Nondestructive Detection of Neutral Glycosphingolipids with Lipophilic Anionic Fluorochromes and Their Employment for Preparative High-Performance Thin-Layer Chromatography

Müthing, Johannes; Kemminer, Sven E.

doi:10.1006/abio.1996.0275

Cited by 23 publications

(14 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Orcinol or resorcinol stained GSL bands can be quantified by densitometric scanning at incident light with a wavelength of 550 and 580-620 nm, respectively. For preparative TLC, GSLs have to be localized by nondestructive reagents such as the fluorochrome primulin (direct yellow) (Skipski, 1975) or others Müthing & Kemminer, 1996). GSLs can then be recovered by scraping the visualized bands and extracting the GSLs from the silica.…”

Section: A Basicsmentioning

confidence: 99%

Advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry

Müthing

Distler

2009

Mass Spectrom. Rev.

Self Cite

View full text Add to dashboard Cite

Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including infectious diseases and the development of cancer. Knowledge of the number and sequence of monosaccharides and their anomeric configuration and linkage type, which make up the principal items of the glyco code of biologically active carbohydrate chains, is essential for exploring the function of GSLs. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of novel biochemical and biophysical technologies. Mass spectrometry (MS) takes part in the network of collaborations to further unravel structural and functional aspects within the fascinating world of GSLs with the ultimate aim to better define their role in human health and disease. However, a single-method analytical MS technique without supporting tools is limited yielding only partial structural information. Because of its superior resolving power, robustness, and easy handling, high-performance thin-layer chromatography (TLC) is widely used as an invaluable tool in GSL analysis. The intention of this review is to give an insight into current advances obtained by coupling supplementary techniques such as TLC and mass spectrometry. A retrospective view of the development of this concept and the recent improvements by merging (1) TLC separation of GSLs, (2) their detection with oligosaccharide-specific proteins, and (3) in situ MS analysis of protein-detected GSLs directly on the TLC plate, are provided. The procedure works on a nanogram scale and was successfully applied to the identification of cancer-associated GSLs in several types of human tumors. The combination of these two supplementary techniques opens new doors by delivering specific structural information of trace quantities of GSLs with only limited investment in sample preparation.

show abstract

Section: A Basicsmentioning

confidence: 99%

Advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry

Müthing

Distler

2009

Mass Spectrom. Rev.

Self Cite

View full text Add to dashboard Cite

show abstract

“…1.14366.001 cholesterol enzymatic, E.Merck, Darmstadt, Germany) (12) and phospholipids bythe method of Stewart (13). Individual phespholipid fractions ~mre separated by high performance thin layer chromatography (HPTLC) by a modified method of Jd~zmes Mulhing (14). (HPTLC piate~ 10X20cm, precoated with 0.2mm Silica gel 60 on aluminum, No.94069, Aldrich, USA).…”

Section: Material8 and Methodsmentioning

confidence: 99%

Comparative analysis of RBC membrane lipids in thalassemia, and iron deficiency anemia in relation to hypochromia and oxidant injury

