Glycosylation of Human Plasma Clusterin Yields a Novel Candidate Biomarker of Alzheimer's Disease

Specific glycosylated peptides of clusterin are found associated with hippocampal atrophy. The glycosylation of clusterin from human plasma was comprehensively analyzed and characterized using mass spectrometry (MS)-based glycoproteomics analysis. All six known N-glycosylation sites are covered, three in the alpha subunit (alpha 64N, alpha 81N and alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). More detailed structural characterization of clusterin glycopeptides was also performed, demonstrating the presence of glycosylated peptides and their corresponding glycans. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we have determined the differences in the glycoforms associated at each of the different glycosylation sites in plasma clusterin obtained from subjects of low hippocampal atrophy (n = 13) and high hippocampal atrophy (n = 14). In our pilot study, the beta 64N site shows the most significant regulations between clinical groups. Eight beta 64N glycoforms are significantly reduced in patients with high atrophy compared with those with low atrophy, which demonstrates the utility of clusterin isoforms as diagnostic and prognostic Alzheimers disease (AD) markers. These results provide a novel and robust workflow suitable for rapid verification of specific clusterin glycoforms with utility as AD biomarkers.