Mass Spectrometry & Spectroscopy
MS detection of Alzheimer’s blood-based biomarkers From a few drops of blood – the fi rst successful detection of plasma Aβ by MS Dr Naoki Kaneko, Shimadzu Corporation
Amyloid β (Aβ) accumulation in the brain is considered as the earliest sign of Alzheimer’s disease (AD) pathology. Recently developed disease-modifying therapies (DMTs) target patients with early-stage AD, hence blood-based biomarkers are needed for a detection of Aβ accumulation. We combined immunoprecipitation (IP) with mass spectrometry (MS) to develop an IP-MS technique that resulted in the fi rst successful detection of plasma Aβ by MS. This technique revealed a high concordance between a composite biomarker of two plasma Aβ ratios and amyloid PET status. As advances are made in DMTs, blood-based biomarkers will become increasingly valuable due to their key role for screening patients, monitoring drug effects and diagnosis.
Plasma amyloid β biomarker assay – creating the early bird for AD diagnosis
Alzheimer’s disease (AD) is the main cause of dementia, accounting for 60 to 70% of all dementia cases. An accumulation of amyloid β (Aβ) in the brain is considered as the earliest sign in the continuum of AD pathological changes and starts 20 to 30 years prior to symptom onset (Figure 1) [1]. Therefore, a biomarker refl ecting the AD pathology plays a key role in the early diagnosis of AD. The detection methods for Aβ biomarker were amyloid positron emission tomography (PET) and immunoassay for cerebrospinal fl uid (CSF) Aβ1-42 and Aβ1-42/1-40.
Amyloid MS: IP-MS methodology using MALDI-TOF MS
Immunoassays have been used in plasma Aβ biomarker research since 1996. However, many studies of plasma Aβ as a biomarker produced confl icting reports regarding the status of plasma Aβ due to its analytical diffi culty, resulting in sceptical views about its utility for many years [5]. MS detects different peptides by separating them according to the mass with high sensitivity. Thus, MS can accurately and simultaneously detect even similar peptides such as Aβ1-38, Aβ1-40, Aβ1-42, etc. However, when a sample contains large amounts of impurities, the strong signal from these impurities drowns out the weak signal from analytes only present in small amounts. Blood plasma samples typically contain large amounts of impurities. This has prevented the use of mass spectrometry to measure biomarkers in plasma samples with high sensitivity.
The introduction of an antibody-based immunoprecipitation (IP) step into the sample preparation enabled a great success: selectively separating and concentrating only Aβ. An IP-MS method for detecting Aβ was already reported in 1996 and could readily detect Aβ in samples of cell culture supernatant and CSF [6], but blood has many more impurities and lower Aβ concentrations than culture supernatant and CSF, preventing the detection of Aβ in blood by IP-MS.
Refi ning the conditions for IP enabled the fi rst detection of Aβ1-42 and Aβ1-40 in human plasma
Optimised conditions used during IP helped to overcome these issues. After examining the preparation of antibody-coated beads, surfactants and eluate compositions during IP, a matrix solution composition was selected, appropriate for samples after IP to establish IP-MS methodology for plasma Aβ analysis using MALDI-TOF MS (AXIMA Performance) (Figure 2).
Figure 1. AD progression model.
However, amyloid PET requires a large PET equipment and is expensive, and CSF Aβ causes more invasiveness of collecting CSF. Therefore, there was a need for a blood-based biomarker that can be measured easily. In recent years, the clinical utility of plasma biomarkers has been reported with the signifi cant technological advancements for plasma biomarker measurement. Main technologies that measure plasma Aβ are an immunoprecipitation coupled with mass spectrometry (IP-MS) and a sandwich immunoassay employing two kinds of antibodies. A head-to-head study for comparison of plasma Aβ assays has reported a higher accuracy in IP-MS than in the immunoassay [2, 3, 4].
In the following passage, amyloid MS technique using IP-MS methodology is highlighted with matrix-assisted laser desorption/ionisation time of fl ight MS (MALDI-TOF MS) and high performance of plasma Aβ biomarkers in clinical studies.
Figure 2. Amyloid MS: IP-MS methodology for plasma Aβ peptides.
LAB ASIA - AUGUST 2024
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