Höglinger et al. 2017

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

Study details

Authors

Doris Höglinger, André Nadler, Per Haberkant, Joanna Kirkpatrick, Martina Schifferer, Frank Stein, Sebastian Hauke, Forbes D. Porter, Carsten Schultz

Journal

PNAS

https://doi.org/10.1073/pnas.1611096114

Abstract

Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.

Lipid probes utilized

Trifunctional Sphingosine

Trifunctional Diacylglycerol

Trifunctional 8-3 Fatty Acid

Cell line analyzed

HeLa

Uncaging & Crosslinking timeline

Lipid Probe	Uptake time	Uncaging time	Interaction time	Crosslinking time
Sph	5 min	2.5 min	0 min	2.5 min
DAG	15 min	2.5 min	0 min	2.5 min
8-3 FA	15 min	2.5 min	0 min	2.5 min

Mass spectrometry quantification method

Peptide spectral counting

Data visualization

Heat map

Heat map depicts the average PSM count for two replicates each of UV-irradiated samples treated with trifunctionalized Sph, DAG, and FA. Color denotes the

---

Venn Diagram

A Venn diagram depicts the overlapping identification of proteins between the three lipid probes. There are 41 proteins uniquely identified after enrichment with tf-Sph, 130 proteins uniquely identified after enrichment with tf-DAG, and no proteins uniquely identified to tf-8:3-FA.

Gene Ontology Analysis

In beta: GO analysis still under development

Cell Compartment

Molecular Function

Biological Process

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

Select overlap regions to see co-enriched proteins:

Enriched to Sph only Enriched to DAG only Enriched to Sph and DAG Enriched to Sph and FA Enriched to DAG and FA Enriched to all probes

Data download