Floris, Federico (2017) Pushing the limits of two-dimensional mass spectrometry. PhD thesis, University of Warwick.

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Abstract

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) is a data independent tandem mass spectrometry technique that allows direct correlation between precursor and fragment ions without the necessity of any sort of isolation prior to fragmentation. Two-dimensional mass spectrometry (2D MS) experiments were conceived in the 1980s from inspiration of two-dimensional nuclear magnetic resonance (2D-NMR), but the development of the technique was stopped because of the insufficient computational capabilities of that time. In 2010, with the progress in computer technology and the improvements in FT-ICR MS instrumentation and methods, the interest in developing 2D MS was renewed. Since then, 2D FT-ICR MS showed to be a valuable platform for the analysis of small molecules, macromolecules such as peptides and glycopeptides, and complex mixtures deriving from tryptic digestion of proteins of increasing sizes. All the proteomics studies performed with 2D MS used the bottom-up (BUP) approach.

The main goal of this Ph.D. research work was to develop two-dimensional mass spectrometry for the challenges of contemporary mass spectrometry, therefore pushing the limits of the technique towards the most interesting, cutting-edge research grounds, by optimising the technique, developing 2D MS analysis methods, and finally constructing improved instrumentation act to better perform 2D MS experiments.

On this purpose, the rising importance of top-down proteomics (TDP) led to the development of 2D FT-ICR MS for the analysis of intact proteins. To realise this first project, 2D MS was optimised for TDP analysis using calmodulin (CaM) as a model, and infrared multiphotondissociation (IRMPD) as fragmentation technique. The study compared the two-dimensional BUP and TDP analysis of CaM with standard one-dimensional tandem mass spectrometry, and showed that the use of 2D MS allowed to obtain comparable cleavage coverages, with a consistent saving in sample and time. The study affirmed the suitability of 2D MS for top-down proteomics, still leaving space for more developments allowing its optimisation. The total cleavage coverage of CaM was later implemented with the use of BUP 2D electron-capture dissociation (ECD) MS.

In order to improve the cleavage coverages obtained in top-down two-dimensional mass spectrometry, another technique was developed, called MS/2D MS. The technique makes use of an extra fragmentation step before two-dimensional mass spectrometry, achieved through external isolation of a charge state of interest, and collisional fragmentation in a hexapole-based collision cell before the ions are sent to the ICR-cell. The output of MS/2D MS is a single 2D mass spectrum with information equivalent to MS3 about the entire mixture. The technique has been successfully developed using ubiquitin (Ubi) and calmodulin as models, and showed new uses of the features exclusive of 2D MS, such as the use of precursor ion scans to assign the protein terminus of many precursors ions at once, and the analysis of internal fragments formed by the first dissociation. Furthermore, as long as extra fragmentation steps can be performed, a deeper investigation of the analytes can be achieved, in an experiment collectively called MSn /2D MS.

After the development of 2D MS for TDP, another method was developed, implementing the use of two-dimensional mass spectrometry for another type of complex mixtures: polymers. An extensive 2D MS study was performed on a simple mixture of homopolymers such as PMMA, and on two complex mixtures commonly used as drug excipients, polysorbate 80 and TPGS. The study demonstrated again that single 2D MS experiments give results equivalent to multiple MS/MS experiments targeted at the different precursors. Furthermore, it allowed the high resolution tandem mass spectrometry of polysorbate 80, representing the first kind of study of that type because of the challenges of isolating single analytes from the polymer mixture without the use of a previous separation, such as chromatography. The use of neutral-loss lines was particularly useful in this study, allowing to discriminate between oleated, linoleated, and non-esterified species. Finally, 2D mass spectra of polymers showed characteristic diagonal lines, specific for each polymeric mixture, representing some sort of fingerprint of the mixture.

The last part of the thesis is dedicated to the optimisation of 2D FT-ICR MS for ECD MS, the application of the technique to another class of compounds, glycans and glycan-derivatives, and finally to the development of a more capacious ICR-cell.

The research performed in this thesis showed that 2D FT-ICR MS is a viable data independent mass spectrometry technique for top-down proteomics and polymer analysis; that the technique is fast, and it possesses unique features that improve ion assignments, such as precursor ion scans and neutral-loss lines. Finally, it opens for two-dimensional mass spectrometry the path to glycomics and to an improved 2D MS analysis of even more complex mixtures, thanks to the development of a novel ICR-cell.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QC Physics Q Science > QD Chemistry
Library of Congress Subject Headings (LCSH):	Fourier transformations, Mass spectrometry, Cyclotrons, Peptides -- Analysis, Glycopeptides -- Analysis
Official Date:	November 2017
Dates:	Date Event November 2017 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Extent:	344 leaves : illustrations, charts
Language:	eng

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