An online nano‐LC‐ESI‐FTICR‐MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid

Abstract

Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), mainly long b-fragments are observed, limiting the possibility to determine variations such as amino acid variants or post-translational modifications (PTMs) within the N-terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann-Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O-glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top-down MS-based method.

Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure

“…106 It was used to identify novel α-synuclein isoforms in brain tissue and amyloid-β in cerebral spinal fluid. [107][108][109] These results demonstrated the utility of MS for structural studies. These proteins have been shown to have varied posttranslational modifications, which have been detected and characterized by mass spectrometry.…”

The role of protein glycosylation in Alzheimer disease

“…Kituzame et al showed that one additional O-glycan is present in the longer splice variant, APP770, in COS cells [26]. Several additional O-glycosylation sites in APP have recently been identified in human-derived CSF: Ser597, Ser606, Ser611, Thr616, Thr634, Thr635, Ser662, and Ser680 (numbering as in the canonical sequence, APP770, without the signal peptide) [27,28]. The roles of these O-glycans are elusive, although it has been proposed that APP processing by a-secretase, b-secretase and c-secretase occurs after O-glycosylation of APP, and that O-glycosylated APP is preferentially secreted [26,29].…”

Glycation vs. glycosylation: a tale of two different chemistries and biology in Alzheimer’s disease

“…Deletion of the glycan domain of APP as well as treatment of hippocampal neurons with tunicamycin [79] indicate that N-glycans are required for the proper axonal sorting and the secretion of APP [80]. The O-glycosylation of APP has also been reported to be important in the function of APP by several authors [81][82][83][84][85][86][87][88]. We found that APP is modified with sialylated O-glycans in brain endothelial cells and that the modified APP is preferentially cleaved and secreted [86], indicating that APP O-glycosylation regulates the APP processing.…”