Structural profiling of individual glycosphingolipids in a single thin-layer chromatogram by multiple sequential immunodetection matched with Direct IR-MALDI-o-TOF mass spectrometry

TY - JOUR

T1 - Structural profiling of individual glycosphingolipids in a single thin-layer chromatogram by multiple sequential immunodetection matched with Direct IR-MALDI-o-TOF mass spectrometry

AU - Souady, Jamal

AU - Soltwisch, Jens

AU - Dreisewerd, Klaus

AU - Haier, Jörg

AU - Peter-Katalinić, Jasna

AU - Müthing, Johannes

PY - 2009/11/15

Y1 - 2009/11/15

N2 - The thin-layer chromatography (TLC) immunoenzyme overlay assay is a widely used tool for antibody-mediated identification of glycosphingolipids (GSLs) in mixtures. However, because the majority of GSLs is left unexamined in a chromatogram of a single assay, we developed a novel method that permits detection of various GSLs by sequential multiple immunostaining combined with individual coloring of GSLs in the same chromatogram. Specific staining was achieved by means of primary anti-GSL antibodies, directed against lactosylceramide, globotriaosylceramide, and globotetraosylceramide, in conjunction with alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary antibodies together with the appropriate chromogenic substrates. Triple coloring with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-AP, Fast Red-AP, and 3,3'-diaminobenzidine (DAB)-HRP resulted in blue, red, and black precipitates, respectively, following three sequential immunostaining rounds. Structures of antibody-detected GSLs were determined by direct coupling of TLC with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. This combinatorial technique was used to demonstrate structural GSL profiling of crude lipid extracts from human hepatocellular cancer. This powerful technology allows efficient structural characterization of GSLs in small tissue samples and marks a further step forward in the emerging field of glycosphingolipidomics.

AB - The thin-layer chromatography (TLC) immunoenzyme overlay assay is a widely used tool for antibody-mediated identification of glycosphingolipids (GSLs) in mixtures. However, because the majority of GSLs is left unexamined in a chromatogram of a single assay, we developed a novel method that permits detection of various GSLs by sequential multiple immunostaining combined with individual coloring of GSLs in the same chromatogram. Specific staining was achieved by means of primary anti-GSL antibodies, directed against lactosylceramide, globotriaosylceramide, and globotetraosylceramide, in conjunction with alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary antibodies together with the appropriate chromogenic substrates. Triple coloring with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-AP, Fast Red-AP, and 3,3'-diaminobenzidine (DAB)-HRP resulted in blue, red, and black precipitates, respectively, following three sequential immunostaining rounds. Structures of antibody-detected GSLs were determined by direct coupling of TLC with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. This combinatorial technique was used to demonstrate structural GSL profiling of crude lipid extracts from human hepatocellular cancer. This powerful technology allows efficient structural characterization of GSLs in small tissue samples and marks a further step forward in the emerging field of glycosphingolipidomics.

KW - Antibodies

KW - Carcinoma, Hepatocellular

KW - Chromatography, Thin Layer

KW - Glycosphingolipids

KW - Humans

KW - Liver Neoplasms

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

U2 - 10.1021/ac901948h

DO - 10.1021/ac901948h

M3 - SCORING: Journal article

C2 - 19908908

VL - 81

SP - 9481

EP - 9492

JO - ANAL CHEM

JF - ANAL CHEM

SN - 0003-2700

IS - 22

ER -