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

212857

Sigma-Aldrich

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

204218

Sigma-Aldrich

Rhodium, powder, 99.95% trace metals basis

06300

Supelco

Aluminum oxide, activated, neutral, Brockmann Activity I

82762

Supelco

Methanol, analytical standard

517747

Sigma-Aldrich

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

717975

Sigma-Aldrich

Methanol, NMR reference standard

GF37207166

Rhodium, wire reel, 0.1m, diameter 1.0mm, as drawn, 99.9%

GF64213680

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

GF06539979

Rhodium, wire reel, 0.1m, diameter 0.5mm, as drawn, 99.9%

GF12568013

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

GF87742659

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

GF37380089

Rhodium, wire reel, 0.2m, diameter 0.25mm, hard, 99.9%

GF22634120

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

GF74452316

Rhodium, wire reel, 0.05m, diameter 0.5mm, as drawn, 99.9%

GF49946417

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

GF38835737

Rhodium, wire reel, 0.2m, diameter 0.12mm, as drawn, 99.9%

GF33789550

Rhodium, foil, not light tested, 50x50mm, thickness 0.001mm, permanent polyester support, 99.85%

GF15499413

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

GF80841146

Rhodium, foil, 25x25mm, thickness 0.15mm, as rolled, 99.9%

GF96356559

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

GF17496973

Rhodium, wire reel, 0.2m, diameter 0.5mm, as drawn, 99.9%

GF44569907

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

GF40539405

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

GF04978963

Rhodium, foil, not light tested, 10x10mm, thickness 0.012mm, as rolled, 99.9%

GF35442727

Rhodium, foil, not light tested, 25x25mm, thickness 0.0005mm, permanent polyester support, 99.85%

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%

GF71687357

Rhodium, microfoil, disks, 10mm, thinness 0.1μm, specific density 124μg/cm², permanent mylar 3.5μm support, 99.9%

GF17514312

Rhodium, foil, not light tested, 25x25mm, thickness 0.00025mm, permanent polyester support, 99.9%

GF49036204

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