TY - JOUR
T1 - 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
AU - Oe, Tomoyuki
AU - Ackermann, Bradley L.
AU - Inoue, Koichi
AU - Berna, Michael J.
AU - Garner, Carlos O.
AU - Gelfanova, Valentina
AU - Dean, Robert A.
AU - Siemers, Eric R.
AU - Holtzman, David M.
AU - Farlow, Martin R.
AU - Blair, Ian A.
PY - 2006
Y1 - 2006
N2 - The 40 and 42 amino-acid residue forms of amyloid beta (Aβ 1-40 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/L (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.
AB - The 40 and 42 amino-acid residue forms of amyloid beta (Aβ 1-40 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/L (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.
UR - http://www.scopus.com/inward/record.url?scp=33845591072&partnerID=8YFLogxK
U2 - 10.1002/rcm.2787
DO - 10.1002/rcm.2787
M3 - Article
C2 - 17117458
AN - SCOPUS:33845591072
SN - 0951-4198
VL - 20
SP - 3723
EP - 3735
JO - Rapid Communications in Mass Spectrometry
JF - Rapid Communications in Mass Spectrometry
IS - 24
ER -