peptides from the microtubule‐binding region (MTBR) were markedly more abundant in AD, indicating aggregation of these species (Fig.1). Compared to the TBS fractions, the SI fraction was enriched in double and triple‐phospho peptides (example in Fig.2), which were markedly increased in the AD group as compared to the other non‐AD tauopathies or the control.ConclusionThe novel assay here developed can identify isoform differences across tauopathies. The parallel investigation of TBS and SI fractions showed higher abundancy of phosphorylations and MTBR region in the SI fraction, possibly reflecting this fraction to contain an interphase between small aggregates and immunohistochemical inclusions. While the measurement of multiply phosphorylated peptides could clearly differentiate AD from the other tauopathies, none of them could discriminate between the non‐AD tauopathies. This further indicate that multiply phosphorylated peptides are highly disease relevant species for AD.