Combinatory Data-Independent Acquisition and Parallel Reaction Monitoring Method for Deep Profiling of Gangliosides

Ganglioside
is an important class of lipid species involved in
intercellular signaling and various diseases, especially for neurodegenerative
diseases. Systematic ganglioside profiling is challenging because
of their naturally low abundance and highly diverse species. Herein,
a new data-independent acquisition and parallel reaction monitoring
(DIA/PRM) method with superior sensitivity was developed. The untargeted
DIA acquisition consecutively records all the precursor ion and fragment
ions at the same time, while the targeted PRM analysis with versatile
higher collisional dissociation generates full MS/MS spectra for structure
elucidation and verification. As compared with traditional data-dependent
acquisition (DDA), the DIA/PRM method unbiasedly detected the majority
of abundant ganglioside species and as low as 50 pg of ganglioside
in an untargeted manner. Gangliosides in four kinds of biological
samples including the mouse brain, mouse plasma, HeLa cell, and human
colon cancer tissue were systematically identified, and low-abundance
ganglioside species were further extended on the basis of linear chromatography
retention rules of the most frequently detected ganglioside species.
A total of 383 ganglioside features were defined with 329 of them
derived from 32 ganglioside species. Taking advantage of the high-resolution
MS analysis, rare ganglioside species were further elucidated according
to their characteristic fragment ions and neutral losses. In total,
18 gangliosides with a ceramide carbon number from 20 to 25 and modified
gangliosides, including 18 acetylated, 8 diacetylated, 1 phosphorylated,
36 N-glycolyneuraminic acid (NeuGc)-containing, and 7 di-NeuGc-containing
gangliosides, were newly identified. The developed DIA/PRM method
therefore generated a rich ganglioside resource for further functional
exploration and is a unique alternative for DDA analysis for global
ganglioside profiling in various biological systems.