Cross-Validation of Lipid Structure Assignment Using Orthogonal Ion Activation Modalities on the Same Mass Spectrometer

The onset and progression of cancer is associated with
changes
in the composition of the lipidome. Therefore, better understanding
of the molecular mechanisms of these disease states requires detailed
structural characterization of the individual lipids within the complex
cellular milieu. Recently, changes in the unsaturation profile of
membrane lipids have been observed in cancer cells and tissues, but
assigning the position(s) of carbon–carbon double bonds in
fatty acyl chains carried by membrane phospholipids, including the
resolution of lipid regioisomers, has proven analytically challenging.
Conventional tandem mass spectrometry approaches based on collision-induced
dissociation of ionized glycerophospholipids do not yield spectra
that are indicative of the location(s) of carbon–carbon double
bonds. Ozone-induced dissociation (OzID) and ultraviolet photodissociation
(UVPD) have emerged as alternative ion activation modalities wherein
diagnostic product ions can enable de novo assignment
of position(s) of unsaturation based on predictable fragmentation
behaviors. Here, for the first time, OzID and UVPD (193 nm) mass spectra
are acquired on the same mass spectrometer to evaluate the relative
performance of the two modalities for lipid identification and to
interrogate the respective fragmentation pathways under comparable
conditions. Based on investigations of lipid standards, fragmentation
rules for each technique are expanded to increase confidence in structural
assignments and exclude potential false positives. Parallel application
of both methods to unsaturated phosphatidylcholines extracted from
isogenic colorectal cancer cell lines provides high confidence in
the assignment of multiple double bond isomers in these samples and
cross-validates relative changes in isomer abundance.