This thesis describes the development of a new type of two-dimensional mass spectrometry: partial covariance-based two dimensional mass spectrometry, or pC-2DMS. Using a commercially available linear ion trap mass spectrometer and the statistical technique of partial covariance mapping, the ability to identify fragment ions originating from the same or consecutive decomposition pathways of the same molecule is demonstrated. As will be shown, this information is beneficial to analytical and mechanistic studies of trapped biopolymers. After initial development on peptide molecules, the generality of the pC-2DMS technique has been confirmed by extension to whole protein molecules as well as DNA and RNA oligonucleotide sequences.
This work represents the first time partial covariance mapping has been used for structural analysis, the first application of a covariance mapping technique to trapped ions, the first time a covariance mapping technique has been applied to molecular species as large as molecules on the order of kilodaltons and the first demonstration of the correlation of secondary consecutive fragmentation products by a covariance mapping technique.
Within pC-2DMS, the total ion count is used as a single fluctuating parameter in the partial covariance formula to suppress spurious correlations due to scan-to-scan fluctuations in a number of different experimental parameters, which cause quasi-uniform fluctuations across all of the tandem mass spectrum.
The applications demonstrated in this thesis include the resolution of mixtures of different combinatorially modified structural isomers (shown here for structural isomers of histone H4 fragment 4-17), an orders-of-magnitude reduction in the false positive rate for matching fragment ion signals in database search algorithms, resolution of charge state for highly charged fragment ions without the need for high mass accuracy resolution of the isotopic envelope and the in silico separation of strongly overlapping fragment ion spectra from different co-isolated and co-fragmented molecular species.Open Acces
