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Mass spectrometry-based glycoproteomics has gone through some incredible developments over the last few years. Technological advances in glycopeptide enrichment, fragmentation methods, and data analysis workflows have enabled the transition of glycoproteomics from a niche application, mainly focused on the characterization of isolated glycoproteins, to a mature technology capable of profiling thousands of intact glycopeptides at once. In addition to numerous biological discoveries catalyzed by the technology, we are also observing an increase in studies focusing on global protein glycosylation and the relationship between multiple glycosylation sites on the same protein. It has become apparent that just describing protein glycosylation in terms of micro- and macro-heterogeneity, respectively the variation and occupancy of glycans at a given site, is not sufficient to describe the observed interactions between sites. In this perspective we propose a new term, meta-heterogeneity, to describe a higher level of glycan regulation: the variation in glycosylation across multiple sites of a given protein. We provide literature examples of extensive meta-heterogeneity on relevant proteins such as antibodies, erythropoietin, myeloperoxidase and a number of serum and plasma proteins. Furthermore, we postulate on the possible biological reasons and causes behind the intriguing meta-heterogeneity observed in glycoproteins.

Erythropoietin (EPO), a glycoprotein hormone of ∼34 kDa, is an important hematopoietic growth factor, mainly produced in the kidney and controls the number of red blood cells circulating in the blood stream. Sensitive and rapid recombinant human EPO (rHuEPO) detection tools that improve on the current laborious EPO detection techniques are in high demand for both clinical and sports industry. A sensitive aptamer-functionalized biosensor (aptasensor) has been developed by controlled growth of gold nanostructures (AuNS) over a gold substrate (pAu/AuNS). The aptasensor selectively binds to rHuEPO and, therefore, was used to extract and detect the drug from horse plasma by surface enhanced Raman spectroscopy (SERS). Due to the nanogap separation between the nanostructures, the high population and distribution of hot spots on the pAu/AuNS substrate surface, strong signal enhancement was acquired. By using wide area illumination (WAI) setting for the Raman detection, a low RSD of 4.92% over 150 SERS measurements was achieved. The significant reproducibility of the new biosensor addresses the serious problem of SERS signal inconsistency that hampers the use of the technique in the field. The WAI setting is compatible with handheld Raman devices. Therefore, the new aptasensor can be used for the selective extraction of rHuEPO from biological fluids and subsequently screened with handheld Raman spectrometer for SERS based in-field protein detection.

This thesis developed a new method for measuring extremely low amounts of organic and biological molecules, using Surface enhanced Raman Spectroscopy. This method has many potential applications, e.g. medical diagnosis, public health, food provenance, antidoping, forensics and homeland security. The method development used caffeine as the small molecule example, and erythropoietin (EPO) as the large molecule. This method is much more sensitive and specific than currently used methods; rapid, simple and cost effective. The method can be used to detect target molecules in beverages and biological fluids without the usual preparation steps.

Hypoxic ischemic brain injury (HIBI) is a common cause of neonatal mortality and morbidity. Approximately 60 % of preterm babies and 2% of full term infants suffer from asphyxia. Asphyxia related death accounts for approximately 23% of neonatal mortality annually. Many therapeutic interventions show promise in the laboratory but fail in clinics. A thorough understanding of mechanisms by which promising therapeutic intervention confers its neuroprotection is necessary to promote smoother transitions from bench to the bedside. Erythropoietin (EPO), a hematopoietic growth factor that increases oxygen availability during hypoxia/ischemia is associated with cell survival and neuroprotection in: in vivo and in vitro models of hypoxia ischemia. However these studies hold limited clinical translations because the underlying mechanism remains unclear and the key molecules involved in EPO-induced neuroprotection are still to be determined. Thus the central aim of this proposal is: to determine the key mediators of EPO-induced neuroprotection and the mechanisms by which this occurs in: in vitro and in vivo models of hypoxia ischemia. Two alternate pathways of EPO-induced neuroprotection were elucidated in vitro using oxygen and glucose deprivation to mimic hypoxia ischemia. Our findings showed that EPO treatment resulted in an upregulation of tissues inhibitor of matrix metalloproteinase (TIMP)-1 and inhibition of hypoxia inducible factor (HIF-1α) both of which subsequently decreased in matrix metalloproteinase (MMP)-9 and promoted neuroprotection. MMP-9 inhibition was associated with neuroprotection in both pathways thus, inferring that inhibition of MMP-9 is one of the primary mechanisms of EPO-induced neuroprotection. We also observed that EPO-induced neuroprotection was reversed by inhibition of TIMP-1 or prolyl hydroxylase (PHD)-2. This lends to the conclusion that TIMP-1 and PHD-2 are the key mediators of EPO-induced neuroprotection in: in vivo and in vitro models of hy

Hypoxic ischemic brain injury (HIBI) is a common cause of neonatal mortality and morbidity. Approximately 60 % of preterm babies and 2% of full term infants suffer from asphyxia. Asphyxia related death accounts for approximately 23% of neonatal mortality annually. Many therapeutic interventions show promise in the laboratory but fail in clinics. A thorough understanding of mechanisms by which promising therapeutic intervention confers its neuroprotection is necessary to promote smoother transitions from bench to the bedside. Erythropoietin (EPO), a hematopoietic growth factor that increases oxygen availability during hypoxia/ischemia is associated with cell survival and neuroprotection in: in vivo and in vitro models of hypoxia ischemia. However these studies hold limited clinical translations because the underlying mechanism remains unclear and the key molecules involved in EPO-induced neuroprotection are still to be determined. Thus the central aim of this proposal is: to determine the key mediators of EPO-induced neuroprotection and the mechanisms by which this occurs in: in vitro and in vivo models of hypoxia ischemia. Two alternate pathways of EPO-induced neuroprotection were elucidated in vitro using oxygen and glucose deprivation to mimic hypoxia ischemia. Our findings showed that EPO treatment resulted in an upregulation of tissues inhibitor of matrix metalloproteinase (TIMP)-1 and inhibition of hypoxia inducible factor (HIF-1α) both of which subsequently decreased in matrix metalloproteinase (MMP)-9 and promoted neuroprotection. MMP-9 inhibition was associated with neuroprotection in both pathways thus, inferring that inhibition of MMP-9 is one of the primary mechanisms of EPO-induced neuroprotection. We also observed that EPO-induced neuroprotection was reversed by inhibition of TIMP-1 or prolyl hydroxylase (PHD)-2. This lends to the conclusion that TIMP-1 and PHD-2 are the key mediators of EPO-induced neuroprotection in: in vivo and in vitro models of hy