Desarrollo de metodologías analíticas para la separación, detección y caracterización de biomarcadores glicoproteicos en muestras biológicas
The work presented in this thesis is focused on the development and optimization of analytical methodologies for the glycosylation analysis and characterization of therapeutically and biologically relevant glycoproteins. One of the first glycosylation studies carried out involved the detection and c...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2017 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/462823 |
| Acceso en línea: | http://hdl.handle.net/10803/462823 |
| Access Level: | acceso abierto |
| Palabra clave: | Marcadors bioquímics Marcadores bioquímicos Biochemical markers Glicoproteïnes Glucoproteínas Glycoproteins Ciències Experimentals i Matemàtiques 543 |
| Sumario: | The work presented in this thesis is focused on the development and optimization of analytical methodologies for the glycosylation analysis and characterization of therapeutically and biologically relevant glycoproteins. One of the first glycosylation studies carried out involved the detection and characterization of human transferrin (hTf) glycopeptide glycoforms by capillary liquid chromatography coupled to mass spectrometry (CapLC-MS). The aim was not only to characterize hTf glycosylation but also to verify whether its glycosylation was altered in individuals with chronic alcoholism and congenital disorders of glycosylation (CDG), a rare genetic disease, and identify which glycoforms were altered due to these two diseases. In the case of chronic alcoholism, several hTf glycopeptide glycoforms were observed to be altered, mainly, the H5N4S2, which decreased in relative abundance, and the non-glycosylated glycoform (peptide glycoform), which increased. Regarding the CDGs, the aim was to explore the data obtained with the analysis of both hTf glycopeptides glycoforms from control, CDG-I or CDG-II serum samples. However, the originated data set was too large and complex due to the number of samples. Consequently, two multivariate data analysis methods, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were evaluated for the classification of the different samples and for providing a novel insight into hTf glycopeptide glycoforms alteration in CDGs. The following work involved the analysis of mouse transferrin (mTf), as a suspected altered glycoprotein in collagen induced arthritis (CIA), an inflammatory and autoimmune disease, which resembles human rheumatoid arthritis (RA). In this case, an increase in fucosylation and glycan branching was observed in sera from mice with CIA+ versus control. Additionally, the effect of a specific gene was evaluated to assess its contribution to the aggravation or amelioration of CIA. The results showed that mice deficient in CD38 developed an attenuated CIA, that is, the increase in fucosylation and glycan branching was not as pronounced as in standard mice with CIA. Additionally, a methodology based on capillary electrophoresis coupled to mass spectrometry (CE-MS) was also evaluated as an alternative method to CapLC-MS for the analysis of the glycosylation of proteins. However, the use of the mandatory anionic surfactant to correctly perform the glycoprotein digestion proved detrimental to CE-MS analysis. Therefore, some approaches regarding sample treatment were evaluated to eliminate the anionic surfactant, being the µElution plates, 96-well plates with Oasis® HLB (WATERS®) stationary phase, the only method that provided proper sample clean-up and complete elimination of the remaining surfactant. Another interesting advantage of using CE as the separation technique prior MS detection is the fact that the electrophoretic migration behavior of ions can be easily predicted using the classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (me vs q/Mα). In this thesis, the classical semiempirical relationships were used to predict and model the migration behavior of peptides and glycopeptides originated from the digestion of human recombinant erythropoietin (rhEPO). Simulated electropherograms were elaborated with these models, which matched almost perfectly the experimental electropherogram. Results were later validated predicting the migration and simulating the separation of a different set of rhEPO glycopeptides and also hTf peptides and glycopeptides, which also agreed with the experimental electropherograms. Complexity of glycan structures can escalate rather quickly when we take into account that some monosaccharide units can have different orientation or be bound to different carbohydrates, giving rise to the formation of isomeric glycan structures. For instance, the sialic acid residue can have two types of linkage in complex N-glycans, depending on its orientation, referred to as α-23 or α-26 linkages. For that reason, the next work presented in this thesis aimed to assess the capability of ion mobility mass spectrometry (IM-MS) for the separation of isomeric glycoconjugates of mTf due to the type of sialic acid linkage. Straightforward separation of isomers was achieved with the analysis of glycans, as opposed to the glycopeptides, which was relatively more complicated, or the intact glycoprotein, which was not possible. Positive results were obtained with the analysis of the free glycans not only in standard samples but also in serum samples from control mice and CIA+ mice, acknowledging the applicability of the developed methodology to the study of real complex samples as well as its potential for the analysis of other glycoproteins. |
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