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

PHR1372

Supelco

Methanol, Pharmaceutical Secondary Standard; Certified Reference Material

GF45819995

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

GF87742659

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

GF22634120

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

GF12568013

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

GF04978963

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

GF33789550

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

GF24911726

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

GF74452316

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

GF17496973

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

GF54466937

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

GF83497652

Rhodium, wire reel, 0.05m, diameter 1.0mm, as drawn, 99.9%

GF72510364

Rhodium, wire reel, 0.5m, diameter 0.25mm, hard, 99.9%

GF57675561

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

GF37380089

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

GF44569907

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

GF26710680

Rhodium, wire reel, 0.1m, diameter 0.12mm, as drawn, 99.9%

GF27499045

Rhodium, wire reel, 0.1m, diameter 0.25mm, hard, 99.9%

GF96356559

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

GF06539979

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

439193

Sigma-Aldrich

Methanol, suitable for HPLC, gradient grade, suitable as ACS-grade LC reagent, ≥99.9%

34885

Sigma-Aldrich

Methanol, suitable for HPLC, gradient grade, ≥99.9%

646377

Sigma-Aldrich

Methanol, HPLC Plus, ≥99.9%

676780

Sigma-Aldrich

Methanol, ACS reagent, ≥99.8%

M1775

Sigma-Aldrich

Methanol, Absolute - Acetone free

154903

Sigma-Aldrich

Methanol, ACS spectrophotometric grade, ≥99.9%

179957

Sigma-Aldrich

Methanol, Laboratory Reagent, ≥99.6%

179337

Sigma-Aldrich

Methanol, ACS reagent, ≥99.8%

24229

Sigma-Aldrich

Methanol, puriss., meets analytical specification of Ph Eur, ≥99.7% (GC)

32213

Sigma-Aldrich

Methanol, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.8% (GC)