Uncovering new proteins in Alzheimer's disease patients by quantitative proteomics: blood-based markers and their connection to Amyloid-beta plaques

Alzheimer"s disease (AD) is the most common neurodegenerative disease-causing dementia. The disease mechanisms are still unclear, and thus, further research is needed for a better understanding of the pathology associated to the disease and for its early diagnosis. Studying proteins involved in AD pathogenesis can provide insights and identify potential AD markers, as well as therapeutic targets of intervention. Here, we have analyzed protein dysregulation in AD tissues compared to healthy individuals and patients with Vascular Dementia (VD) and Frontotemporal Dementia (FTD)"s tissues to identify key proteins in AD pathogenesis. Two proteomics strategies were used: TMT 10-plex quantitative proteomics using an Orbitrap Q Exactive and label free (LFQ) analysis of a pull-down with in vitro synthesized Aβ fibers using an Orbitrap Astral mass spectrometer.

For proteomics analyses, frozen left prefrontal cortex tissue samples from AD, VD, and FTD patients, and healthy individuals were used. TMT 10-Plex quantitative proteomics was performed with AD patients and healthy individuals and VD and FTD patients as controls. For the pull-down assays, in vitro synthesized biotinylated Aβ fibers were incubated with protein extracts from AD patients and healthy individuals. In addition, fibers induced from scramble Aβ peptides were used as negative controls of the assays.

Among the 3281 proteins identified by TMT, 15 and 154 proteins were found statistically upregulated and downregulated, respectively, in AD patients compared to controls. After bioinformatics analysis, 10 dysregulated proteins in AD were selected for further validation and to elucidate their role as blood-based biomarkers. Additionally, about 332 proteins were observed as potential interactors of Aβ fibers, with 18 proteins selected for validation.

The dysregulation of selected targets in AD patients was confirmed at mRNA and protein level. Importantly, two proteins showed potential as blood-based diagnostic biomarkers of AD, and seven candidate proteins were validated in vitro by WB and ex vivo using FFPE tissue of AD patients and controls and immunofluorescence and immunohistochemistry as novel Aβ plaques interactors, highlighting their role in AD development and progression.