Advances in Structural Elucidation of Small Molecules via High Performance Mass Spectrometry

Qualitative and quantitative analyte analysis is omnipresent: from quality control in food, pharmaceutical, biotechnology industries, to new molecular syntheses, detection of explosives and drugs, as well as natural compound mixture analysis, such as crude oils or animal fluids. Of particular interest are analytes associated with human health and energy, both of synthetic and natural origin. Thus, a selection of synthetic analytes related to ongoing efforts in drug design and alternative energy sources, has been made for this Thesis. Additionally, for natural analytes, peptides and crude oil were selected as analytes of choice due to their central role in human health and energy sources, respectively. Mass spectrometry was chosen as the method of choice for analyte investigation (elemental composition and structural feature determination) due to its sensitivity, ability to obtain data on thousands of analytes simultaneously, and use of radical and photo chemistry to probe molecular structure. The thesis is split into three major parts, first investigating synthetic analytes, then peptides and, finally, crude oil fractions. In the first part, use of photoionization is discussed as an alternative to other common analyte ionization methods as most selected synthetic targets do not ionize with the latter. A rough guideline, based on analyte structural composition, is proposed to determine analyte photoionization response. In-source analyte reactions are analysed and used as indicators of various functional group presence. Finally, upper mass boundary on analytes is considered and extended with photoionization. In the second part, peptide-ion/electron and ion/radical anion interactions in tandem mass spectrometry experiments, along with in silico modelling are used to put forth a new mechanistic proposal, which is fundamentally different to all the other mechanistic proposals related to this type of fragmentation process. The proposal involves charge-driven, rather than radical driven peptide radical fragmentation. It is also additionally significantly influenced by the local 3D structure of the peptide. The proposal may be of particular interest when considering radiation damage effects on key biological systems. In the third part, several methods are proposed aimed at enhancing crude oil (and other complex mixture) analysis on a new type of high resolution mass spectrometer, the Orbitrap Elite FTMS, including maximising number of identifiable analytes and evaluating key parameters of mass spectra of complex mixtures. Additionally, a data visualization method is proposed, based on analyte heteroatom content, which creates a graphical representation of all the identified components in the complex mixture and how changes in bulk properties of the sample get reflected on its compositional makeup.

Peptide Structure and Radical Chemistry in the Gas Phase Revealed by Product Ion Abundance Analysis in Electron Capture and Transfer Dissociation Mass Spectrometry

Hisham Ben Hamidane

Peptide and protein ion abundances in mass spectrometry (MS) and tandem mass spectrometry (MS/MS) may provide orthogonal, or complementary, information to their mass measurements. Collision induced dissociation (CID), the most commonly used MS/MS technique, demonstrates high variability of fragmentation pathways and their relative product abundances as a function of experimental parameters. Moreover, a reliable prediction of relative abundances of CID products is possible only for relatively small systems, but not for larger biomolecules. Recently developed radical-induced fragmentation techniques, e.g., electron capture and transfer dissociation (ECD/ETD), appear to demonstrate more repeatable and reproducible fragmentation patterns. However, it has been reported that ECD/ETD product ion abundance distribution for peptides is rather flat and extracting additional peptide sequence information is yet to be done. In the present work, an attempt is made to understand the peptide's ECD/ETD product ion abundance formation fundamentals and to reveal the possible implications of this source of information for improving peptide and protein structural characterization. Specifically, we have shown that ECD/ETD of peptides is sensitive to the chemical environment in terms of peptide primary structure (amino acid nature) and possibly higher order structure (hydrogen bond connectivity). After demonstrating the analytical validity of the ECD/ETD product ion abundance methodology, its parameter dependence and its applicability to different experimental setups, results will be presented and rationalized along two main axes of comprehension: gas phase peptide radical chemistry and structure. Whereas fundamental aspects of ECD/ETD (mechanism, thermodynamics) can be addressed with the former, additional ECD/ETD yield modulations were observed and presumably relate to structural properties of ions in the gas phase. ECD specificity will be demonstrated on the class of amphipathic peptides, where periodicity is revealed in product ion abundance and correlated to peptide primary and secondary structure. To decipher between both contributions, sequence variations of amphipathic peptides were probed by ECD and correlated to gas phase structures suggested by ion mobility mass spectrometry and supported by molecular dynamics computations. After probing amino acid side chain influence, unraveling the chemical environment specificity of ECD/ETD led us to investigate peptide backbone modifications. Peptides containing beta amino acids revealed two main effects in ECD and ETD. We demonstrate that individual substitutions yield c and z type fragments only for amino acids that provide sufficient z• radical stabilization, whereas multiple substitutions shift the preferential fragmentation pathway to the otherwise minor a-type ion formation pathway indicating possible change in peptide protonation. Finally, understanding the partitioning of radical ions between N- and C-terminal fragments led us to investigate ion internal energy dependence. The extent of hydrogen atom transfer, the [c+z] complex lifetime and secondary fragmentation pathways (w-type ion formation) are directly modulated by ion internal energy as demonstrated by decoupling ETD from charge reduced CID. To summarize, ECD/ETD product ion abundance methodology developed in the thesis advances biomolecule radical chemistry understanding and provides a potentially useful methodology for peptide and protein structural characterization.

EPFL2011

Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS

Luca Fornelli, Yury Tsybin

The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure-function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019620, 1758-1767, 2012.

Amer Soc Biochemistry Molecular Biology Inc2012

Peptide Structure and Radical Chemistry in the Gas Phase Revealed by Product Ion Abundance Analysis in Electron Capture and Transfer Dissociation Mass Spectrometry

Hisham Ben Hamidane

EPFL2011