One of the best ways to diagnose breast cancer early or to predict therapeutic response is to use serum biomarkers. Unfortunately, for breast cancer, we do not have effective serological biomarkers. We hypothesized that novel candidate tumor markers for breast cancer may be secreted or shed proteins that can be detected in tissue culture supernatants of human breast cancer cell lines. A two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) strategy was utilized to identify and compare levels of extracellular and membrane-bound proteins in the conditioned media. Proteomic analysis of the media identified in excess of 600, 500 and 700 proteins in MCF-10A, BT474 and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6 and 10 (ranging in concentration from 2-50 µg/L), as validated by ELISA and confidently identified HER-2/neu in BT474 cells. Sub-cellular localization was determined based on Genome Ontology (GO) for the 1,139 proteins, of which 34% were classified as extracellular and membrane-bound. Tissue specificity, functional classifications and label-free quantification were performed. The levels of eleven promising molecules were measured in biological samples to determine its discriminatory ability for control versus cases. This screen yielded activated leukocyte cell adhesion molecule (ALCAM) as a promising candidate. The levels of ALCAM, in addition to the classical breast cancer tumor markers carbohydrate antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) were examined in 300 serum samples by quantitative ELISA. All three biomarkers effectively separated cancer from non-cancer groups. ALCAM, with area under the curve (AUC) of 0.78 [95% CI: 0.73, 0.84] outperformed CA15-3 (AUC= 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC= 0.63 [95% CI: 0.56, 0.70]). The incremental values of AUC for ALCAM over that for CA15-3 were statistically significant (Delong test, p <0.05). Serum ALCAM appears to be a new biomarker for breast cancer and may have value for disease diagnosis.

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A growing interest in sustainable development has made efficient utilisation of starting materials and, if they occur, by-products become increasingly important. Vast amounts of by-products are generated by the forestry and food industry. Incineration for energy production is one way to make use of these by-products but some of them contain compounds that would have an increased value if they were extracted, so called “high value species”. The by-products are often very complex, so reliable methods for analysis of the high value species are required in the development of processes to utilise them. A wide range of compounds can be analysed using chromatographic separation coupled to mass spectrometry, making it a powerful tool in the evaluation of methods for extracting high value species from industry by-products. This thesis is based on four studies of potential high value species. In the first study, methods were developed to differentiate isobaric flavonoids and then use this knowledge to determine the identity of the flavonoids in three different plant extracts. In the second study, three different methods to extract betulin from birch bark were evaluated regarding extracted amount and purity of betulin. One of the methods was then investigated in industrial scale using a model approach. In the third study, the flavonoid contents of lovage were determined and other major extracted compounds were investigated by high performance liquid chromatography coupled to electrospray ionisation mass spectrometry. Gas chromatography and supercritical fluid chromatography were used to obtain complementary information about major components. In the fourth study, high resolution mass spectrometry utilising two different types of fragmentation was used with the purpose of overcoming the shortcomings of the methods developed in the first study. The results indicated that it would be possible to develop methods compatible with chromatographic separation for differentiating different types of isobaric substituents. The ability of performing sequential fragmentation was used to investigate some isobaric aglycones by creating spectral trees, and unique pathways were found for each of them.

Electrospray Ionization (ESI) is a technique that permits the soft ionization of large proteins and biomolecules without fragmenting them, which allows them to be characterized via Mass Spectrometry (MS). It has the potential of permitting the identification of transient intermediate products in biological processes in situ, which would provide great insight to researchers in the growing fields of proteomics and metabolomics. However, this application presents a technical challenge in that most relevant biochemistry occurs in aqueous solutions with high salt content, which makes successful identification of analytes by ESI-MS difficult. This thesis presents the design, fabrication, and characterization of a microfabricated dialysis module that could alleviate this issue by desalting samples inline between sampling and electrospraying interfaces. Its small volume (~10 nL) minimizes sample transit time and, thus, optimizes ESI-MS analysis temporal resolution. A preliminary analytical model of dialysis elucidates the key performance parameters and sets the guidelines for consideration in its design. The device is then microfabricated in a cleanroom environment using techniques that have been well established by the microelectronics industry such as E-beam evaporation and Reactive Ion Etching. The system efficiency is demonstrated experimentally by assessing its salt removal effectiveness as a function of sample residence time. Mass spectrometry analyses of proteins in solutions with high salt content further corroborate its performance.

This research is directed at developing the interplay of experimental and computational methods in the area of biochemical mass spectrometry. The experimental method is that of electrospray ionization mass spectrometry (ESI-MS). The computational methods employed are those of semi-empirical quantum mechanics and molecular modeling.The use of Electrospray Mass Spectrometry was developed to investigate whole proteins and the non-covalent complexes that may be formed with small molecules. This method provides the soft ionization needed to accurately determine a noncovalently bound complex's mass with an error of less than 0.1 %. An original design electrospray ionization source (ESI) and a syringe pump have been built to fulfill the goals of the research. The ESI source design has been published in The Journal of the American Society for Mass Spectrometry, (1993, 4, 964-967).In this work the protein selected was cytochrome c and its variants from different species. The small molecules chosen were a broad class of structures known as crown ethers. With the ESI technique the proteins are prepared in an acidic solution that fully protonates the solvent-exposed basic amino acid residues. This provides the protein with many positive charges which makes the analysis by ESI mass spectrometry possible with a single quadrupole instrument, an Extrel ELQ 400. The mass of the protein is divided by the number of positive charges. The small molecules, the crown ethers, were chosen due to their ability to bind ammonium ion and protonated amino groups. This binding is non-covalent, hydrogen bonds stabilize the complex formation. Because this complex is non-covalent in nature the charge of proteins does not change. To aid in the interpretation of our electrospray mass spectra we have originated a new kind of linear plot for use with ESI data. It was found that in using the ESI technique that ion currents representing non-covalent complexes of cytochrome c and crown ethers could be observed in the mass spectra. The measurements of the total ion counts of peaks in the mass spectra allowed binding constants to be calculated. This had not been reported before in the literature.The accurate weight determination and the characteristic charge distribution in the ESI mass spectrum provides data suitable for computer modeling. The nature of the protein's positive charges in ESI had not been well defined. The experimentally determined binding constants allowed comparison to results from computational chemistry and molecular modeling. This result shows that the binding occurs at specific, protonated amino acid residues. The calculations performed yielding the Heats of Formation (OH f) for protonated amino acids complexed with crown ethers indicates that the OH binding of crowns increases from histidine, to arginine, to lysine. The use of a 3D model of cytochrome c from crystallographic data provided in the Brookhaven Protein Database and the SYBYL molecular modeling program allows a structural correlation to be made between the 3D model of the protein and protein/crown ether complex. The stoichiometric ratios of bound crown ether to protein determined from experiment, along with the computational results, have been used to rationalize a protein molecular model that allows predictions to be made about the potential for binding of other small molecules. / Department of Chemistry