Pane, Francesca (2016) Quantitative analysis in proteomics and metabolomics. [Tesi di dottorato]

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Testo QUANTITATIVE ANALYSIS IN PROTEOMICS AND METABOLOMICS_FrancescaPane.pdf Download (3MB) | Anteprima

Abstract

The quantification of differences between two or more states, physiological or pathological, in order to identify differentially expressed analytes in complex mixtures is among the most important objectives of proteomics and metabolomics. Mass spectrometry (MS) is one of the key analytical technology on which the emerging ‘‘-omics’’ approaches are based. It may provide detection and quantization of thousands of proteins and biologically active metabolites from a tissue, body fluid or cell culture working in a ‘‘global’’ or ‘‘targeted’’ manner, down to ultra-trace levels. Selected reaction monitoring (SRM) performed on triple quadrupole mass spectrometers has been actually the reference quantitative technique to analyze different molecules with high selectivity, sensitivity and a wide dynamic range. In SRM analysis, two stages of mass selection, i.e., selection of a precursor ion and monitoring of specific product ion(s) derived from the precursor, provide the highest analytical specificity applicable to complex mixture analysis. The combination of m/z setting for the first and third quadrupole is referred to as ‘transition'. New methodologies for selective quantitative analysis of targeted metabolites and proteins based on LC-MS/MS in Selected Reaction Monitoring scan mode. are developed. This approach has shown to be a valuable tool to identify altered pathways in pathological conditions and useful for quantitative measurements of a specific subset of known analytes such as same hormone and TDC, oxylipins, H2S sulfur metabolites, D, L Asp and NMDA and proteins as IDE and TSH. The SRM strategies optimized in this thesis focused on 1) identification of a set of proteins and metabolites of interest capable of satisfying a specific biological or clinic request 2) selection of transitions maximizing sensitivity and selectivity 3) optimization of SRM transitions by tuning acquisition parameters of the mass spectrometer 4) validation of the transitions in biological matrix to account to unspecific contributions of fragment ions background 5) quantification by the external standard method with the realization of specific calibration curves. The analysis in SRM mode (Selected Reaction Monitoring), has allowed us to achieve the specificity and selectivity to quantify the analytes of interest in relatively complex matrices, also at very low concentration with good reproducibility and accuracy.

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