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bioRxiv · 10.1101/2024.07.04.602093

A pilot study for deciphering post-translational modifications and proteoforms of tau protein by capillary electrophoresis-mass spectrometry

Fang, F.·Xu, T.·Chien Hagar, H.-T.·Hovde, S.·Kuo, M.-H.·Sun, L.
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Abstract

Abnormal accumulation of tau proteins is one pathological hallmark of Alzheimer{square}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 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 53 phosphorylation sites of tau-0N3R in total, which is about 30% 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 birds-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 a native-like cIEF-MS condition, 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.

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Fang, F., Xu, T., Chien Hagar, H.-T., Hovde, S., Kuo, M.-H., Sun, L.. 2024-07-08. A pilot study for deciphering post-translational modifications and proteoforms of tau protein by capillary electrophoresis-mass spectrometry. https://doi.org/10.1101/2024.07.04.602093

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