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The most common form of dementia is Alzheimer’s disease. According to the amyloid hypothesis, the disease is preceded by an accumulation of the amyloid-β (Aβ) protein, which leads to downstream events including activation of microglia, inflammation, synaptic dysfunction, and neuronal loss. The objective of this research is to address the physiology of Aβ in humans by measuring its in vivo metabolic rates.
A method was previous developed in our laboratory for measuring the in vivo synthesis and clearance rates of total Aβ within the cerebrospinal fluid (CSF) of normal healthy volunteers using a metabolic label of 13C6 leucine. After a spinal tap, the total Aβ peptide pool was isolated from the CSF by immunoprecipitation, followed by mass spectrometry. As in selected reaction monitoring experiments, we use signals obtained from fragmentation of precursor peptide ions containing unlabeled and labeled leucine, but with a full scan tandem MS, instead of using precursor ions for quantitation as done in isotope-labeling experiments.
Total Aβ in human CSF was measured by ELISA methods to be about 5000–20,000 pg/mL (~2 pmol total and 20 fmol 1% labeled). The metabolism of Aβ was measured by determining the percent 13C6 leucine labeling in Aβ in CSF samples taken every hour over a 36-h time course. The incorporated label was not detected until the fifth hour of label infusion, followed by an increase to a steady state at 20 to 24 h, then a decrease over the last 12 h.
In vivo metabolic protein labeling can be measured with high sensivtivity (low fmol labeled peptides), accuracy (R2 = 0.99), and reproducibility. These technical advances using common commercially available instruments may be applied to other proteins of interest and adapted to measuring total amounts of proteins or percent labeled proteins, for absolute and relative quantitation respectively.