Plasma Proteome and Phosphoproteome Analysis Across Alzheimer’s Disease Stages by LC–MS/MS

Abstract

Blood-based molecular features associated with the Alzheimer’s disease (AD) continuum have attracted growing interest as minimally invasive manifestations of systemic AD-related biology. In this study, integrated whole-proteome and phosphoproteome profiling of human plasma was performed across four clinically defined stages: cognitively normal amyloid-negative (CONneg), cognitively normal amyloid-positive (CONpos), mild cognitive impairment (MCI), and Alzheimer’s disease dementia (AD). Parallel data-dependent acquisition (DDA) and data-independent acquisition (DIA) LC–MS/MS workflows were implemented to evaluate acquisition performance and characterize stage-associated plasma proteomic alterations. DIA achieved greater proteome and phosphoproteome coverage, reduced missingness, and improved quantitative reproducibility relative to DDA, providing a more robust foundation for downstream biological analyses. DIA-based differential expression analysis revealed organized stage-associated functional patterns rather than abrupt qualitative transitions. Immune- and extracellular matrix (ECM)/adhesion-related processes were already evident in CONpos individuals, expanded in MCI to include lectin-pathway complement activation and acute-phase features, and further extended in AD to encompass humoral immune responses, terminal complement components, leukocyte-associated functions, and oxidative stress–related processes. A subset of proteins, including complement-associated components, ECM/adhesion-related proteins, and metabolic regulators, displayed consistent directional changes across multiple stage comparisons, delineating reproducible systemic features along the AD continuum. Phosphoproteomic profiling revealed complementary regulatory alterations across disease stages. Phosphopeptide-level changes exhibited coherent and stage-consistent organization along the AD continuum. A broad set of phosphopeptides showed pronounced decreases in AD, with overlapping alterations already observable at earlier pathological stages. These decreased phosphopeptides were predominantly derived from proteins associated with cytoskeletal organization, platelet/coagulation pathways, and membrane- or vascular interface–related functions, indicating coordinated downregulation of phosphorylation across structural and circulatory protein groups. Collectively, this study provides a stage-resolved characterization of plasma proteomic and phosphoproteomic alterations across the AD continuum, supported by DIA-based profiling, and establishes an integrated framework for interpreting systemic molecular changes in Alzheimer’s disease.