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

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Abstract
The 40 and 42 amino-acid residue forms of amyloid beta (Aβ1–40 and Aβ1–42) 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/mL (200 pg/mL) for Aβ1–42 and 92 fmol/mL (400 pg/mL) for Aβ1–40. An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Aβ1–42 in human CSF (r2 = 0.915), although less correlation was observed for Aβ1–40 (r2 = 0.644). Mean CSF Aβ1–42 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). Aβ1–40 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 Aβ1–42 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 Aβ1–40 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 Aβ1–42 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. Copyright © 2006 John Wiley & Sons, Ltd.