Volcano plots depict the relative enrichment of each protein versus control (x-axis) and the statistical significance of each protein following a Student’s t-test (y-axis, -log10 transformed). The left panel depicts the results of a pulldown using trifunctionalized phosphatidic acid; the right panel depicts the results of the pulldown using trifunctionalized phosphatidylethanolamine. Black proteins are unenriched or depleted in the presence of probe, Purple proteins are enriched to the probe but fall below a Fold-change or p-value threshold of significance (p < 0.05 & logFC > 1), and Orange proteins surpass the threshold of significance (p < 0.05 & logFC > 2).
Kundu et al. 2024
Investigation of Glycosylphosphatidylinositol (GPI)‐Plasma Membrane Interaction in Live Cells and the Influence of GPI Glycan Structure on the Interaction
Study details
Journal
Chemistry - A European Journal https://doi.org/10.1002/chem.202303047
Abstract
Glycosylphosphatidylinositols (GPIs) need to interact with other components in the cell membrane to transduce transmembrane signals. A bifunctional GPI probe was employed for photoaffinity‐based proximity labelling and identification of GPI‐interacting proteins in the cell membrane. This probe contained the entire core structure of GPIs and was functionalized with photoreactive diazirine and clickable alkyne to facilitate its crosslinking with proteins and attachment of an affinity tag. It was disclosed that this probe was more selective than our previously reported probe containing only a part structure of the GPI core for cell membrane incorporation and an improved probe for studying GPI‐cell membrane interaction. Eighty‐eight unique membrane proteins, many of which are related to GPIs/GPI‐anchored proteins, were identified utilizing this probe. The proteomics dataset is a valuable resource for further analyses and data mining to find new GPI‐related proteins and signalling pathways. A comparison of these results with those of our previous probe provided direct evidence for the profound impact of GPI glycan structure on its interaction with the cell membrane.
Lipid probes utilized
Bifunctional Glycosylphosphatidylinositol (bf-GPI)
Bifunctional phosphatidyl moiety with &-D-glucoside (Control)
Cell line analyzed
Uncaging & Crosslinking timeline
| Lipid Probe | Uptake time | Uncaging time | Interaction time | Crosslinking time |
|---|---|---|---|---|
| bf-GPI | 180 min | NA | NA | 15 min |
| Control | 180 min | NA | NA | 15 min |
Mass spectrometry quantification method
Precursor ion intensity Label Free Quantitation (LFQ), performed using Proteome Discoverer
Additional sample preparation ?
Data visualization
Ranked-order plots depict the relative enrichment of each protein versus control (y-axis) from lowest to highest. The left panel depicts the results of a pulldown using trifunctionalized phosphatidic acid; the right panel depicts the results of the pulldown using trifunctionalized phosphatidylethanolamine. Black proteins are unenriched or depleted in the presence of probe, Purple proteins are enriched to the probe but fall below a Fold-change or p-value threshold of significance (p < 0.05 & logFC > 1), and Orange proteins surpass the threshold of significance (p < 0.05 & logFC > 2).
MA plots depict the average abundance of each protein in the crosslinked and uncrosslinked conditions (x-axis) versus the log2-transformed fold-change between the crosslinked and uncrosslinked conditions (y-axis). Black proteins are unenriched or depleted in the presence of probe, Purple proteins are enriched to the probe but fall below a Fold-change or p-value threshold of significance (p < 0.05 & logFC > 1), and Orange proteins surpass the threshold of significance (p < 0.05 & logFC > 2).
Data exploration
Check the boxes below to filter the dataset by which lipid probe was used in the pulldown and by significance thresholds.