These authors contributed equally to this work.
Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions
Article first published online: 12 MAR 2013
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Special Issue: FOCUS ON INTEGRATIVE PROTEOMICS
Volume 13, Issue 8, pages 1334–1338, April 2013
How to Cite
Tian, R., Jin, J., Taylor, L., Larsen, B., Quaggin, S. E. and Pawson, T. (2013), Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions. Proteomics, 13: 1334–1338. doi: 10.1002/pmic.201200410
Colour Online: See the article online to view Fig. 2 in colour.
- Issue published online: 12 APR 2013
- Article first published online: 12 MAR 2013
- Accepted manuscript online: 11 FEB 2013 04:08AM EST
- Manuscript Accepted: 24 NOV 2012
- Manuscript Revised: 12 NOV 2012
- Manuscript Received: 5 SEP 2012
- CIHR Postdoctoral Fellowship
- Lipid–protein interaction;
- Mass spectrometry;
Gangliosides are ubiquitous components of cell membranes. Their interactions with bacterial toxins and membrane-associated proteins (e.g. receptor tyrosine kinases) have important roles in the regulation of multiple cellular functions. Currently, an effective approach for measuring ganglioside-protein interactions especially in a large-scale fashion is largely missing. To this end, we report a facile MS-based approach to explore gangliosides extracted from cells and measure their interactions with protein of interest globally. We optimized a two-step protocol for extracting total gangliosides from cells within 2 h. Easy-to-use magnetic beads conjugated with a protein of interest were used to capture interacting gangliosides. To measure ganglioside-protein interaction on a global scale, we applied a high-sensitive LC-MS system, containing hydrophilic interaction LC separation and multiple reaction monitoring-based MS for ganglioside detection. Sensitivity for ganglioside GM1 is below 100 pg, and the whole analysis can be done in 20 min with isocratic elution. To measure ganglioside interactions with soluble vascular endothelial growth factor receptor 1 (sFlt1), we extracted and readily detected 36 species of gangliosides from perivascular retinal pigment epithelium cells across eight different classes. Twenty-three ganglioside species have significant interactions with sFlt1 as compared with IgG control based on p value cutoff <0.05. These results show that the described method provides a rapid and high-sensitive approach for systematically measuring ganglioside-protein interactions.