An Improved SPE-LC–MS–MS Platform for the Simultaneous Quantification of Multiple Amyloid Peptides in Cerebrospinal Fluid for Preclinical or Biomarker Discovery

Erin E. Chambers¹, Mary E. Lame², and Diane M. Diehl¹, ¹Waters Corporation and ²Pfizer, Neuroscience Research Unit

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. A previous Waters application note (720003682en) described in detail the development of a fast, flexible SPE-LC–MS–MS platform for the quantification of multiple aβ peptides from human or monkey CSF for use in a biomarker or preclinical discovery setting. In this work, the mass spectrometry platform has been updated from the Xevo TQ MS to the Xevo TQ-S mass spectrometry system. This change facilitated both a 4× reduction in required sample size and a 4–5× increase in assay sensitivity. This work focuses on methods for the 1–38, 1–40, and 1–42 aβ, Table I.

Table I: Sequence, MW, and pI Information for Amyloid Peptides

Experimental Conditions

SPE-LC–MS–MS Conditions

LC system: Waters ACQUITY UPLC System

Column: ACQUITY UPLC BEH C18 300 Å, 2.1 × 150 mm, 1.7 µm, Peptide Separation Technology

SPE: Oasis MCX µElution 96-well plate, 50 µL human or animal CSF

MS system: Waters Xevo TQ-S, ESI+

Figure 1: Representative ESI+ MS-MS spectrum for amyloid 1-42 with fragment sequence ions labeled.

Results and Conclusions

• An improved SPE-UPLC–MS–MS bioanalytical method was developed and validated for the simultaneous quantification of multiple amyloid β peptides in human CSF.

• MS was performed in positive ion mode since CID of the 4+ precursor ion yielded several distinct product ions corresponding to inherently specific b sequence ions (representative spectrum shown in Figure 1).

• UPLC separation of the three amyloid β peptides is shown in Figure 2.

Figure 2: Representative UPLC–MS–MS analysis of amyloid 1-38, 1-40, and 1-42 peptides extracted from artificial CSF + 5% rat plasma.

• The increased sensitivity of the Xevo TQ-S triple quadrupole mass spectrometer facilitated the use of 4× less sample and a 4–5× improvement in quantification limits (Table II).

• Average basal levels and RSD values for all 3 aβ peptides in two sources of human CSF are shown in Table III and are lower or equal to 5%.

• Overspiked QC samples were prepared in triplicate in two sources of pooled human CSF at 0.04, 0.075, 0.15, 0.2, 0.8, 2, and 6 ng/mL. Accuracy and precision values met the regulatory criteria for LC–MS–MS assays. Results from QC sample analysis are shown in Table IV. Average deviation from expected is 2.3%.

• The method described herein eliminates time-consuming immunoassays or immunoprecipitation steps for preclinical work.

• 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.

Table II: Comparison of Standard Curve and QC Range Using Xevo TQ and TQ-S MS

Table IV: Average Deviation Values for all Overspike QC Samples

Table III: Baseline Levels of Amyloid β Peptides in Two Sources of Pooled Human CSF

Copyright 2010: ACQUITY UPLC, Oasis, The Science of What's Possible, Xevo are trademark of Waters Corporation.

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