Alzheimer's disease (AD) is one kind of irreversible neurodegenerative disease (NDD), which affects daily life and also normal social function [40]. As life expectancy increases and the population ages, the overall prevalence of Alzheimer's disease (AD) is anticipated to rise continuously across the globe, posing a significant burden, particularly in developing countries. While some treatments can remit the symptoms of AD, there remains a pressing demand to raise the understanding of pathogenesis to facilitate the development of disease-modifying therapy [31]. Moreover, the current therapy strategies merely alleviate symptoms and almost incurable for AD. Consequently, there is an urgent need for novel detection targets and innovative medications to prevent or effectively treat AD.

The most essential aim of proteomics is to comprehensively identify and quantify the entire complement of proteins, along with their modifications, within the biological samples of interest [14]. Recently, the revolution in the mass spectrometer design and information biology algorithms have led to rediscovery and progress of other revolutionary method, such as data independent acquisition (DIA) [36]. While it overlaps with the scope of data-independent acquisition (DDA) in terms of identification, it nonetheless achieves the reproducible quantification characteristic associated with multiple reaction monitoring (MRM) or its modern incarnation known as parallel reaction monitoring (PRM) [3]. The DIA analysis, aided by spectral libraries derived from prior DIA tests or ancillary DDA data, enables the identification and quantification of peptides with reproducibility and accuracy comparable to that achieved by MRM/PRM, except for peptides with very low abundance that may be obscured by stronger signals [27]. The outlook in future is vigorous and different researches are focusing on new bioinformatics techniques to deal with the issues and promote the reproducibility and recognition scope of DIA [32]. In this current study, we have employed DIA in our work to maximize the proteomic data in AD progression [23].

PRM is a targeted MS method which not only simultaneously monitors the whole fragment ions of the chosen precursor ions, but also permits the acquisition of comprehensive MS/MS spectra for every exact peptide [24,27]. The identification of peptides is enhanced by the progress of hybrid high-resolution mass spectrometers, which provide superior specificity and quantitative accuracy [9]. The technique is well-suited for the research encompassing disease markers, drug targets, host-pathogens and underlying disease mechanism [37].

The chemical compounds and active ingredients come from Chinese herbal medicine have been used in treating dementia for nearly a thousand years. Potential active ingredients have been recognized to put off the development of dementia, promoting the cognitive dysfunction and remit the mental and behavior disorders [2]. Tetrahydroxy stilbene glycoside (TSG), an active component come from Polygonum multiflorum, has garnered more and more interests as a therapeutic option for the NDDs [33,38]. Numerous studies have consistently corroborated the pharmacological functions of it in facilitating brain injury repair, particularly Parkinson's disease [25,10]. Additionally, TSG remitted the learning and memory capabilities in AD rats and aged mice as well [42]. Nonetheless, the accurate mechanisms underlying the positive functions within TSG in AD remain poorly elucidated, especially on proteins or peptides.

Using the DIA phosphorylated proteomics technique, we aimed to identify and analyze the phosphorylated proteins in NDDs. The raw data were analyzed by various bioinformatics methods to detect the phosphorylation events related to TSG. Furthermore, the PRM conducted a thorough examination of the DIA data. Additionally, we identified the key metabolite classes revealing from the analyses to the important biological pathways relevant in AD to clear the promising functions of the molecules and the interactions in causing the symptom onset. Meanwhile, we have clarified the alterations in proteins, protein phosphorylation, phosphorylation kinases, and internal connections within the context of AD onset and TSG intervention.