Partial synthesis of ganglioside and lysoganglioside lipoforms as internal standards for MS quantification

Abstract

Nonnatural ganglioside and lysoganglioside lipoforms 2693 MATERIALS AND METHODS MaterialsAll chemicals were of analytical grade or the highest purity available. Water (H 2 O) used for buffers and solutions was purifi ed by an ultrapure H 2 O system (EASYpure UV/UF D8612, Werner Reinstwassersysteme, Leverkusen, Germany). Solvents used in reactions of oxygen-and moisture-sensitive compounds were in anhydrous form. 1-Propanol, methanol (MeOH), pyridine, and acetic anhydride were degassed before use. The native mixture of bovine brain gangliosides, Cronassial ® , which consists of 21% GM1, 40% GD1a, 16% GD1b, 19% GT1b, and 4% other gangliosides ( 25 ), was available in our lab. Sphingolipid ceramide N -deacylase (SCDase) and ␤ -galactosidase from bovine testes were from Sigma-Aldrich (Schnelldorf, Germany). Triton™ X-100, sodium taurodeoxycholate, Grubbs catalyst 2nd generation, HoveydaGrubbs catalyst 2nd generation, and Stewart-Grubbs catalyst were also from Sigma-Aldrich. For normal phase (NP) column chromatography, silica gel 60 (0.040-0.063 mm or 0.015-0.040 mm) from Merck (Darmstadt, Germany) was used. For desalting and reversed phase (RP) column chromatography, LiChroprep ® RP-18 (0.040-0.063 mm) from Merck was used. For TLC analysis, highperformance TLC (HPTLC) silica gel 60 F 254 and HPTLC silica gel 60 RP-18 plates from Merck were used. For anion-exchange chromatography, DEAE Sephadex™ A-25 from Amersham Pharmacia Biotech AB (Uppsala, Sweden) was used. Purifi cationDesalting was performed by the method of Wiliams and McCluer ( 26 ). The usage of polar eluents for column chromatography causes signifi cant contamination of column material in the products. We reduced this contamination to a minimum by the following method. NP silica gel columns of a very small column volume (approximately 1 ml column volume per 5 mg of product) were prepared in fi ltration columns from Supelco (Bellefonte, PA) using a mobile phase in which the substance is retained on the column. The substance was applied to the column and washed with three column volumes of the same solvent. Then, the substance was eluted by an appropriate solvent of higher polarity. C-NMR spectroscopy, Bruker Avance 300, 400, or 500 instruments (Billerica, MA) were used. For exchange of exchangeable protons by deuterium, gangliosides were dissolved in CD 3 OD and the solvent was removed in a stream of nitrogen. This process was repeated three times before measurement. For EI-MS, a MAT 95 XL sector fi eld instrument (Thermo Finnigan MAT GmbH, Bremen, Germany) or a MAT 90 sector fi eld instrument gangliosidosis ( 22 ). Both substances are potential biomarkers for these diseases ( 23 ), and the lysoganglioside standards prepared in this work can be used for their quantitative analysis. Analytical proceduresQuantifi cation of gangliosides by MS, including quantitative imaging MS, requires calibration substances. Hereby, calibration is defined as the establishment of a correlation between analyte concentration and mass spectrometer response. Three principle meth...

Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites

“…The following internal standards (Matreya LLC) were used: 25 pmol GB3 (d18:1/18:0-D 3 ), 25 pmol GM3 (d18:1/18:0-D 3 ), 25 pmol GM2 (d18:1/18:0-D 3 ), and 25 pmol GM1 (d18:1/18:0-D 3 ) semi-synthesized in-house, following a protocol described in the ref. 81 . For cells, residual amounts of GM1 were recovered from the aqueous phase of the 2-step extraction by a C8 functionalized solid-phase extraction 82 .…”

Apprehending ganglioside diversity: a comprehensive methodological approach

“…Some have used GM3d3 for various GG classes (GM3, GM2, GD3, and GD2) in human plasma ( 36 ) or GM1d3 for GM2 in cells and plasma ( 47 ), while others have used phospholipids ( 30,31 ). Recently, authors reported the synthesis of GG-bearing nonnatural FAs for the major classes of mammalian brain and also some tumorassociated classes, which will be valuable tools to use as internal standards for MS analyses ( 48 ). For quantifi cation purposes, authors used commercial standards they considered to be representative of the GG molecular species present in their samples to spike their extracts.…”