Pilot Study for\nDeciphering Post-Translational Modifications\nand Proteoforms of Tau Protein by Capillary Electrophoresis-Mass Spectrometry

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