Covariance mapping spectroscopy of ultrafast laser induced biomolecular dissociation
File(s)
Author(s)
Ayers, Ruth Kathleen
Type
Thesis
Abstract
This thesis reports the development of and first results from femtosecond laser-induced ionisation/dissociation (fs-LID) two-dimensional partial covariance mass spectrometry (2D-PC- MS) of biomolecules.
Collision induced dissociation (CID) 2D-PC-MS is first extended to oligonucleotides. 2D- PC-MS fragment–fragment correlations are shown to give more sequence-specific information for oligonucleotides than the individual fragment analysis of 1D MS/MS. This is particularly relevant in the important case of modified oligonucleotides, where common sequencing methods can struggle to discover the nature and location of chemical modifications.
The experimental development of fs-LID 2D-PC-MS runs in parallel with predictive simulations of the laser–ion cloud overlap in our experimental system, which inform the experimental parameters required for success. The discovery that contaminant free measurements are possible in negative ion mode, combined with oligonucleotides being more amenable to negative ion mode MS, the relevant experience from oligonucleotide CID 2D-PC-MS and the fact that, to the best of our knowledge, fs-LID activated MS of oligonucleotides was unprecedented, lead to a focus on fs-LID 2D-PC-MS of oligonucleotide anions.
Novel 2D-PC-MS molecular diagrams are developed to give insights into the fragmentation patterns of phosphorylated peptides and oligonucleotides. These diagrams visually represent the relative probabilities of a molecule taking the fragmentation pathways found to be strongest when correlated fragment pairs are ranked by their 2D-PC-MS significance score. Comparing 2D-PC-MS molecular diagrams for CID and fs-LID activation elucidated mechanistic differences in the fragmentation behaviour between the statistical bond-breaking of CID and the ultrafast fragmentation triggered by fs-LID. A comparison of the fs-LID fragmentation pathways around different riboses finds that the nature of the ribose greatly influences the local fragmentation behaviour. In a wider context, investigating laser–biomolecule interaction mechanisms could contribute to the understanding of light–biomolecule interactions such as sunlight interacting with living cells.
Collision induced dissociation (CID) 2D-PC-MS is first extended to oligonucleotides. 2D- PC-MS fragment–fragment correlations are shown to give more sequence-specific information for oligonucleotides than the individual fragment analysis of 1D MS/MS. This is particularly relevant in the important case of modified oligonucleotides, where common sequencing methods can struggle to discover the nature and location of chemical modifications.
The experimental development of fs-LID 2D-PC-MS runs in parallel with predictive simulations of the laser–ion cloud overlap in our experimental system, which inform the experimental parameters required for success. The discovery that contaminant free measurements are possible in negative ion mode, combined with oligonucleotides being more amenable to negative ion mode MS, the relevant experience from oligonucleotide CID 2D-PC-MS and the fact that, to the best of our knowledge, fs-LID activated MS of oligonucleotides was unprecedented, lead to a focus on fs-LID 2D-PC-MS of oligonucleotide anions.
Novel 2D-PC-MS molecular diagrams are developed to give insights into the fragmentation patterns of phosphorylated peptides and oligonucleotides. These diagrams visually represent the relative probabilities of a molecule taking the fragmentation pathways found to be strongest when correlated fragment pairs are ranked by their 2D-PC-MS significance score. Comparing 2D-PC-MS molecular diagrams for CID and fs-LID activation elucidated mechanistic differences in the fragmentation behaviour between the statistical bond-breaking of CID and the ultrafast fragmentation triggered by fs-LID. A comparison of the fs-LID fragmentation pathways around different riboses finds that the nature of the ribose greatly influences the local fragmentation behaviour. In a wider context, investigating laser–biomolecule interaction mechanisms could contribute to the understanding of light–biomolecule interactions such as sunlight interacting with living cells.
Version
Open Access
Date Issued
2022-03
Date Awarded
2022-10
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Marangos, Jonathan
Frasinski, Leszek
Sponsor
Engineering and Physical Sciences Research Council (EPSRC)
Grant Number
EP/L016524/1
Publisher Department
Physics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)