Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions

Ruijun Tian*, Jing Jin, Lorne Taylor, Brett Larsen, Susan E. Quaggin, Tony Pawson

*Corresponding author for this work

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1334-1338
Number of pages5
JournalProteomics
Volume13
Issue number8
DOIs
StatePublished - Apr 1 2013

Fingerprint

Gangliosides
Mass spectrometry
Mass Spectrometry
Proteins
G(M1) Ganglioside
Bacterial Toxins
Vascular Endothelial Growth Factor Receptor-1
Retinal Pigments
Retinal Pigment Epithelium
Receptor Protein-Tyrosine Kinases
Cell membranes
Hydrophobic and Hydrophilic Interactions
Membrane Proteins
Immunoglobulin G
Cell Membrane

Keywords

  • Ganglioside
  • Lipid-protein interaction
  • Mass spectrometry
  • Technology

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Tian, Ruijun ; Jin, Jing ; Taylor, Lorne ; Larsen, Brett ; Quaggin, Susan E. ; Pawson, Tony. / Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions. In: Proteomics. 2013 ; Vol. 13, No. 8. pp. 1334-1338.
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Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions. / Tian, Ruijun; Jin, Jing; Taylor, Lorne; Larsen, Brett; Quaggin, Susan E.; Pawson, Tony.

In: Proteomics, Vol. 13, No. 8, 01.04.2013, p. 1334-1338.

Research output: Contribution to journalArticle

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