Applications Of Liquid Chromatography Tandem Mass Spectrometry For The Analysis Of Complex Mixtures

From inside our bodies to the environment we live in, there are a wide variety of unknown compounds that have been difficult to identify and characterize through current methods. We aim to study these compounds by liquid chromatography (LC) high-resolution tandem mass spectrometry (MS/MS). LC-MS/MS has become the gold standard for the analysis of complex mixtures due to its capabilities to decrease spectral overlap and high mass measurement accuracy. Proteins are of particular interest biologically because they give insight into various diseases and the health of our biological processes. Analysis of these proteins is hindered by the current methods implemented in laboratories due to the size and characteristics of these compounds and their natural behavior outside of biological systems. Just as import as what is inside the human body is what is in the environment, specifically the Earth's oceans. Just like proteins in the body, the Earth's oceans give us a picture into the health of the environment. We know that human activity has severally impacted our atmosphere and water sources. We aim to understand the connections with pollution and the effects it has on oceans, a major water source on Earth. First a broad overview of LC-MS/MS is presented in Chapter 1. The first section discusses the MS instrumentation used for the studies in the following chapters, the 14- and 21-Tesla Fourier Transform Ion Cyclotron Mass Spectrometer (14- and 21-T FT ICR MS). The custom-built system is designed to produce the highest resolution data for compound identification and structural information. Our facilities are also equipped with various liquid chromatography (LC) systems that are used to separate mixtures and decrease sample complexity. Lastly, a brief introduction to various complex mixtures analyzed by LC-MS. The chapters that follow cover the specific studies performed with the knowledge of LC-FT ICR-MS and compound identifications. Chapter 2 discusses a study into the benefits and limitations on the application of isotopic depletion for cultured proteins. Isotopic depletion is a method used to deplete compounds of various heavy isotopes of elements present, most commonly carbon, nitrogen, hydrogen, oxygen, and sulfur. Heavy isotopes effect MS experiments in multiple ways which are investigated in the study for proteins derived from multiple cell lines (E. coli and C. elegan). This work is novel in the application and analysis of isotopically depleted C. elegans proteins The methods used for the biological applications can be applied to the analysis of marine samples which is discussed in Chapters 3 and 4. Chapter 3 investigates the composition of sinking particles in the California Coastal Current, specifically metabolites. Sinking particles play a role in the regulation of biological processes in the water column but additionally have shown to regulate greenhouse emissions. We take an even deeper look with analysis of marine sediments to see how far pollutants travel down the water column and are stored at the bottom of the ocean, in Chapter 4. Marine sediment samples were collected from the Oregon continental shelf at established sample stations. The analysis of these environmental samples by LC-MS/MS is still in the developmental phase. Our work aims to develop novel LC-MS/MS methods that can give a fuller picture into environmental impacts of pollution. The overall goal is to expand the capabilities of LC-MS/MS experiments to complex mixtures. LC-MS/MS shows its true capabilities and potential in the experiments discussed here. From 300 Da to 40 kDa in size, LC-MS/MS can efficiently analyze a wide dynamic range of compounds which is critical to broad applicability. With this work, future experiments can analyze and identify a wide range of compounds by high-throughput LC-MS/MS analysis.