Amyloid-beta isoform metabolism quantitation by stable isotope-labeled kinetics

Abstract

Abundant evidence suggests a central role for the amyloid-beta (Aβ) peptide in Alzheimer's disease (AD) pathogenesis. Production and clearance of different Aβ isoforms have been established as targets of proposed disease-modifying therapeutic treatments of AD. However, previous studies used multiple sequential purification steps to isolate the isoforms individually and quantitate them based on a common mid-domain peptide. We created a method to simultaneously purify Aβ isoforms and quantitate them by the specific C-terminal peptides in order to investigate Aβ isoform physiology in the central nervous system. By using standards generated from in vitro metabolic labeling, the relative quantitation of four peptides representing total amount of Aβ (Aβ-Total), Aβ38, Aβ40, and Aβ42 were achieved both in cell culture and in human cerebrospinal fluid (CSF). Standard curves for each isoform demonstrated good sensitivity with very low limits of detection and high accuracy. Because the assay does not require antibody development for each Aβ isoform peptide, significant improvements in the throughput and accuracy of isoform quantitation were achieved.

Keywords: Alzheimer’s disease; Amyloid-beta isoforms; Cerebrospinal fluid; Mass spectrometry; Relative quantitation.

Figure 1

Schematic diagram of the quantitative isoform characterization and kinetics assay, by using stable isotope labeling kinetics (SILK) technique. Aβ peptide isoforms were purified from the same sample source (media or CSF) using common epitope antibody bound to beads (HJ5.1) (A) and digested with Metalloendopeptidase (Lys-N) to generate isoform specific peptides (B). The digest was separated and quantitated by LC/SRM (C), resulting in unique SRM spectra from each peptide (D). The chromatographic elution profile of the various peptides as detected by SRM is as shown in (E). The least hydrophobic peptide common to all isoforms elutes first, followed closely by the peptide for Aβ38, then Aβ40 and Aβ42. Isotopic enrichment of each Aβ isoform was calculated as the ratio of the SRM peak area for labeled peptide to unlabeled peptide. This relative quantitation of chromatographic SRM peak ratios was computed for each time point of CSF collection, to generate a graph of Aβ isoform kinetics. Leucine residues (L) (colored gold) indicate ¹³C₆-Leucine labeling sites in the peptides. The unique C-terminal residues were highlighted in different colors to distinguish one unique isoform peptide from the other. Aβ42 is colored red, Aβ40 green and Aβ38 blue. The spectra were illustrated in colors to match those colors used for the C-terminal peptides they were generated from, with their corresponding gold-colored spectra from ¹³C₆-Leucine labeling.

Figure 2

The SRM chromatogram of Aβ isoforms detected from media standards labeled to 10% ¹³C₆-Leucine incorporation. The ion chromatograms demonstrate the elution profiles of the C-terminal peptides generated from the various Aβ isoforms from the same CSF sample, and aligned below the total ion count (TIC) plot (A). The peptides were eluted off the LC column based on their hydrophobicity. The first to elute was the peptide common to all isoforms, followed closely by the peptide for Aβ38, then Aβ40 and Aβ42. Peaks of ion pairs of unlabeled and ¹³C₆-Leucine labeled peptides, as detected by SRM were plotted below the TIC plot with their retention times. Among the 4 different peptides monitored were theAβ-Total, common peptide contributed by all major isoforms of Aβ, which eluted first (B). This was followed by the C-terminal peptides for Aβ38 (C), then Aβ40 (D), and Aβ42 (E). Isotopic enrichment of each Aβ isoform was calculated as the ratio of the area of the SRM peak for labeled peptide divided by the area of the unlabeled peptide. Sample labeled to 10% ¹³C₆-Leucine labeling was used as an example to show that the endogenous and ¹³C₆-Leucine labeled pairs co-elute.

Figure 3

Calibration curve plots of measured vs. predicted ratios of labeled Aβ isoform standards representing Aβ-Total (A), Aβ38 (B), Aβ40 (C) and Aβ42 (D). Labeled media was serially diluted (0, 1.25%, 2.5%, 5%, 10% and 20%) with unlabeled media to generate six sets for use in the production of standards for these curves. Aβ isoforms were immunoprecipitated from the media, Lys-N digested and the unique fragments were analyzed on TSQ Vantage MS. There were four biological replicates for each sample set. Due to very low errors in measurement, the point markers were not shown. Indicated instead were the standard deviations at the point marker locations. Measured ratio of labeled to unlabeled peptides (mean ± standard deviation between replicates), of each set are shown on the Y-axis with the predicted values on the X-axis. The linear regression lines show an accuracy (slope) of at least 0.9 and a precision (R²) of at least 0.999.

Figure 4

SILK profiles of Aβ isoforms to demonstrate the relative quantitation of production and clearance of Aβ isoforms in healthy human’s CSF over 36 hours. The isotopic enrichment time courses of Aβ 38 (blue squares), 40 (green triangles) and 42 (red triangles) are presented for each Aβ isoform, in relation to total Aβ (black circles). The kinetics curve shows there was no detectable incorporation of label in the first 4 hours. This was followed by an increase in percent labeled Aβ (production phase) at hours 5 to 15, which plateaued near steady-state levels of labeled leucine (up to about 10% at time points of between 18 to 20 hours), before decreasing (clearance phase) over the last 12 hours of the study (from time point 21 to 32 hours). The steady-state value (10%) was estimated as the average amount of labeled leucine in the CSF measured during labeling. From these curves, the Fractional Synthesis Rate (FSR) and Fractional Clearance Rate (FCR) of each isoform could be calculated as previously described [21]. The kinetic curves of Aβ38, Aβ40, and Aβ42 quantitated from CSF were compared to show the relationship between Aβ isoform metabolic kinetics.