Azide-tagged sphingolipids: new tools for metabolic flux analysis.

Azide-tagged sphingolipids: new tools for metabolic flux analysis.

Chembiochem : a European journal of chemical biology (2015-02-14)

María Garrido, José Luis Abad, Gemma Fabriàs, Josefina Casas, Antonio Delgado

ABSTRACT

Several diseases involve alterations in sphingolipid metabolism, so the development of tools for the analysis of sphingolipid metabolic fluxes is of interest. In this work, ω-azidosphingolipids 1-3 have been synthesized and tested as tracers in live cells. The synthesis starts from (S)-Garner's aldehyde and uses bromide or tosyloxy precursors for the introduction of the azido group into the sphingoid base. Studies in HGC-27 cells showed that probes 1-3 compete with the natural metabolites and are incorporated into sphingolipid pathways without affecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited by means of click reactions with different azadibenzocyclooctyne tags. This allows the mass spectrometric characterization of azidosphingolipidomes in pooled samples from different cell populations after independent treatments, providing proof of concept of the applicability of this technology in sphingolipid metabolic flux analysis.

MATERIALS

Product Number

Brand

Product Description

717975

Sigma-Aldrich

Methanol, NMR reference standard

204218

Sigma-Aldrich

Rhodium, powder, 99.95% trace metals basis

82762

Supelco

Methanol, analytical standard

212857

Sigma-Aldrich

Rhodium on alumina, extent of labeling: 5 wt. % loading, matrix alumina support, powder

517747

Sigma-Aldrich

Aluminum oxide, mesoporous, MSU-X (wormhole), average pore size 3.8 nm

06300

Supelco

Aluminum oxide, activated, neutral, Brockmann Activity I

34860

Sigma-Aldrich

Methanol, suitable for HPLC, ≥99.9%

PHR1372

Supelco

Methanol, Pharmaceutical Secondary Standard; Certified Reference Material

676780

Sigma-Aldrich

Methanol, ACS reagent, ≥99.8%

M1775

Sigma-Aldrich

Methanol, Absolute - Acetone free

179337

Sigma-Aldrich

Methanol, ACS reagent, ≥99.8%

154903

Sigma-Aldrich

Methanol, ACS spectrophotometric grade, ≥99.9%

179957

Sigma-Aldrich

Methanol, Laboratory Reagent, ≥99.6%

GF24911726

Rhodium, foil, not light tested, 25x25mm, thickness 0.012mm, as rolled, 99.9%

GF22634120

Rhodium, foil, not light tested, 50x50mm, thickness 0.00075mm, permanent polyester support, 99.85%

GF44569907

Rhodium, foil, 25x25mm, thickness 0.125mm, as rolled, 99.9%

GF49946417

Rhodium, microfoil, disks, 10mm, thinness 0.50μm, specific density 620μg/cm², permanent mylar 3.5μm support, 99.99%

GF36379345

Rhodium, foil, light tested, 15x15mm, thickness 0.05mm, as rolled, 99.9%

GF49036204

Rhodium, foil, not light tested, 25x25mm, thickness 0.00075mm, permanent polyester support, 99.85%

GF15499413

Rhodium, foil, not light tested, 25x25mm, thickness 0.006mm, as rolled, 99.9%

GF14938005

Rhodium, microfoil, disks, 25mm, thinness 0.1μm, specific density 124μg/cm², permanent mylar 3.5μm support, 99.9%

GF87742659

Rhodium, foil, not light tested, 25x25mm, thickness 0.001mm, permanent polyester support, 99.85%

GF57675561

Rhodium, foil, light tested, 25x25mm, thickness 0.05mm, as rolled, 99.9%

GF82366342

Rhodium, foil, not light tested, 50x50mm, thickness 0.00025mm, permanent polyester support, 99.9%

GF54466937

Rhodium, wire reel, 0.5m, diameter 0.12mm, as drawn, 99.9%

GF64213680

Rhodium, wire reel, 0.05m, diameter 0.12mm, as drawn, 99.9%

GF12568013

Rhodium, foil, light tested, 25x25mm, thickness 0.05mm, as rolled, x-ray quality, 99.9%

GF40539405

Rhodium, foil, 25x25mm, thickness 0.1mm, as rolled, 99.9%

GF45819995

Rhodium, wire reel, 0.05m, diameter 0.25mm, hard, 99.9%

GF96356559

Rhodium, foil, light tested, 25x25mm, thickness 0.075mm, as rolled, 99.9%