Theory

Some of the basic theory that guides our research


Our focus: Our research focuses on the design, construction, and application of advanced technologies for structural mass spectrometry, in particular, for studies in structural proteomics, chemical biology, and biophysics. To identify and structurally characterize biomolecules from complex samples, we perform rapid (µs-ms) two-dimensional gas-phase separations using ion mobility-mass spectrometry (IM-MS), which provides separations on the basis of apparent surface area (ion-neutral collision cross section) and mass-to-charge (m/z), respectively. Biomolecular structural information is interpreted by comparing experimentally obtained collision cross-sections in the context of those obtained via molecular dynamics simulations.

Theoretical depiction of conformation space fo biomolecules Figure 1: (A) An illustration of the conformation space separation of different classes of biomolecules. Note that the indicated trends in the correlation function of collision cross section and m/z for particular molecular classes are for qualitative illustration purposes. (B) Structural motifs (secondary structural elements, intramolecular solvation of post-translational modifications, etc.) that give rise to deviation from the average correlation function within a particular molecular class. Specific cases are indicated for peptides.


In the analysis of complex biological materials, IM-MS provides a significant advantage over contemporary MS in that the regions in which signals appear in 2D conformation-space correspond with specific molecular class, i.e. the correlation of collision cross section with m/z varies as nucleotides/carbohydrates< peptides/ proteins < lipids/surfactants (Figure 1(A)). Deviations from where a particular signal is predicted to occur can provide additional information including: (i) identification of sights of post-translational modification, (ii) characterization of secondary, tertiary, and quaternary structural motifs, and (iii) rapid screening for analyte-ligand binding interactions (Figure 1(B)). The separation of analytes on the basis of molecular class provides significant advantages as a proteomics tool, because signals arising from concomitant, non-peptidic, materials are readily separated from the peptides of interest. For example, Figure 2 shows a representative IM-MS plot of 2D conformation-space for an HPLC fraction of an E. Coli whole-cell lysate. Signals arising from surfactant contaminant in the sample preparation are easily identified on the basis of structure and can thus be eliminated when searching peptide m/z signals against proteomic and genomic databases for high-confidence level protein identification.

Example spectra for separation of concomitant species
Figure 2: (A) Plot of IM-MS conformation space for a representative E. Coli whole-cell lysate fraction measured by HPLC-MALDI-IM-TOFMS. Two distinct trends of correlated arrival time distribution (which is the observable measured and subsequently transformed to collision-cross section) vs. m/z are obtained corresponding to non-peptidic concomitant species and tryptic peptides, respectively (indicated by dashed-lines for clarity). (B) Integrated mass spectra for all ATD-space (top), non-peptidic concomitant signals (middle), and peptides (bottom). Several intense signals in the comprehensive mass spectrum corresponding to concomitant species are indicated by "*".

Importantly, IM-MS can provide detailed structural information for conformational sub-populations of the same analyte (Figure 3), i.e. the relative abundances of different biomolecular conformations can be readily determined. By measuring the change in relative abundance of specific structural conformations as a function of IM separation temperature, thermodynamic and kinetic parameters can also be determined for relative structural sub-populations, or for structural transitions, respectively (i.e. via van't Hoff or Arrhenius plots). Note that this procedure can also be used to quantify the thermodynamic consequences of stepwise addition of solvent, or small molecules, on the prevailing molecular structure.























Figure 3: Experimental IM-MS conformation-space for a model peptide exhibiting two distinct structural sub-populations. Structures obtained by molecular dynamics simulations indicate helical and compact structures consistent with these results.

Key areas of investigation and development include: (i) What are the prevailing influences of post-translational modifications (e.g. glycosylation, phosphorylation, ubiquination, sumoylation, etc.) on protein secondary and tertiary structure? (ii) To what extent does stepwise hydration of a protein change analyte structure, i.e. what is the relative magnitude of stepwise addition of water on solvated structure? (iii) Development of imaging IM-MS instrumentation for multidimensional imaging/characterization of biomarker species from microarrays or thin tissue sections. (iv) Construction of advanced laser optical strategies for high-throughput screening of drug-ligand interactions.