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

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.