Our research focuses on developing and applying novel analytical methods, based on gas-phase radical ion
chemistry, to the structural characterization of biological molecules. This work is interdisciplinary and performed by both Analytical Chemistry and Chemical Biology graduate students as well as postdoctoral researchers with expertise in Physical and Inorganic Chemistry. Specifically, we are interested in two tandem mass spectrometry techniques: electron capture dissociation (ECD) and electron detachment dissociation (EDD), which produce radical cations and anions, respectively, by attaching or detaching electrons from even-electron biomolecular ions created by electrospray ionization (see Scheme 1).
Scheme 1. Fragmentation routes in infrared multiphoton dissociation (top), electron capture dissociation (middle), and electron detachment dissociation (bottom).
Gas-Phase Radical Ion Chemistry for Structural Probing of Nucleic Acids
In research funded by the American Society for Mass Spectrometry, the Petroleum Research Fund, and the National Science Foundation, we are characterizing and applying ECD and EDD in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer to the structural characterization of nucleic acids and their complexes with small molecules. Recent results from our laboratory have shown that both ECD and EDD can provide extensive oligonucleotide sequence information, complementary to that obtained from other tandem mass spectrometry techniques (see Figure 1). We have also found that EDD, analogous to ECD, can be soft in that higher order structure can be preserved following backbone cleavage. We are exploring the utility of the latter characteristic for elucidation of nucleic acid structure and folding. This work adds to the fundamental knowledge of gas phase radical ion chemistry and has the potential to significantly impact crucial areas as diverse as basic biology, the molecular basis of life processes, drug discovery, and forensic science.
Figure 1. ECD (top) and EDD (bottom) both result in complementary bond cleavages compared to infrared multiphoton dissociation (IRMPD), which is based on slow heating of even-electron ions. The latter technique produces more secondary cleavages, involving nucleobase and water loss.
Integration of ECD and IRMPD with Proteomics
In research supported by the Searle Scholars Program, we incorporate ECD and IRMPD with proteomics to integrate the unambiguous characterization of protein posttranslational modifications. Such modifications play key roles in directing protein function. ECD can cleave peptide backbone bonds with retention of weakly-bound modifications. Thus, modifications can be localized while simultaneously obtaining amino acid sequence information. By contrast, the main pathways in IRMPD are often loss of and cleavage within modifications. IRMPD therefore can identify the presence of modifications, and provide complementary structural information. Our work aims at improving the ECD sensitivity and we have so far found two promising strategies. First, ionization by divalent metal ion adduction allows ECD (which requires at least two charges) to be performed on neutral oligosaccharides and acidic peptides. Second, we have pioneered a novel strategy for enriching phosphorylated peptides from proteolytic peptide mixtures, based on selective binding to zirconia (see Fig. 2). This approach extends the applicability of ECD for phosphoproteomics. We have also found an altered IRMPD behavior for high mannose-type glycans: both glycan and peptide bonds are cleaved. This feature needs to be incorporated in current approaches for predicting glycan fragmentation (i.e. pattern recognition), important for identification of disease-related aberrant glycosylation patterns via glycoproteomic strategies.
Figure 2. Negative mode electrospray ionization FT-ICR mass spectra from a tryptic digest of alpha-casein obtained prior to phosphopeptide enrichment (top) and following ZrO2 (middle) and TiO2 (bottom) phosphopeptide enrichment. All peaks labeled with numbers correspond to phosphopeptides. Highly selective enrichment of singly phosphorylated peptides (e.g. #7) is observed following ZrO2 enrichment whereas multiply phosphorylated peptides (e.g. #6) are selective enriched by TiO2.
ECD of Deuterated Peptides
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 structural resolution of this approach is limited by the size of proteolytic peptides and gas-phase fragmentation has been proposed as a means to increase resolution. However, most dissociation methods involve heat and will therefore promote hydrogen scrambling and loss of site-specific information. We have recently shown that the N-terminal product ions (c-type ions) from ECD can provide site specific hydrogen exchange rates
Figure 3. Time evolution of the isotopic distributions of the ECD product ions, c232+ and c222+, following deuterium to hydrogen exchange of melittin. The amide hydrogen exchange rate of Arginine 24 can be derived from these two consecutive ions.