Our research focuses on applying state-of-the-art mass spectrometric techniques to the following areas: 1) identification and characterization of protein posttranslational modifications; 2) mapping macromolecular contact surfaces; 3) exploration of the gas-phase fragmentation behavior of various biomolecules following ion-electron interactions; and 4) probing covalent intermediates in the catalysis of non-ribosomal peptide synthetases and polyketide synthases. A major goal is to excel in both analytical technique development and biologically relevant problem solving.
Electron capture dissociation (ECD) can cleave backbone bonds with retention of weakly-bound posttranslational modifications, thereby allowing their localization while simultaneously resulting in amino acid sequence information. By contrast, the main dissociation pathways in slow-heating techniques, such as infrared multiphoton dissociation (IRMPD), are loss of and cleavage within modifications. IRMPD can therefore identify the presence of modifications, and provide complementary structural information compared with ECD. However; one drawback of ECD is that multiply positively charged precursor ions are required, which can pose a challenge for acidic species such as phospho- and sulfopeptides. Alternative negative ion mode activation techniques are therefore of great interest to us, including electron detachment dissociation (EDD) and negative ion ECD (niECD), which we recently discovered. niECD provides identical structural information as conventional ECD but with the added advantage of operating in negative ion mode. In addition, niECD involves charge increase rather than decrease and thus ensures high fragmentation efficiency and improved product ion signal abundance. We incorporate these ion-electron and ion-photon reactions into the field of proteomics to specifically target modified proteins.
Solution-phase hydrogen/deuterium exchange in combination with mass spectrometric detection of proteolytic peptides is a valuable tool for characterization of protein-protein interactions. The exchange rates of amide hydrogens at contact surfaces generally slow down several orders of magnitude compared to hydrogens accessible to the solvent. We utilize the ultrahigh resolution (m/Delta mFWHM of several million) and ppm mass accuracy of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to improve peptide assignment, protein sequence coverage, and mass increase measurements. We also apply this technology to characterize protein-nucleic acid and protein-carbohydrate interactions, and explore the possibility of employing ECD to increase structural resolution.
Finally, we are interested in extending the radical ion chemistry of ECD/niECD and other techniques based on ion-electron interactions (e.g., electron induced dissociation (EID)) to structural characterization of a larger variety of biological molecules, such as oligonucleotides, oligosaccharides, metabolites, and lipids. Fragmentation patterns of both positive and negative ions are investigated, and should provide insights for a deeper understanding of these processes.
Song, H.; Hakansson, K., Electron Detachment Dissociation and Negative Ion Infrared Multiphoton Dissociation of Electrosprayed Intact Proteins. Anal. Chem. 2012, 84, 871?876.
Yoo, H. J.; Wang, N.; Zhuang, S. (undergraduate author); Song, H.; Hakansson, K., Negative Ion Electron Capture Dissociation: Radical-Driven Fragmentation of Charge-Increased Gaseous Peptide Anions. J. Am. Chem. Soc. 2011, 133, 16790?16793.
Yoo, H. J.; Hakansson, K., Determination of Double Bond Location in Fatty Acids by Manganese Adduction and Electron Induced Dissociation. Anal. Chem. 2010, 82, 6940?6946.
Kalli, A.; Hakansson, K., Electron Capture Dissociation of Highly Charged Proteolytic Peptides from Lys N, Lys C and Glu C Digestion. Mol. Biosyst. 2010, 6, 1668-1681.
Gu, L.; Wang, B.; Kulkarni, A.; Geders, T. W.; Grindberg, R. V.; Gerwick, L. G.; Hakansson, K.; Wipf, P.; Smith, J. L.; Gerwick, W. H.; Sherman, D. H., Metamorphic Enzyme Assembly in Polyketide Diversification. Nature 2009, 459, 731-735.