Liquid Chromatography Tandem Mass Spectrometry Analysis of Tau Phosphorylation

Hyperphosphorylation, aggregation, and formation of neurofibrillary tangles of the microtubule-associated protein tau have been implicated in the pathogenesis of Alzheimer’s disease (AD) and other tauopathies. Phosphorylation and dephosphorylation of tau regulate its attachment or detachment from microtubules (Yoshiyama et al., J Neurol Neurosurg Psychiatry 84(7):784–795, 2013). The abnormal hyperphosphorylation of tau, however, disrupts its proper binding to microtubules and induces microtubule disassembly. Accumulation of unbound tau, then, results to aggregate and neurofibrillary tangle formation. Knowing the mechanism behind the abnormal phosphorylation of tau, therefore, is important to understanding the pathogenesis of neurodegenerative diseases. A protocol that employs nanospray liquid chromatography tandem mass spectrometry (nanoLC-MS3) for the analysis of tau phosphorylation by the checkpoint kinases, Chk1 and Chk2, in vitro, is provided here. Technical details on phosphorylation, protein digestion, peptide desalting, reversed-phase liquid chromatography (RPLC), mass spectrometry, and proteomics data analysis are discussed.