Structural Proteomic Profiling of Cerebrospinal Fluids to Reveal Novel Conformational Biomarkers for Alzheimer’s Disease

Abstract

Alzheimer's disease (AD) is the most common representation of dementia, with brain pathological hallmarks of protein abnormal aggregation, such as with amyloid beta and tau protein. It is well established that posttranslational modifications on tau protein, particularly phosphorylation, increase the likelihood of its aggregation and subsequent formation of neurofibrillary tangles, another hallmark of AD. As additional misfolded proteins presumably exist distinctly in AD disease states, which would serve as potential source of AD biomarkers, we used limited proteolysis-coupled with mass spectrometry (LiP-MS) to probe protein structural changes. After optimizing the LiP-MS conditions, we further applied this method to human cerebrospinal fluid specimens collected from healthy control, mild cognitive impairment (MCI), and AD subject groups to characterize proteome-wide misfolding tendencies as a result of disease progression. The fully tryptic peptides embedding LiP sites were compared with the half-tryptic peptides generated from internal cleavage of the same region to determine any structural unfolding or misfolding. We discovered hundreds of significantly up-and down-regulated peptides associated with MCI and AD indicating their potential structural changes in AD progression. Moreover, we detected 53 structurally changed regions in 12 proteins with high confidence between the healthy control and disease groups, illustrating the functional relevance of these proteins with AD progression. These newly discovered conformational biomarker candidates establish valuable future directions for exploring the molecular mechanism of designing therapeutic targets for AD.

Probing Protein Structural Changes in Alzheimer’s Disease via Quantitative Cross-linking Mass Spectrometry

“…The K251−K260 interaction occurs within the C-terminal regions, which is implicated in oligomer formation and amyloid β (Aβ) binding. 37 Our quantitative findings highlight the consistency in the abundance of this linkage across multiple sample sets for comparison (Figure 5a), suggesting that the potential pathological binding between ApoE and heparan sulfate (HS) remains unaffected under the MCI stage. 53 The underlying hypothesis of HS-assisted accumulation and induction with the tau protein is not supported.…”

“…Intralink analysis provides insights, indicating that specific interactions within the helix structures are retained in both conditions: ApoE K90–K113 and ApoE K251–K260. The K251–K260 interaction occurs within the C-terminal regions, which is implicated in oligomer formation and amyloid β (Aβ) binding . Our quantitative findings highlight the consistency in the abundance of this linkage across multiple sample sets for comparison (Figure a), suggesting that the potential pathological binding between ApoE and heparan sulfate (HS) remains unaffected under the MCI stage .…”

“…While numerous publications have reported the CSF proteomic and corresponding pathological alterations during AD progression, the structural information within the protein complex or docking domains remains elusive. − Hence, our quantitative XL-MS workflow, executed through Skyline, presents a novel strategy highlighting site-specific interactions between CSF biomarkers. The cross-linking data were processed according to the methodology outlined in the Experimental Section.…”

The application of NLRP3 inflammasome inhibition in Alzheimers disease therapeutics: Clinical benefits, applications, current limitations, and future development

“…With the development of new screening techniques such structural proteomic profiling [22], the screening of AD genetic changes and studies on potent target sites for NLRP3 inflammasome inhibitors would be greatly helpful for the progression of drug development and discovery of NLRP3 inhibitors. Additionally, the development of the CRISPR technology has enabled the utilization of CRISPR in the diagnosis and therapeutics in AD.…”

Convolutional Neural Networks for Alzheimer’s Disease

“…These biomarkers are essential for early diagnosis, tracking disease progression, and monitoring the effectiveness of potential treatments [8]. There are several types of biomarkers associated with Alzheimer's disease, as shown in Figure 2: Biomarkers in Cerebrospinal Fluid (CSF): Analysis of cerebrospinal fluid, which surrounds the brain and spinal cord, can reveal specific biomarkers associated with Alzheimer's disease [9]. Two key biomarkers in CSF are elevated levels of tau protein and decreased levels of beta-amyloid [10].…”