Chemical utilization of unoccupied mass spectral space for proteome analysis

Date of Completion

January 2009


Chemistry, Analytical




Mass spectrometry-based proteomics is a powerful tool routinely applied to address various biological and clinical problems. Sample preparation before mass spectrometry has a large impact on proteomics, which reduces the enormous complexity of the protein mixtures obtained from biological systems. Various chemical strategies have been used for different purposes to improve protein identification and characterization by mass spectrometry. In this dissertation three chemical methods have been developed, aiming at utilizing unoccupied mass spectral space to facilitate proteome analysis. ^ Two of the methods target phosphate-specific fragment ions, termed as phosphoryl marker ions, taking advantage of their large mass defects. In one project, an oxygen stable isotope is incorporated into the structure of a phosphoryl marker ion; this is the first method that substitutes stable isotope on pre-existing peptidyl phosphates. By measuring labeled marker ions in unoccupied spectral space, sequence-independent quantitation of protein tyrosine phosphorylation can be achieved with minimum inference. The second project designs and generates the first positive phosphoserine marker ion via chemical modification. The new phosphoserine marker ion can be detected in unoccupied spectral space with positive ion mode mass spectrometry for specific identification of phosphoserine peptides in complex protein digests. In analogy to the utilization of unoccupied spectral space via naturally occurring phosphorylation, the principle of unoccupied spectral space has been expanded to the analysis of general proteomes via chemical modification in the third project. A novel method of fragment ion mass defect labeling is developed for versatile proteome analyses. With this method, N-terminal ions of modified peptides produced from proteomes can be detected in unoccupied spectral space due to significantly shifted mass defects. Different types of fragment ion mass defect labeling produce modified N-terminal ions with distinct preferences and all of the ions will facilitate proteome analyses for diverse purposes. ^