Structural Analysis of Complex Molecular Systems by High-Resolution and Tandem Mass Spectrometry

In this chapter, we describe the advances in high-resolution and tandem mass spectrometry applied for structural analysis of complex molecular systems. Focus is made on Fourier transform mass spectrometers (FTMS) because of their ability to routinely provide resolving power (resolution), which is above 100 000, and correspondingly high mass accuracy, which is higher than 2 ppm. The advantages of using these analytical capabilities have been demonstrated for analysis of extremely complex mixtures of molecules of diverse nature and size. What are the recent and envisioned steps in FTMS method and technique development? What are the novel application areas that await these developments? We describe the state-of-the-art in ion cyclotron resonance (ICR) and Orbitrap FTMS and showcase the advantages of FTMS applications for petroleomics and intact protein mass measurements. We briefly overview the current methods of ion activation and dissociation as most widely employed in tandem mass spectrometry (MS/MS) for peptide and protein structure analysis. Finally, we discuss the analytical limitations of the state-of-the-art FTMS and offer a brief introduction to some of the possible solutions to these limitations. Particularly, we describe the achieved and envisioned benefits of advanced signal processing methods aimed specifically at FTMS improvement.

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

Fourier transform mass spectrometry at the uncertainty principle limit for improved qualitative and quantitative molecular analyses

Anton Kozhinov

Nowadays, among the instrumentation park of mass spectrometry, Fourier transform mass spectrometers (FTMS), including ion cyclotron resonance (ICR) and Orbitrap FTMS, provide the highest analytical performance for accurate measurements and mass resolution. Nevertheless, molecular analysis in currently challenging research areas, such as life and environmental sciences, necessitates further improvement of these analytical characteristics. In recent decades, a regular approach to address the problem behind the analytical performance was using increased electromagnetic fields. However, per a given increase of their magnitudes, that approach is currently requiring more and more resources, hence demonstrating its limited feasibility for tomorrow. Therefore, only a qualitative breakthrough in the underlying methodology may then lead to the next series of developments enabling improved molecular analysis. The present research is dedicated to the fundamental question behind the analytical performance in FTMS. Specifically, this thesis represents an interdisciplinary study aimed at improved molecular analysis in FTMS-based applications, achieved via better comprehension of the uncertainty principle in FTMS, viz. its dependence on the measurement scheme, including ion traps, signal processing, and data analysis, as well as its influence on achievable analytical characteristics in FTMS. To start, the uncertainty principle for measurements in FTMS has been investigated, asserting a limit to the precision with which complementary physical quantities in FTMS, e.g. detection period and scale of frequency details to resolve, can be measured. Specifically, two corollaries of the uncertainty principle are considered, viz. resolution performance and performance for accurate measurements in FTMS. Importantly, the uncertainty principle shows dependence on the particular measurement scheme employed. For instance, signal detection, signal processing, and data analysis of the standard measurement scheme impose their own restrictions to the resulting uncertainty principle, thus leading to the current limitations. However, the ultimate limitations in the analytical characteristics in question are defined by the uncertainty principle limit due to physics of ion motion in the mass analyzer. Hence, with corresponding developments in data analysis methods, methods for signal processing, and designs of ion traps, novel measurement schemes have been implemented, where the quantitative form of the uncertainty principle is modified towards the ultimate limit. Finally, the implemented solutions have been evaluated in the context of FTMS-based analysis of crude oil fractions, protein identification and characterization, quantitative proteomics, and analysis of isotopic fine structures of peptides. To conclude, the achieved success of this work should considerably contribute to the currently challenging analytical applications of FTMS.

EPFL2015

Fourier transform mass spectrometry at the uncertainty principle limit for improved qualitative and quantitative molecular analyses

Anton Kozhinov

EPFL2015