Single-subject Proteomic Signatures in Alzheimer’s Disease Reflect Clinical Phenotypes and Distinguish Asymptomatic from Symptomatic Cases

INTRODUCTION: Alzheimer's Disease (AD) exhibits considerable inter-individual variability in clinical presentation, neuropathological burden, and underlying molecular processes. Conventional cohort-based analyses of omics molecular data often mask individual-level heterogeneity, limiting insights into the precision therapeutic strategies. To address this challenge, we developed INDIGO (INdividual-level DIfferential GenOmics), a computational framework that quantifies molecular deviations for each individual relative to healthy controls, enabling subject-specific profiling of diseaseassociated alterations in proteomic data, with a framework that is readily applicable to other omics modalities. METHODS: We applied INDIGO to dorsolateral prefrontal cortex (DLPFC) proteomic data from ROSMAP cohort (N = 610). Protein level deviations were aggregated into gene set activity scores for KEGG pathways and curated AD Biodomain annotations. Functional alterations across AD and Asymptomatic AD (AsymAD) individuals were evaluated and correlated with clinical metrics including APOE4 genotype, Braak stage, CERAD and MMSE scores. Graph-based clustering was used to identify molecularly distinct subgroups based on shared patterns of functional dysregulation. RESULTS: Limited overlap was observed between cohort-level differential expression analysis and INDIGO single-subject analyses. Individual deviations in various processes, including metabolic, immune and epigenetic pathways, exhibited sex- and disease stage-specific patterns. Amyloid clearance and immune activation were strongly associated with APOE4 dosage, higher amyloid and tau burden and cognitive decline, whereas upregulation of mitochondrial and synaptic modules correlated positively with preserved cognitive function. By linking individuals through concordant directional proteomic changes, we identified molecularly coherent subgroups that transcend conventional diagnostic boundaries and include both AD and AsymAD subjects. Each subgroup displayed distinct functional signatures and defined by a unique set of key regulatory proteins. DISCUSSION: These results demonstrate that single-subject omics profiling can resolve individual molecular signatures aligned with clinical and neuropathological variation in AD. By linking molecular heterogeneity with disease phenotypes, INDIGO provides a scalable framework for precision modeling and novel therapeutic target discovery.