Exploring Fundamental Aspects of Proteomic Measurements: Increasing Mass Measurement Accuracy, Streamlining Absolute Quantification, and Increasing Electrospray Response

Abstract

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers unparalleled performance in terms of resolving power and mass measurement accuracy. In order to realize the highest achievable mass measurement accuracy for a given FT-ICR MS system, frequency shifts due to space-charge effects must be accounted for via external or internal calibration methods. Herein, a dual electrospray ionization source was coupled to a hybrid quadrupole Fourier transform ion cyclotron resonance mass spectrometer and utilized to incorporate internal calibrant to yield high mass measurement accuracy results. Frequency shifts from space-charge effects were also counteracted by accounting for total ion population and relative ion population on a MALDI-FT-ICR MS and a hybrid LTQ-FT-ICR MS equipped with automatic gain control. This was achieved through the use of multiple linear regression and artificial neural networks. These experiments resulted in mean mass measurement accuracies in the parts-per-billion range. C-reactive protein (CRP) is an important clinical marker for inflammation, atherosclerosis, and has also been observed to be upregulated in patients with epithelial ovarian cancer (EOC). This dissertation contains results from a protein cleavage isotope dilution mass spectrometry method developed for the absolute quantification of CRP from human plasma. A total of 110 human plasma samples were analyzed including 54 samples from patients with EOC. The results were compared to a CLIA certified ELISA test which showed high correlation but different absolute values, which suggested different reference ranges for different analytical techniques. In addition, a correlation was observed between the stage of diagnosis of cancer and CRP concentrations. Chemical tags have long been utilized with mass spectrometry for a multitude of purposes, extending the effectiveness of the measurements dramatically. Electrospray ionization has been shown to preferentially ionize more hydrophobic species. Four new iodoacetamide derivatives, which react with the amino acid cysteine, were reacted with three peptides to determine their ability to increase electrospray response. This resulted in increases up to 2000-fold compared to alkylation of peptides with iodoacetamide. The combination of these results will aid future studies to identify and quantify proteins and peptides which contain cysteine and can be expanded to all peptides using amine-specific chemistry.