Parallel global proteome and glycaproteome of human brain reveal altered glycation of key proteins linked to Alzheimer's disease

Although amyloid-beta (Aβ) deposition and tau neurofibrillary tangles in brain are hypothesized as a central force driving Alzheimer"s disease (AD) pathogenesis, the mechanism underlying the development pathology is unknown. Protein glycation with primary amine groups of proteins reacting with aldehyde or reactive carbonyl group of a sugar affects structure, bioactivity and molecular stability. While increased glycation has been reported in AD brains, omic studies have never been done. Here we analyzed proteome and glycaproteome of brains from AD patients and cognitively normal individuals in parallel to explore roles of glycation in AD pathology, and to identify potential new biomarkers and novel therapeutic targets.

Proteins were extracted from human brains of five controls and five AD patients. TMT10-plex labeling was used for global proteomics and boronate affinity enrichment of glycapeptides was performed for glycaproteome. All samples were analyzed by nanoLC-MS/MS on Orbitrap Eclipse and data processed by Proteome Discoverer.

In the global proteome, 530 proteins of the 4,848 that were quantified with statistical differences between AD and controls. Confident quantification of 814 glycapeptides from 395 glycaproteins revealed six different glycated forms including Nε-fructosyl, on both Lys and Arg residues were determined.

Glycation peptides were consistently increased in the AD samples. While 105 unique glycated peptides from 73 unique glycated proteins were significantly increased (p<0.05) in AD patients, no glycapeptides were significantly decreased. Glycation of several key proteins associated with AD were increased including amyloid-beta precursor protein (APP), Tau,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glial fibrillary acidic protein (GFAP), fibrinogen and synaptophysin. APP is the precursor protein that when cleaved generates the β-amyloid peptides, and was found with Nε-fructosyl modification on its key K612 residue. This is remarkable as we found previously the same site of APP was modified by succinylation, which is linked to form amyloid plagues. Increased glycation was found on K311, K353, K369 and K375 of Tau protein. K311 was succinylated promoting pathological tau aggregation to tangles and impairing microtubule assembly. The increased glycation fold change over the change of their protein abundance suggests that glycapeptides may represent sensitive biomarkers of AD. Our data also suggest there appears crosstalk between glycation and other post-translation modification such as succinylation in potential regulation of AD pathogenesis.