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

• Published in: Journal of proteome research 2024-11, Vol.23 (11), p.5085-5095
• Main Authors: Fang, Fei, Xu, Tian, Chien Hagar, Hsiao-Tien, Hovde, Stacy, Kuo, Min-Hao, Sun, Liangliang
• Format: Article
• Language: English
• Subjects: Acetylation; Alzheimer Disease - metabolism; Chromatography, Reverse-Phase - methods; Electrophoresis, Capillary - methods; Humans; Isoelectric Focusing - methods; Phosphorylation; Pilot Projects; Protein Processing, Post-Translational; Proteomics - methods; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Tandem Mass Spectrometry - methods; tau Proteins - analysis; tau Proteins - chemistry; tau Proteins - metabolism
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/acs.jproteome.4c00587

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.