Matching IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay and its clinical application for tracing tumor-associated glycosphingolipids in hepatocellular and pancreatic cancer

TY - JOUR

T1 - Matching IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay and its clinical application for tracing tumor-associated glycosphingolipids in hepatocellular and pancreatic cancer

AU - Distler, Ute

AU - Hülsewig, Marcel

AU - Souady, Jamal

AU - Dreisewerd, Klaus

AU - Haier, Jörg

AU - Senninger, Norbert

AU - Friedrich, Alexander W

AU - Karch, Helge

AU - Hillenkamp, Franz

AU - Berkenkamp, Stefan

AU - Peter-Katalinić, Jasna

AU - Müthing, Johannes

PY - 2008/3/15

Y1 - 2008/3/15

N2 - Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including the development of cancer. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of modern mass spectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay for the structural characterization of GSLs. We matched three complementary methods including (i) TLC separation of GSLs, (ii) their detection with oligosaccharide-specific proteins, and (iii) in situ MS analysis of protein-detected GSLs. The high specificity and sensitivity is demonstrated by use of antibodies, bacterial toxins, and a plant lectin. The procedure works on a nanogram scale, and detection limits of less than 1 ng at its best of immunostained GSLs were obtained. Furthermore, only crude lipid extracts of biological sources are required for TLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associated GSLs in human hepatocellular and pancreatic tumors. Thus, the in situ TLC-IR-MALDI-MS of immunolabeled GSLs opens new doors by delivering specific structural information of trace quantities of GSLs with only a limited investment in sample preparation.

AB - Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including the development of cancer. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of modern mass spectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay for the structural characterization of GSLs. We matched three complementary methods including (i) TLC separation of GSLs, (ii) their detection with oligosaccharide-specific proteins, and (iii) in situ MS analysis of protein-detected GSLs. The high specificity and sensitivity is demonstrated by use of antibodies, bacterial toxins, and a plant lectin. The procedure works on a nanogram scale, and detection limits of less than 1 ng at its best of immunostained GSLs were obtained. Furthermore, only crude lipid extracts of biological sources are required for TLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associated GSLs in human hepatocellular and pancreatic tumors. Thus, the in situ TLC-IR-MALDI-MS of immunolabeled GSLs opens new doors by delivering specific structural information of trace quantities of GSLs with only a limited investment in sample preparation.

KW - Carbohydrate Sequence

KW - Chromatography, Thin Layer

KW - Glycosphingolipids

KW - Liver Neoplasms

KW - Molecular Sequence Data

KW - Pancreatic Neoplasms

KW - Sensitivity and Specificity

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

KW - Spectrophotometry, Infrared

U2 - 10.1021/ac702071x

DO - 10.1021/ac702071x

M3 - SCORING: Journal article

C2 - 18278947

VL - 80

SP - 1835

EP - 1846

JO - ANAL CHEM

JF - ANAL CHEM

SN - 0003-2700

IS - 6

ER -