Integrated Structural and Glycoproteomic Profiling Reveals Protein Conformational Remodeling and Biomarkers Across Alzheimer’s Disease Progression

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and protein misfolding, yet the structural dynamics of proteins and their post-translational modifications during disease progression remain poorly understood. Here, we present an integrated structural and glycoproteomic analysis of paired serum and cerebrospinal fluid (CSF) samples from individuals across three clinical stages: normal cognition, mild cognitive impairment, and AD. Using limited proteolysis mass spectrometry (LiP-MS) combined with high-field asymmetric waveform ion mobility spectrometry and data-independent acquisition, we identified 54 proteins exhibiting structural alterations, two of which (clusterin and ceruloplasmin) showed structural changes in both serum and CSF. Furthermore, our findings reveal potential crosstalk between protein structural changes and N-glycosylation, supported by correlations between LiP-derived structural features and glycosylation patterns in key proteins, such as haptoglobin and kininogen-1. This study demonstrates that integrating structural proteomics with glycoproteomics in matched serum and CSF samples enhances biomarker discovery and provides novel insights into the molecular mechanisms of AD. Our approach offers a powerful platform for identifying robust, minimally invasive biomarkers and for understanding post-translational modification-induced protein remodeling in neurodegenerative diseases.