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Waters Application Note
Erin E. Chambers,1 Mary E. Lame2 and Diane M. Diehl,11 Waters Corporation, Milford, Massachusetts, USA, 2 Pfizer, Neuroscience Research Unit, Groton, Connecticut, USA.
Fast, flexible platforms for peptide quantification are needed, particularly for a discovery setting. This type of methodology would be especially advantageous in the case of amyloid beta (aβ ) peptides. The deposition/formation of insoluble aggregates, or plaques, of aβ peptides in the brain is considered to be a critical event in the progression of Alzheimer's Disease (AD) and thus has the attention of many researchers. Historically, quantification of aβ peptides in biological fluids has relied mainly on the use of immunoassays, such as ELISA. These assays are time consuming, expensive to develop and labour intensive. They are subject to cross reactivity and an individual assay is required for each peptide. To meet the throughput requirements and constant flow of demands for new peptide methods in a discovery setting, there is a need for a highly specific yet flexible methodology based on an LC–MS–MS platform. In this work, this platform is coupled with selective sample preparation for the simultaneous quantification of multiple a peptides. This work focuses on methods for the 1-38, 1-40 and 1-42 aβ peptides, in support of preclinical studies.
Figure 1: Sequence, MW and pI information for amyloid Î² peptides.
LC system: Waters ACQUITY UPLC system
Column: ACQUITY UPLC BEH C18, 300 Å, 2.1 × 150 mm, 1.7 µm, Peptide Separation Technology
SPE device: Oasis MCX µElution 96-well plate
MS system: Waters Xevo TQ MS, ESI+
MS was performed in positive ion mode because CID of the 4+ precursor ion yielded several distinct product ions corresponding to inherently specific b sequence ions (representative spectrum shown in Figure 2).
Figure 2: Representative ESI+ MSâMS spectrum for amyloid Î² 1-42 with fragment sequence ions labelled.
Separation of the three amyloid β peptides is shown in Figure 3.
Figure 3: UPLCâMSâMS analysis of amyloid Î² 1-38, 1-40 and 1-42 peptides extracted from artificial CSF + 5% rat plasma.
SPE was performed using Oasis MCX, a mixed-mode sorbent, to enhance selectivity of the extraction. The sorbent relies on both reversed-phase and ion exchange retention mechanisms to selectively separate the aβ fraction from other high abundance polypeptides in complex CSF samples. The Oasis µElution plate (96-well format) provided significant concentration while eliminating evaporation and reconstitution, minimizing peptide losses.
Figure 4: Representative chromatogram showing basal levels of amyloid Î² 1-42 extracted from 3 sources of human and 1 source of monkey CSF.
N15-labelled internal standards were used for each peptide. Standard curves for all 3 aβ peptides were linear (1/x weighting) from 0.1 to 10 ng/mL in artificial CSF + 5% rat plasma. Basal levels of amyloid β 1-42 extracted from 3 sources of human and 1 source of monkey CSF are shown in Figure 4. Overspike QC samples were prepared in 3 sources of pooled human CSF and 1 source of pooled monkey CSF at 0.2, 0.8, 2 and 6 ng/mL. Accuracy and precision values for all 3 peptides met the regulatory criteria for LC–MS–MS assays. Representative results from QC sample analysis are shown in Table 1.
Table 1: Representative results from analysis of QC samples prepared in pooled human CSF, source 3.
• An SPE-UPLC–MS–MS bioanalytical method was developed and validated for the simultaneous quantification of multiple amyloid β peptides in human and monkey CSF.
• 96 samples can be extracted and ready for injection in < 30 minutes, providing the sample prep throughput required for pre-clinical and clinical studies.
• The method described herein eliminates time-consuming immunoassays or immunoprecipitation steps for pre-clinical work.
• This approach also allows one assay for the simultaneous measurement of several different amyloid β peptides from a single sample. This single assay provides a high degree of selectivity and specificity in a high-thoughput format while still achieving the high sensitivity required for low level endogeneous amyloid β peptides.
• The use of a single UPLC–MS–MS assay represents a significant advantage over an ELISA assay, which would require multiple assays with multiple antibodies to quantify each of the relevant peptides.
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