Strategy for absolute quantification of phospho‐Tau epitopes in cerebrospinal fluid by mass spectrometry

Abstract

AbstractBackgroundMass spectrometry is used as reference method for protein quantification by measuring tryptic peptides derived from the target protein relative to a stable isotope‐labeled standard. Existing targeted mass spectrometric methods to measure total tau (tTau) and phosphorylated tau (pTau) species in cerebrospinal fluid (CSF) rely either on immunoaffinity enrichment of the protein or exploit the unusual property of tau being soluble in 2.5% perchloric acid (PA), to precipitate abundant sample proteins. Both strategies, however, carry risk of introducing bias: immunoaffinity enrichment relies on the specificity of the used antibodies, which may not have equal affinity for all of the large number of processed or modified forms of tau, and PA may partially precipitate some tau forms. To address this problem, we have developed an antibody‐ and PA‐free method to measure tau and pTau epitopes in CSF by targeted mass spectrometry.MethodCSF samples (100 µl) from healthy individuals (HC) and Alzheimer’s disease (AD) patients were spiked with stable isotope‐labeled tau peptide standards containing the phospho‐epitopes pTau181, pTau231, pTau217, pTau205, and non‐phosphorylated Tau212‐221, and subjected to tryptic digestion. Desalted samples were fractionated by strong anion‐ and strong cation‐ exchange (SAX and SCX, respectively) solid phase extraction with step‐wise pH elution. The fractions were analyzed by parallel reaction monitoring on a nano‐flow LC‐MS on a Quadrupole‐Orbitrap hybrid mass spectrometer.ResultSeveral tau peptides were detected with both SAX and SCX, including pTau181, pTau217, pTau205, and Tau212‐221. The reproducibility of the new assay was assessed, and its performance was compared with PA‐based sample preparation and immunoassays for tau and pTau in a pilot study of AD patients and controls.ConclusionThe method described here allows for the detection of multiple tau/pTau peptides, while avoiding the need for selective protein isolation methods such as immunoaffinity enrichment or PA precipitation, thereby eliminating bias toward more/less soluble tau species.