Sanghani

Haldankar

Shalia

et al. 2001

Indian J Clin Biochem

View full text Add to dashboard Cite

The effect of an intrinsic defect in the red cell and pronounced hypochromia on oxidative damage to RBC membrane lipids was compared in bata-thalassemia and iron deficiency anemia (IDA), which have a vaded etiology but equivalent low hemoglobin content. The study was planned to correlate the etiology of the disorders to the severity of lipid imbalance and RBC hemolysis in membranes of both the conditions. Results Indicated a fall of lysophosphatidylcholine~LPC), phosphatidylethanolemine(PE) and the unsaturated to saturated fatty acid ratlo in both conditions, while phosphat~ylcholine(PC) Increased only in thalassemia. However, irrespective of the disease, sphingomyelin(SM), total cholesterol and phospholipid levels elevated and the hydrogen peroxide stress test Indicated Increased susceptibility of both pathologic RBCs to peroxidation. Present findings Indicate that IDA and thalassemla allow for considerable amounts of oxidative damage to membrane Iipicls, irrespective of their etiologias, and thus point hypochromia as an Important contributor for Inducing lipid Imbalance and RBC hemolysis. KEYWORDS:Hypochromia, intnnsic defect, oxidation, RBC membrane, lipid imbalance. UClrRODUCTIONAn i,~,b--=cellular defect in the red blood cell (RBC) can accentuate oxygen radical formation and risk damage to cellular components (1,2). On the other hand, there is some evidence that hypochromia may fadlitate oxidation of the membrane by reducing the buffering protection of hemoglobin (3,4). The med'~misms facilitating oxidative damage to red cells in thalassemia are multifactodal and result from the presence of excess unpaired globin chains and high i,~;~-~ellular content of nonhemogtobin iron, in addition to hypochromia (2,3,5). However, in iron deficiency anemia (IDA), hypochromia is the lone factor that may enhance oxidant-induced threat to the RBC membrane (6). The accumulat|on of activated oxygen can paternally cause hemolysis (3,5,7). Neverlh~ess it is not clear whether the presence of an =intrinsic" defect in the RBC or "hypochromia" is the more Author for correspondence: Dr. V.A.Haldankar, Professor and Head, Department of Biochemistry, at the above address. important factor as far as lipid damage and hemolysis is concerned.In order to correlate the etiology of the disorders to the severity of lipoperoxidation, lipid imbalance and RBC hemolysis, we conducted a comparative inves~gation between beta thalassemia hornozygous and IDA membrane-lipid profiles. MATERIAL8 AND METHODSIn the present study, 25-transfusion dependent, non-spienectomized (non-spx) beta thalassemia homozygous children (5-12 years) and 35 patients afflicted with IDA ( 22-40 years) attending the hematology out patient department of B.Y.L.Nair Charitable hospital were included with their consent. Age and se~ matched 20 normal children and 30 adult volunteers formed the control groups for thalassemia and IDA respectively. In thalassemia patients, blood v~s collected prior to blood tra~fusion, in an effort to minimize the influence of donor blood ce...

show abstract

“…2(a), the major neutral GSLs of mouse hybridoma cells are Lc2Cer and Gb3Cer. This GSL mixture was selected for the ensuing bacteria‐binding assays on the basis of its high content of Gb3Cer 16. Both GSLs separate in double bands, due to heterogeneous fatty acid composition of the ceramide moiety.…”

Section: Resultsmentioning

confidence: 99%

“…A reference mixture of neutral GSLs composed of lactosylceramide (Lc2Cer) and globotriaosylceramide (Gb3Cer) was prepared from mouse‐mouse hybridoma cells 15, 16. A neutral GSL mixture from human erythrocytes, containing Gb4Cer, Gb3Cer and Lc2Cer as major compounds, has been recently described 17.…”

Section: Methodsmentioning

confidence: 99%

Application of thin‐layer chromatography/infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry to structural analysis of bacteria‐binding glycosphingolipids selected by affinity detection

Müsken

Souady

Dreisewerd

et al. 2010

Rapid Comm Mass Spectrometry

Self Cite

View full text Add to dashboard Cite

Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin-layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL-specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria-specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time-consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo-series neutral GSLs recognized by P-fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR-MALDI-o-TOF-MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate-specific pathogens involved in human infectious diseases.

show abstract

Nondestructive Detection of Neutral Glycosphingolipids with Lipophilic Anionic Fluorochromes and Their Employment for Preparative High-Performance Thin-Layer Chromatography

Cited by 23 publications

References 4 publications

Advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry

Advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry

Comparative analysis of RBC membrane lipids in thalassemia, and iron deficiency anemia in relation to hypochromia and oxidant injury

Application of thin‐layer chromatography/infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry to structural analysis of bacteria‐binding glycosphingolipids selected by affinity detection

Contact Info

Product

Resources

About