Thomas et al. 2025

Trifunctional lipid derivatives: PE’s mitochondrial interactome

Authors

Alix Thomas, Rainer Mueller, Ana Kojic, Scotland E. Farley, Frank Stein, Per Haberkant, Carsten Schultz

Journal

Chemical Communications

https://doi.org/10.1039/D4CC03599B

Abstract

Phosphatidylethanolamine (PE) is a ubiquitous lipid species in higher eukaryotes which resides preferentially in mitochondria. Here, we synthesized a multifunctionalized PE derivative (1) de-signed to identify PE-binding proteins in intact cells through photo-crosslinking and subsequent isolation and proteomic analysis of the PE-protein conjugates. Due to its aromatic caging group, 1 is initially concentrating in perinuclear membranes. After uncaging, rapid translocation to mitochondria was observed. Hence, the tool is useful for tracking PE location and for determining the PE interactome. A trifunctional phosphatidic acid (PA) derivative in compari-son was rapidly metabolized and is hence more limited in its use.

Lipid probes utilized

Phosphatidic Acid Phosphatidic Acid

Phosphatidylethanolamine PPhosphatidylethanolamine

Cell line analyzed

HeLa

Uncaging & Crosslinking timeline

Lipid Probe Uptake time Uncaging time Interaction time Crosslinking time
PA 60 min 5 min 15 min 5 min
PE 60 min 5 min 15 min 5 min

Mass spectrometry quantification method

16-channel Tandem Mass Tagging (TMT16)

ProteomeXchange title: Trifunctional lipid derivatives: PE’s mi-tochondrial interactome

ProteomeXchange accession: PXD059960

PubMed ID: Not applicable

Publication DOI: 10.1039/D4CC03599B

Project Webpage: http://www.ebi.ac.uk/pride/archive/projects/PXD059960

FTP Download: https://ftp.pride.ebi.ac.uk/pride/data/archive/2025/01/PXD059960

Additional sample preparation ?

Data visualization

Volcano plots depict the relative enrichment of each protein versus non-crosslinked 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 enriched candidates are defined as proteins with a false discovery rate less than 0.2 and a fold change of at least 1.5-fold, and Orange enriched hits are defined as proteins with a false discovery rate less than 0.05 and a fold change of at least 2-fold in the +UV over the -UV).

Ranked-order plots depict the relative enrichment of each protein versus non-crosslinked 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 enriched candidates are defined as proteins with a false discovery rate less than 0.2 and a fold change of at least 1.5-fold, and Orange enriched hits are defined as proteins with a false discovery rate less than 0.05 and a fold change of at least 2-fold in the +UV over the -UV).

[1] "Data not yet provided."

Gene Ontology Analysis

In beta: GO analysis still under development

Figure 1: GO Dot plots display the enrichment of GO terms among the proteins enriched to the probe. For these analyses, only proteins categorized as “enriched candidates” and “enriched hits” were subject to GO analysis. The Cell Compartment analysis assesses whether the list of enriched proteins contains a statistically significant number of proteins in the same cellular region; appropriately, the Molecular Function and Biological Process analyses does the same for molecular function and biological process, respectively. Click here for more information about Gene Ontology analysis.

Data exploration and download

Check the boxes below to filter the dataset by which lipid probe was used in the pulldown and by significance thresholds.

Data download