Targeting the human glycoproteome New enrichment protocols and mass spectrometric analyses reveal unique and novel glycosylation sites
Year: 2012
Type: dissertation
Abstract: Glycosylation is one of the most common and structurally diverse posttranslational\nmodifications of proteins. Given that protein glycosylation is\ninvolved in various cellular processes, the characterization of site-specific\nN- and O-linked glycosylations is becoming increasingly important.\nHowever, current mass spectrometry-based technologies, i.e. proteomics\nand glycomics, are unable to resolve the site-specific glycosylation pattern\nof glycoproteins.\nThe primary aim of this thesis was to develop glycoproteomic techniques\nfor mass spectrometric analysis of glycoproteins. A sialic acid captureand-\nrelease method, based on hydrazide chemistry, for selective\nenrichment of N- and O-linked glycopeptides from complex biological\nsamples was developed. Enriched glycopeptides were separated by\nreversed phase liquid chromatography and analyzed by Fourier transform\nion cyclotron mass spectrometry (FTICR MS) utilizing collision induced\ndissociation (CID) and electron capture dissociation (ECD) fragmentation\ntechniques.\nInitially, both N- and O-glycopeptides from sialylated glycoproteins of\nhuman cerebrospinal fluid (CSF) were enriched and characterized.\nSubsequently, a targeted O-glycoproteomics approach was developed,\nallowing for sequence analysis of preferred O-glycosylation sites of\nglycoproteins. The applicability of the sialic acid capture-and-release\nstrategy was further demonstrated for human urine, a technically more\nchallenging biological fluid. The LC-MS/MS analyses revealed unique N- and\nO-glycosylations, many of which were previously unknown, both for\nCSF and urinary glycoproteins. In e.g. CSF, a series of O-glycopeptides\nwith Thr linked O-glycans in the vicinity of the β-secretase cleavage site\nof the amyloid precursor protein (APP) were identified. Additionally,\namyloid beta (Aβ) peptides, originating from APP, were\nimmunoprecipitated from CSF samples for a targeted glycoproteomic\nanalysis. These analyses revealed that a series of Aβ peptides were\nuniquely modified with sialylated O-glycans at a specific Tyr residue. A\nrelative increase of such Tyr O-glycosylated Aβ peptides was observed in\nCSF samples from Alzheimer’s disease (AD) patients compared to non-AD patients, suggesting that these Aβ glycopeptides may potentially be\nused as biomarkers of AD.
Author: Adnan Halim
Institution: University of Gothenburg
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2169142034