What is the ideal sample type for IP-MS Aβ quantification?

The most common sample types for IP-MS Aβ analysis are:

What is the significance of the Aβ42/40 ratio in diagnosing Alzheimer's disease?

The Aβ42/40 ratio is a widely used metric in Alzheimer's diagnostics.

Can IP-MS detect Aβ oligomers?

Yes, one of the advantages of IP-MS is its ability to selectively isolate and quantify soluble Aβ oligomers (which are believed to be neurotoxic intermediates in Alzheimer's disease).

What is the sensitivity of IP-MS for detecting low levels of Aβ in plasma?

IP-MS is highly sensitive and can detect Aβ peptides at sub-picogram levels, depending on the sample type and the instrument used. The sensitivity is further enhanced by the immunoprecipitation step, which concentrates the Aβ peptides from plasma, a complex biological matrix, before they are analyzed by mass spectrometry.

How do you ensure specificity when detecting different forms of Aβ (monomers, oligomers, aggregates)?

Specificity is achieved by using highly selective antibodies that bind only to the target Aβ species. For example, 6E10 targets the first 16 amino acids of Aβ (Aβ1–16), while OC antibody specifically detects Aβ oligomers. Additionally, mass spectrometry allows for the precise detection of different molecular species based on their unique mass-to-charge ratio (m/z), ensuring that only the targeted Aβ forms are measured.

How does the choice of bead (Protein A/G vs. Dynabeads) affect the results?

Both Protein A/G beads and Dynabeads are commonly used for immunoprecipitation. Protein A/G beads are ideal for higher affinity antibody binding, but may require more washing steps to reduce non-specific binding. Dynabeads, on the other hand, offer high stability, uniformity, and are easier to handle for high-throughput workflows. The bead type might affect yield and specificity, so the choice depends on the experimental needs and sample type.

How do you quantify Aβ using IP-MS? Do you use internal standards?

Absolute quantification is performed by spiking samples with stable isotope-labeled (SIL) Aβ peptides before immunoprecipitation. After MS analysis, the amount of Aβ in the sample is compared to the SIL standard to determine the concentration of the endogenous peptides. This allows for precise and reproducible quantification, even in complex biological matrices.

What mass spectrometry methods are used to analyze the Aβ peptides after IP?

The analysis is typically performed using LC-MS/MS (Liquid Chromatography-Mass Spectrometry/Mass Spectrometry). Specifically, methods like Multiple Reaction Monitoring (MRM) or Parallel Reaction Monitoring (PRM) are used to target specific Aβ peptides (e.g., Aβ40, Aβ42) based on their unique mass-to-charge ratios. These techniques allow for highly sensitive and specific detection of endogenous Aβ peptides in complex biological samples.

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IP-MS Absolute Quantification of Amyloid-β Service Case Study

Title: Blood-based biomarkers and plasma Aβ assays in the differential diagnosis of Alzheimer's disease and behavioral-variant frontotemporal dementia

Journal: Alzheimer's Research & Therapy

Published: 2024

Background

The formation of neurotoxic amyloid beta (Ab) oligomers and plaques in specific brain regions is a key event in the pathogenesis of Alzheimer's disease (AD). The 42-amino acid isoform of Ab (Ab1-42) is thought to initiate plaque formation and AD progression. Various isoforms of Ab, such as Ab1-42, Ab1-40, and pGluAb3-42, have been detected in both sporadic AD (SAD) and familial AD (FAD) brains, but the relative importance of these isoforms in disease development is not fully understood. A study using immunoprecipitation and mass spectrometry analyzed Ab isoform patterns in the cerebellum, cortex, and hippocampus of AD subjects, including those with presenilin (M146V) or amyloid precursor protein (KM670/671NL) mutations, SAD patients, and non-demented controls. The results revealed that the dominant Ab isoforms in all brain regions were Ab1-42, pGluAb3-42, Ab4-42, and Ab1-40, with Ab1-42 and Ab4-42 being the primary isoforms in the hippocampus and cortex across all groups, including controls. There were no significant differences in Ab isoform patterns between FAD and SAD patients, suggesting similar amyloid pathology in both forms of AD.

