Abstract
The expression of human chorionic gonadotropin (hCG) is com-monly associated with a variety of cancers including testicular,bladder and gestational trophoblast diseases. All trophoblastictumours secrete very high amounts of hCG however hyperglyco-sylated form of hCG was found to be highly elevated in chorio-carcinoma patients. Current analytical methods, measuringbranched N-linked glycans in hCG, involve use of antibodieswhich may cause uncertainty due to issues of antibody specificity.In this project, highly sensitive and robust method to identify N-linked glycans, based on their specific chemical composition andmass tolerance, was developed. It was applied to variety of hCGforms, including hCG from pregnancy, choriocarcinoma andrecombinant hCG. hCG and its varied forms, with freebhCG,were isolated using IgG-coupled magnetic beads. N-linked gly-cans were then isolated from hCG by denaturation, reduction,alkylation (100°C, 10mM DTT, 20mM IAA) followed by degly-cosylation (PNGase, 37°C, 20h) and purification using porousgraphite carbon tips (Hypercarb). MALDI IT-TOF MS was usedto separate resulting glycans, which were then characterised usingProtein Scape software. Glycan peaks were reported, m/z 900–5,000 (Da), and carbohydrate library with precise glycan searchparameters (GlycoQuest, Bruker) was designed. A series of gly-cans were successfully identified from selected hCG sources, andsignificant differences in repertoire of glycans composition weredetected. Alteration in cellular glycosylation of proteins con-tribute to cancer metastasis, therefore strategies to understandtumour glycome and glycoproteome provide diagnostic potential,and targets for therapeutic intervention. Rapid and efficientdetection method of specific hCG glycoforms, from crudepatients urine samples, could help to speed and improve diagno-sis of hCG associated cancers including choriocarcinoma andnon-trophoblastic malignancies.
Highlights
It has been estimated that only Alzheimer’s disease (AD) alone, the most common form of dementia, will affect approximately 81 million individuals by 2040
There has been a stronger focus on proteomics and metabolomics studies with the hope of increasing our understanding of the underlying mechanisms leading to disease
Technological advancements in high-resolution mass spectrometry and rapid improvements in chromatographic techniques have led to quick expansion of the field of in this research
Summary
It has been estimated that only Alzheimer’s disease (AD) alone, the most common form of dementia, will affect approximately 81 million individuals by 2040. We will present our discovery data on growth hormone (GH) isoforms, differentially expressed proteins (n = 56) between controls and macroadenomas, novel phosphoproteins, and oxidative stress nitroproteins (NS). There is no definitive blood test or simple diagnostic method for many of those diseases so far available. There has been a stronger focus on proteomics and metabolomics (e.g. lipidomic) studies with the hope of increasing our understanding of the underlying mechanisms leading to disease.
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