Pilot Study for Deciphering Post-Translational Modifications and Proteoforms of Tau Protein by Capillary Electrophoresis-Mass Spectrometry

Abnormal accumulation
of tau protein in the brain is one pathological
hallmark of Alzheimer′s disease (AD). Many tau protein post-translational
modifications (PTMs) are associated with the development of AD, such
as phosphorylation, acetylation, and methylation. Therefore, a complete
picture of the PTM landscape of tau is critical for understanding
the molecular mechanisms of AD progression. Here, we offered a pilot
study of combining two complementary analytical techniques, capillary
zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase
liquid chromatography (RPLC)-MS/MS, for bottom-up proteomics of recombinant
human tau-0N3R. We identified 50 phosphorylation sites of tau-0N3R
in total, which is about 25% higher than that from RPLC-MS/MS alone.
CZE-MS/MS provided more PTM sites (i.e., phosphorylation) and modified
peptides of tau-0N3R than RPLC-MS/MS, and its predicted electrophoretic
mobility helped improve the confidence of the identified modified
peptides. We developed a highly efficient capillary isoelectric focusing
(cIEF)-MS technique to offer a bird’s-eye view of tau-0N3R
proteoforms, with 11 putative tau-0N3R proteoforms carrying up to
nine phosphorylation sites and lower pI values from more phosphorylated
proteoforms detected. Interestingly, under native-like cIEF-MS conditions,
we observed three putative tau-0N3R dimers carrying phosphate groups.
The findings demonstrate that CE-MS is a valuable analytical technique
for the characterization of tau PTMs, proteoforms, and even oligomerization.