A proposed key event in the pathogenesis of Alzheimer’s disease (AD) is the formation of neurotoxic amyloid β (Aβ) oligomers and amyloid plaques in specific brain regions that are affected by the disease. The main plaque component is the 42 amino acid isoform of Aβ (Aβ1-42), which is thought to initiate plaque formation and AD pathogenesis. Numerous isoforms of Aβ, e.g., Aβ1-42, Aβ1-40 and the 3-pyroglutamate derivate of Aβ3-42 (pGluAβ3-42), have been detected in the brains of sporadic AD (SAD) and familial AD (FAD) subjects. However, the relative importance of these isoforms in the pathogenesis of AD is not fully understood. Here, we report a detailed study using immunoprecipitation in combination with mass spectrometric analysis to determine the Aβ isoform pattern in the cerebellum, cortex and hippocampus in AD, including subjects with a mutation in the presenilin (M146V) or amyloid precursor protein (KM670/671NL) genes, SAD subjects and non-demented controls. We show that the dominating Aβ isoforms in the three different brain regions analyzed from control, SAD, and FAD are Aβ1-42, pGluAβ3-42, Aβ4-42 and Aβ1-40 of which Aβ1-42 and Aβ4-42 are the dominant isoforms in the hippocampus and the cortex in all groups analyzed, controls included. No prominent differences in Aβ isoform patterns between FAD and SAD patients were seen, underscoring the similarity in the amyloid pathology of these two disease entities.

We present a method to enrich for glycoproteins from proteomic samples. Sialylated glycoproteins were selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of sialic acid glycosidic bonds. Mass spectrometric fragment analysis allowed identification of glycan structures, and additional fragmentation of deglycosylated ions yielded peptide sequence information, which allowed glycan attachment site and protein identification. We identified 36 N-linked and 44 O-linked glycosylation sites on glycoproteins from human cerebrospinal fluid.

These results suggest that CSF Ng is a novel AD biomarker that may be used to monitor synaptic degeneration, and correlates with the rate of cognitive decline in prodromal AD.

Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.