LC-MS Applied Techniques for the Identification and Quantitation of Protein Glycosylation and its Isomeric Conformations

Liquid chromatography mass spectrometry (LC-MS) techniques are widely employed across various omics fields to perform comprehensive qualitative and quantitative analysis. The approach has been successfully employed for the structural analysis of proteins, glycopeptides, glycans, lipids, and metabolites. Among them we focused our research in describing the protein glycosylation and its association in biological processes during the development or progression of different diseases. Glycosylation is one of the most abundant posttranslational modifications in mammalian cells. Regulate the structure and functions of proteins such as protein-protein interaction, cell signaling and trafficking, protein stability, host-pathogen interactions. Aberrant glycosylation has been associated with various diseases such as cancer, neurodegenerative diseases, viral infections, and metabolic disfunctions. Hence, analytical approaches that provide increased accuracy and sensitivity are needed to allow a more comprehensive characterization of glycosylation and a subsequently better understanding of its relationship in biological processes. The dissertation proposes efficient LC-MS based methods that enables the application of proteomics, glycoproteomics, and glycoproteomics analysis in disease related samples, as well standard bodily fluids and glycoproteins. The applied methods have demonstrated to produce extensive description of proteins, glycopeptides and glycans and their isomeric forms. Among the upgraded methodologies we have a proposed a sensitive CSF glycan analysis that used as little as 15 L of starting material. Our method produced better throughput and sensitivity than similar methodologies that utilized sample volumes between 200-300 L. We have stablished methods for the early detection of mild cognitive impairment (MCI) and hepatocellular carcinoma (HCC). In both cases we have reported glycans and glycopeptides with the ability to differentiated between healthy and MCI states, and between cirrhosis and earlyHCC. We also have investigated the N-glycosylation of the S1 protein from eleven variants of the virus SARS-CoV-2. A profound understanding of the N-glycosylation conformations in different variant of SAR-CoV-2 would help to understand the virus pathogenesis and improve the development of vaccine candidates that can elicit biologically relevant immune responses. Additionally, we worked in the optimization of MS-based target methods for the quantitation of glycans and glycopeptides parallel reaction monitoring (PRM). Enabling the identification and quantitation of peptides, glycopeptides, and glycans. Additionally, we discussed the continuity of running projects and future directions in the field of LC-MS based-omics.

Embargo status: Restricted until 09/2025. To request the author grant access, click on the PDF link to the left.