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Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/214704
New analytical strategies based on mass spectrometry for the sensitive analysis of protein and microRNA biomarkers in human biofluids
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[eng] The analysis of molecular biomarkers in biological samples is of great interest for tackling numerous diseases. However, several challenges remain, including the low abundance of these biomarkers, their complex structures, the influence of the sample matrix components, and the limitations in sample volume. In this doctoral thesis, we present innovative strategies based on mass spectrometry (MS) for the separation, detection, characterization, and quantification of intact protein, enzymatically digested protein, and microRNA (miRNA) biomarkers in biofluids.
MS is a highly selective analytical technique that allows the unequivocal identification and detailed structural characterization of the detected compounds. However, most biological samples are complex and demand overcoming challenges like matrix interference and low analyte abundance. This necessitates the application of appropriate sample pretreatments and the on-line coupling of high-performance separation techniques, such as capillary electrophoresis (CE) to MS. CE-MS is considered an excellent technique for the highly efficient separation and characterization of polar and charged biomolecules. However, its ability to detect analytes at low concentration remains a major limitation due to the small sample volume that can be injected in the separation capillary. To address this drawback, the on-line coupling of solid-phase extraction to CE-MS (SPE-CE-MS) enables sample matrix clean-up and improved loadability, resulting in high preconcentration factors and low limits of detection (LODs). In the present thesis, on-line aptamer affinity-solid phase extraction coupled to capillary electrophoresis mass spectrometry and direct mass spectrometry (AA-SPE-CE-MS and AA-SPE-MS, respectively) have been investigated for the sensitive and accurate analysis of protein biomarkers at the intact level in biological fluids. These biomarkers have been α-synuclein (α-syn), which is a potential biomarker of Parkinson's disease (PD), and SARS-CoV-2 nucleocapsid (N) protein, which is an RNA-binding protein in SARS-CoV-2 virus that plays a crucial role in both viral replication and cellular signaling regulation.
Several strategies for high-throughput bottom-up analysis of protein biomarkers have been studied in this doctoral thesis. On-line immobilized enzyme microreactor capillary
electrophoresis-mass spectrometry (IMER-CE-MS) has been shown to be a rapid and efficient approach for bottom-up analysis using trypsin as proteolytic enzyme. However, IMER-CE-MS applicability is limited by the small sample injection volumes and the restricted range of enzymes that can be effectively immobilized. In this thesis, we have investigated for the first time the on-line coupling of AA-SPE with IMER-CE-MS using trypsin as a powerful alternative to increase the sample loadability and decrease the LODs of IMER-CE-MS for the analysis of α-syn in biological fluids. In a separate study, an in-capillary digestion capillary electrophoresis mass spectrometry (ICD-CE-MS) method has been developed as an enhanced alternative to IMER-CE-MS for the analysis of α-syn, using endoproteinase GluC instead of trypsin, which fails to map Ser-129, a phosphorylated site of this protein, which is upregulated in PD.
Regarding research about miRNA biomarkers, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been investigated as a rapid and simple direct MS technique for the analysis of miRNAs, exploring the use of ionic matrices (IMs) as an alternative to conventional matrices. The method was optimized using a standard mixture of miRNAs: miR-21, let-7g, and iso-miR-16 and subsequently applied to direct, multiplex, and untargeted analysis of miRNAs in serum samples.
However, the low concentration of miRNAs in complex biological samples and the potential interference from other molecules, particularly proteins, demands the application of appropriate purification and enrichment strategies. In this thesis, sample preparation for purification of miRNAs from biological samples and analysis by MS-based-techniques were comprehensively and critically reviewed. Then, purification and characterization of exosomes, which are a type of lipid bi-layer membrane extracellular vesicle (EVs) with diameter between 30-150 nm, were investigated. Exosomal miRNAs are the most abundant cargo molecules, and their presence within the exosomes makes them very stable and resistant to degradation. In this thesis, we focused on developing a method for the isolation of EV preparations enriched for exosomes from human serum and a CE method for their characterization
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SALIM, Hiba. New analytical strategies based on mass spectrometry for the sensitive analysis of protein and microRNA biomarkers in human biofluids. [consulta: 5 de desembre de 2025]. [Disponible a: https://hdl.handle.net/2445/214704]