PhD Oral Exam - Dmitri Sitnikov, Chemistry

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Protein precipitation with organic solvents is the most common method used for global metabolomics of human plasma. This standard method has broad selectivity but does not provide good coverage of low abundance metabolites. Increasing the metabolite coverage by parallel analysis of plasma samples prepared using different organic solvents is inefficient due to the low orthogonality of their selectivity. Moreover, there is an increasing demand for more selective sample preparation methods suitable for targeted and untargeted metabolomics analysis of biofluids that can also provide better coverage of metabolites missed by current standard workflows. To achieve these goals, a novel sequence of orthogonal but complementary methods was designed and evaluated after performing a quantitative systematic side-by-side comparison of seven extraction methods with different selectivity to establish the most orthogonal combinations with good analytical performance. This novel sequential solid-phase extraction protocol was introduced to fractionate metabolome into anion, cation, neutral, and zwitterion fractions, followed by a rigorous analytical assessment. The final goal was to evaluate oral fluid as a sample source for targeted metabolomics analysis via the development and validation of the liquid chromatography with tandem mass spectrometry assay and consecutive accumulation of sufficient background knowledge to handle this sample type in future developments.

The side-by-side comparison of solvent precipitation methods confirmed their low orthogonality and was accompanied by severe matrix effects in comparison to more selective methods. The comparison of solid-phase extraction (SPE) and liquid-liquid extraction methods revealed their orthogonal metabolite coverage to other methods. However, further experiments showed that methyl tert-butyl ether incompletely removed lipids, which resulted in the significant and undesirable splitting of lipids between aqueous and organic layers. Thus, the final SPE sequential fractionation protocol combined sample deproteinization by methanol followed by the sequential SPE executed on mixed-mode strong anion-exchange and mixed-mode strong cation-exchange polymeric sorbents. It produced four fractions enriched with anions, cations, neutrals, and zwitterions providing flexibility to analyze the fraction individually or in strategic combinations tailored to various mass spectrometry methods. The final protocol resulted in a 1.6-fold increase in total metabolite coverage comparative to methanol precipitation with a 2-fold increase in the analysis time. The method demonstrated excellent signal repeatability for both targeted (relative standard deviation < 13%) and global (relative standard deviation < 30% for 75% metabolites) metabolomics. Moreover, excellent separation of anion and cation metabolites (< 4% overlap) and small (< 27%) overlap between other pairs of metabolite classes allow a supplementary assignment of ionic properties to an unknown metabolite to aid metabolite identification. The utility of the above sequential solid-phase extraction method for fractionation of polar metabolome can in future extend beyond plasma to other biofluid types, such as oral fluid.

Two model analytes cortisone and cortisol were selected for preliminary study in oral fluid. This method combined methanol precipitation and a short reversed- phase chromatography with tandem mass spectrometry method. The successful development and validation of the method revealed an accurate, precise (<15% variability), and sensitive (low limit of quantitation = 0.31 ng/mL) high-throughput assay, whose selectivity outcompeted an immunoaffinity method, while requiring only 30 µL of oral fluid sample.

In sum, this thesis establishes the first steps towards the adaptation of the sequential SPE method for simultaneous global and targeted metabolomics analyses of plasma and oral fluid. Such studies will benefit from the systemic, more in-depth characterization of metabolite status, enriched scientific value, and faster identification and transition of putative biomarkers from the discovery to the targeted stage. Therefore, my research provided the necessary groundwork for developing and validating universal preparation and analysis workflows, which will permit the feasible execution of pluripotent metabolomics studies.