Quantitative analysis of amyloid β peptides in cerebrospinal fluid of Alzheimer's disease patients by immunoaffinity purification and stable isotope dilution liquid chromatography/negative electrospray ionization tandem mass spectrometry

The 40 and 42 amino-acid residue forms of amyloid beta (Aβ _1-40 and Aβ_1-42) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Aβ peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Aβ peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/ tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [¹⁵N]-labeled Aβ peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/ MS system. The validated method had limits of quantitation of 44 fmol/L (200 pg/mL) for Aβ_1-42 and 92 fmol/mL (400 pg/mL) for Aβ_1-40. An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Aβ_1-42 in human CSF (r² = 0.915), although less correlation was observed for Aβ^1-40 (r² = 0.644). Mean CSF Aβ_1-42 concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/ MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 ± 0.25 ng/mL; n = 7). Aβ_1-40 concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 ± 3.07 ng/mL; n = 7). Consistent with literature reports, mean Aβ_1-42 concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 ± 0.59 ng/mL; n = 7), whereas there was no difference in Aβ_1-40 concentrations between AD patients and normal subjects (mean 5.88 ± 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Aβ_1-42 concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.