Materials & Methods

IP-MS measurement of Aβ38, Aβ40 and Aβ42 in plasma samples (Ulm)

EDTA plasma samples, calibrators and QC samples (490μL each) were mixed with 15N-Aβ38, 15N-Aβ40 and 15N-Aβ42 (rPeptide, Watkinsville, GA, USA) as internal standards, with triethylammonium bicarbonate (TEAB, final 120mM) and Tween 20 (final 0.05%). Magnetic beads (0.5 mg per sample, Thermo 14302D) covalently coupled with 6E10 antibody (Biolegend, 2 μg/mg beads) were added to each sample and incubated on a rotator over night at 4 °C. Beads were washed three times with 500μL 50mM TEAB/0.1% n-Dodecyl-β-D-maltoside and eluted with 25μL 50mM glycine HCl (pH 2.5). The eluted Aβ peptides were digested with 10μL of a TrypN working solution (12.5ng/μL, Protifi, Fairport, NY, USA) for 2.5 h at 37 °C and stopped with 10μL of 0.5% TFA in acetonitrile and stored in the autosampler at 4 °C. A volume of 20μL was injected into a QTRAP6500 mass spectrometer (Sciex) coupled to an Eksigent MicroLC200 and Agilent 1260 pump. Peptides were loaded on an Acclaim PepMap100, C18 trap column (5 μm, 0.3×5 mm, Thermo) using mobile phase A: 0.05% TFA and mobile phase B: 90% acetonitrile, 0.1% ammonium hydroxide and a flow rate of 200μL/min. Separation of peptides was performed on a HALO Fused-Core C18, 100×0.5 mm analytical column (Eksigent, Framingham, MA, USA) at 60 °C and a gradient time of 9.85 min (5–35%B, total run time 17.5 min) with mobile A: 4% DMSO, 0.1% formic acid and mobile phase B: 4% DMSO, 96% acetonitrile, 0.1% formic acid. Peptides were infused into the QTRAP6500 mass spectrometer by electrospray ionization and measured in MRM mode using the following transitions: Aβ38 (aa28-38, 508.3→784.5 (b8+), 508.3→883.5 (b9+), 508.3→653.4 (b7+)); Aβ40 (aa28-40, 607.4→997.6 (b11+), 607.4→548.8 (b12++), 607.4→499.3 (b11++)); Aβ42 (aa28-42, 699.4→598.4 (b13++), 699.4→548.8 (b12++), 699.4→1096.7 (b12+)). Data were analysed using Skyline software v23.1 and for quantification, external calibration curves were generated using the light-to-heavy peak area ratios of calibrator samples and a quadratic function with 1/x² weighting. Calibrator samples (8-point calibration) were prepared in a surrogate matrix (3% bovine serum albumin in PBS) using synthetic Aβ38, Aβ40 and Aβ42 (Sigma) in the range of 1-100pg/mL (Aβ38, Aβ42) and 10-1000pg/mL (Aβ40). Plasma QC samples were included in all runs to monitor performance of measurements. The method was validated in terms of intraassay (1.2–10.5%) and interassay CV (3.4–7.4%), dilution stability (tested for 2- and 4-fold dilution, accuracy 89.9-110.7%), spike-in recovery (20pg/mL for Aβ38 and Aβ42, 200pg for Aβ40, recovery 93.1-100.4%) and stability at room temperature for 2h and up to 3 freeze-thaw-cycles (accuracy 80.2-108.1%). Intraassay CV of QC samples during measurement of patient samples was 1.2–10.5%.

Results

A strong correlation was observed between Aβ40 concentrations in patient samples, as determined by various assays (Fig. 1). The novel assay, referred to as "ULM," performed with IP-MS using ESI-MRM, demonstrated good correlation, with Spearman's rho values of 0.74 (P≤0.0001) when compared to the Shimadzu assay and 0.80 (P≤0.0001) when compared to the Simoa assay. For Aβ42 concentrations, a moderate to good correlation was seen, with Spearman's rho values of 0.61 (P≤0.0001) for the Shimadzu assay and 0.71 (P≤0.0001) for the Simoa platform. In contrast, the correlation for the Aβ42/40 ratio between the ULM assay and the Shimadzu assay was weak, with a Spearman's rho of 0.30 (P≤0.05), and a weak to moderate correlation of 0.46 (P≤0.001) with the Simoa platform's Aβ42/40 ratio. Comparatively, the correlations between the Shimadzu assay and the Simoa platform were modest but slightly lower, with Spearman's rho values of 0.68 (P≤0.0001) for Aβ40, 0.57 (P≤0.0001) for Aβ42, and 0.28 for the Aβ42/40 ratio. However, the latter correlation remained above the threshold of statistical significance (P>0.05).

Figure 1. Scatter plots visualizing the correlations between plasma concentrations of Aβ40 and Aβ42, along with the Aβ42/Aβ40 ratio, across various as says.

Conclusion

To summarize, this study developed an IP-MS assay for Aβ42/40, which showed comparable accuracy to the Shimadzu composite score in distinguishing Alzheimer's disease (AD) from control groups. This new assay outperformed both the Shimadzu Aβ42/40 ratio and the Simoa Aβ42/40 assay. The results indicate that Aβ levels alone do not offer additional diagnostic value when differentiating AD from behavioral variant frontotemporal dementia (bvFTD). Importantly, the combination of pTau181 and GFAP proves to be a powerful tool for the blood-based differential diagnosis of AD and bvFTD.

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