MSACL 2024 Abstract

Analyte Derivatization for Enhancement of Mass Spectrometry Response for Quantitation of β-amyloid Peptides

Ruben Y. Luo (1,2), Fangjun Chen (1), Priscilla S.-W. Yeung (1,2)
(1) Stanford University, Stanford, CA (2) Stanford Health Care, Palo Alto, CA

Fangjun CHEN, MD (Presenter) stanford university >> POSTER (PDF)

Presenter Bio: I am currently a visiting scholar at Stanford University and a technical director in the Mass Spectrometry Laboratory of the Laboratory of Clinical Laboratory, Zhongshan Hospital, Shanghai, China.I have developed many MS methods such as steroids, TDM, Catecholamines and etc.I have published 5 SCI papers about clinical MS.

Relevant Financial Disclosures (within past 24 months, reported on Feb 28, 2024) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and memory impairment. Accurate and early diagnosis of AD is crucial for effective management and intervention strategies. It is known that the presence of β-amyloid (Aβ) plaques, along with neurofibrillary tangles consisting of aggregated tau protein, are hallmark neuropathological features of AD. Therefore, beta-amyloid 1-40 (Aβ40), beta-amyloid 1-42 (Aβ42), total tau, and phosphorylated tau (p-tau) are most widely used diagnostic markers for AD. Numerous studies have demonstrated the utility of Aβ40 and Aβ42 as diagnostic markers for AD.2 The Aβ42/Aβ40 ratio has shown superior diagnostic accuracy compared to individual measurements alone. As amyloid-based therapies are going to become standard of care in clinics, testing of plasma biomarkers for AD and possibly other neurodegenerative diseases will become critical for diagnosis and therapy monitoring.

The diagnostic markers Aβ40 and Aβ42 can be quantitated by Sandwich immunoassays. Although the immunoassays are able to detect the Aβ peptides, the assay performance is limited due to the propensity of non-specific binding of the analytes. This disadvantage caused high intra- and inter-laboratory variability and spurred the development of alternative approaches. On the other hand, mass spectrometry methodologies, more particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS), improves intra- and inter-laboratory precision. LC-MS/MS has exquisite analyte selectivity derived from the use of multiple reaction monitoring (MRM). LC separates analytes in time, and MS/MS enables further selectivity by the analyte’s mass-to-charge (m/z) ratio and subsequent confirmation of the analyte’s primary structure by fragmentation. The challenge to LC-MS/MS quantitation of Aβ peptides in plasma is analytical sensitivity, as the concentrations in plasma can be two orders of magnitude lower than those in cerebrospinal fluid (CSF). Analyte derivatization can be a highly effective and efficient solution to enhance the analytical sensitivity of LC-MS/MS to measure endogenous Aβ40 and Aβ42 in plasma.

Method

Aβ peptides could be derivatized by dansyl chloride (5-N,N-dimethylaminonaphthalene-1-sulfonyl chloride), dabsyl chloride (4-N,N-dimethylaminoazobenzene-4’-sulfonyl chloride), and other sulfonyl chlorides. The derivatization reaction took place on amino groups, phenol groups, and aromatic rings in a peptide. To implement the derivatization reaction, the derivatization reagent was directly added into Aβ peptide samples, and the mixture was incubated at 40°C for 1.5 hr. Ammonium hydroxide in water was added to quench the reagent.

LC-MS/MS analysis was implemented in a Vanquish Flex-Orbitrap Q-Exactive system (Thermo Fisher Scientific). The LC separation was carried out using a C18 column, and a gradient elution of mobile phase A (0.1% ammonium hydroxide in water) and mobile phase B (ACN). The MS analysis was carried out in MRM mode.

Results

Derivatized Aβ peptides were observed in mass spectra, and each peptide contained multiple derivatization groups on lysine side chain, tyrosine side chain, phenylalanine side chain, and peptide N-terminus. Although the derivatization method did not significantly increase the ionization efficiency of Aβ peptides, it significantly enhanced the MRM response by providing higher-abundance signature fragments resulted from the derivatization groups. For example, dansyl chloride derivatization resulted in a signature fragment at 317 m/z.

Conclusion

The novel derivatization method is an effective and efficient approach to enhance the MS response of Aβ peptides. It has a great potential to be applied to the clinical testing of Aβ peptides in plasma samples. Compared with other MS response enhancement methods such as enzymatic digestion, the derivatization method is much easier and faster to implement and more compatible with clinical laboratory settings.