Fast-atom-bombardment mass spectrometry and electron-impact direct-probe mass spectrometry in the structural analysis of oligosaccharides with special reference to the poly(N-acetyl-lactosamine) series.

A comparison has been made of positive- and negative-ion fast-atom-bombardment (FAB) and electron-impact (EI) mass spectrometry for analysis of oligosaccharides and alditols containing alternating and consecutive sequences of neutral and acetamido sugars. Among these were novel chemically synthesized tetrasaccharides with Ii antigen activities. FAB ionization has the advantage that it is applicable to non-derivatized oligosaccharides and it can determine Mr. However, the abundance of fragment ions providing structural information and the amount of material required for analysis (1-50 nmol) varied from sample to sample. In contrast, EI mass spectrometry of 5 nmol of permethylated or peracetylated oligosaccharides reliably gives all the fragment ions formed by cleavage across the glycosidic bonds.     

Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry

Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.     

Nonredundant mass spectrometry: a strategy to integrate mass spectrometry acquisition and analysis

Protein identification using automated data-dependent tandem mass spectrometry (MS/MS) is now a standard procedure. However, in many cases data-dependent acquisition becomes redundant acquisition as many different peptides from the same protein are fragmented, whilst only a few are needed for unambiguous identification. To increase the quality of information but decrease the amount of information, a nonredundant MS (nrMS) strategy has been developed. With nrMS, data analysis is an integral part of the overall MS acquisition and analysis, and not an endpoint as typically performed. In this nrMS workflow a matrix assisted laser desorption/ionization-time of flight-time of flight (MALDI-TOF/TOF) instrument is used. MS and restricted MS/MS data are searched and identified proteins are used to generate an "exclusion list", after in silico digestion. Peptide fragmentation is then restricted to only the most intense ions not present in the exclusion list. This process is repeated until all peaks are accounted for or the sample is consumed. Compared to nanoLC-MS/MS, nrMS yielded similar results for the analysis of six pooled two-dimensional electrophoresis (2-DE) spots. In comparison to standard data-dependent MALDI-MS/MS for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel band analysis, nrMS dramatically increased the number of identified proteins. It was also found that this new workflow significantly increased sequence coverage by identifying unexpected peptides, which can result from post-translational modifications.     

Toll-like receptors in autoimmunity with special reference to systemic lupus erythematosus

The Toll-like receptor (TLR) family plays a fundamental role in host innate immunity by mounting a rapid and potent inflammatory response to pathogen infection. TLRs recognize distinct microbial components and activate intracellular signaling pathways that induce expression of host inflammatory genes. Several studies have indicated that TLRs are implicated in many inflammatory and immune disorders. Extensive research in the past decade to understand TLR-mediated mechanisms of innate immunity has enabled pharmaceutical companies to begin to develop novel therapeutics for the purpose of controlling an inflammatory disease. The roles of TLRs in the development of autoimmune diseases have been studied. TLR7 and TLR9 have key roles in production of autoantibodies and/or in development of systemic autoimmune disease. It remains to be determined their role in apoptosis, in the pathogenesis of RNA containing immune complexes, differential expression of TLRs by T regulatory cells.     

Profiling of human body fluids in healthy and diseased states using gas chromatography and mass spectrometry,with special reference to organic acids

This review summarizes recent advances in the application of gas chromatography and mass spectrometry to the study of human diseases. Emphasis is placed upon the organic acid profiles of the various body fluids. Methods for sample work-up prior to separation and mass spectrometric analysis are reviewed, and artifacts and pitfalls are discussed. Organic acid profiles, obtained with packed or capillary columns attached to mass spectrometers with or without computer systems, have led to the discovery of new normal metabolites, new metabolic disorders, and to new knowledge about a number of other diseases. Stable isotopes and gas chromatography—mass spectrometry are suitable for quantitative analysis of many compounds in the body fluids, and well suited for investigation of metabolic pathways.     

Single-step gas chromatography-mass spectrometry analysis of glycolipid constituents as heptafluorobutyrate derivatives with a special reference to the lipid portion

In a previous work (Zanetta et al. Glycobiology 9, 255-266 (1999)), it was reported that all constituents of gangliosides could be obtained as heptafluorobutyrate derivatives after methanolysis in a single gas chromatography analysis. This report demonstrates that gas chromatography coupled with mass spectrometry in the electron impact mode allows identification and quantification of long-chain bases and fatty acids without interference from monosaccharides. On the basis of ions specific for families and for individual compounds, sphingosines, sphinganines, and phytosphingosines (including ramified, unsaturated, hydroxylated, and etherified compounds) can be identified. Fatty acid methyl esters, including linear, ramified, unsaturated, and hydroxylated species, are identified and quantified in the same way. Possible extensions of this method to the fatty moiety of other lipids (alkylacylglycerol and dimethyl acetal) are discussed.     

Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes

Human lymphocytes were isolated from defibrinated blood by Ficoll-Hypaque centrifugation with erythrocyte hypotonic lysis. Homogenates of mixed lymphocytes were subjected to analytical subcellular fractionation by sucrose gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their marker enzymes: cytosol (lactate dehydrogenase), plasma membrane (5′-nucleotidase), endoplasmic reticulum (neutral α-glucosidase), mitochondria (malate dehydrogenase), lysosomes (N-acetyl-β-glucosaminidase), peroxisomes (catalase). γ-Glutamyl transferase was exclusively localized to the plasma membrane. Leucine amino-peptidase, especially when assayed in the presence of Co²⁺, was also partially localized to the plasma membrane. Experiments with diazotized sulphanilic acid, a non-permeant enzyme inhibitor, showed that these plasma membrane enzymes are present on the cell surface. No detectable alkaline phosphatase was found in the lymphocytes. Acid phosphatase and β-glucuronidase were localized to lysosomes and there was some evidence for lysosomal heterogeneity. Leucine amino peptidase, optimal at pH 8.0, showed a partial localization to intracellular vesicles, possibly lysosomes, especially when assayed in the presence of EDTA. These studies provide a technique for determining the intracellular distribution of hitherto unassigned lymphocyte constituents and serve as a basis for investigating the cell pathology of lymphocytic disorders.     

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.     

Thioredoxin and thioredoxin reductase: Current research with special reference to human disease

Thioredoxin (Trx) and thioredoxin reductase (TrxR) plus NADPH, comprising the thioredoxin system, has a large number of functions in DNA synthesis, defense against oxidative stress and apoptosis or redox signaling with reference to many diseases. All three isoenzymes of mammalian TrxR contain an essential selenocysteine residue, which is the target of several drugs in cancer treatment or mercury intoxication. The cytosolic Trx1 acting as the cells’ protein disulfide reductase is itself reversibly redox regulated via three structural Cys residues. The evolution of mammalian Trx system compared to its prokaryotic counterparts may be an adaptation to the use of hydrogen peroxide and nitric oxide in redox regulation and signal transduction.     

Microscale analysis of glycosphingolipids by TLC blotting/secondary ion mass spectrometry: a novel blood group A-active glycosphingolipid and changes in glycosphingolipid expression in rat mammary tumour cells with different metastatic potentials

The glycosphingolipid compositions of rat mammary tumour cell lines with different metastatic potentials for the lung [a parental tumour cell line (MTC) and its subclones MTLn2 (a non metastatic subclone) and MTLn3 (a subclone with high metastatic potential to the lung)] were studied using a newly developed TLC blotting/secondary ion mass spectrometry system and crude glycosphingolipids obtained from 0.5–1×10⁷ cells of each cell line. G_M3 and G_M2 were the major components of the MTC cell line, but they were very minor components in the MTLn2 and MTLn3 cell lines, G_Dla being the major ganglioside. HexNAc-fucosyl-G_Mla was found in the MTLn2 cells by the TLC blotting/SIMS method, and the terminal sugar linkage was shown to be a blood group A-type structure by immunostaining. These findings suggest that the ganglioside is a novel type of blood group A-active ganglioside, GalNAc1-3(Fuc1-2)G_Mla. No blood group A-active lipid was present in MTLn3 cells, whereas Hex-G_Mla and neutral glycosphingolipids with more than 5 sugar residues were. Abbreviations: TLC, thin-layer chromatography; HPTLC plate, high performance thin-layer chromatography-plate; PVDF, polyvinylidene difluoride; SIMS, secondary ion mass spectrometry; GC-MS, gas chromatography-mass spectrometry; C16:0, hexadecanoic acid; C18:0, octadecanoic acid; C22:0, docosanoic acid; C24:0, tetracosanoic acid; d18:1, 2-amino-4-octadecene-1,3-diol; Hex, hexose; HexNAc,N-acetylhexosamine; Gal, galactose; Glc, glucose; GalNAc,N-acetylgalactosamine; Lac, lactose; NeuAc,N-acetylneuraminic acid; Cer, ceramide; Glob, globoside; iGlob, isogloboside; GlcCer, glucosylceramide; LacCer, lactosylceramide; Gb₃Cer, Gal1-4Gal1-4Glc1-1Cer; Gb₄Cer (Glob), GalNAc1-3Gal1-4Glc1-1Cer; iGb₃Cer, Gal1-3Gal1-4Glc1-1Cer; iGb₄Cer (iBlob), GalNAc1-3Gal1-4Glc1-1Cer; Ganglio-series gangliosides are named according to Svennerholm [1].     

Glycosphingolipids of human large intestine: detailed structural characterization with special reference to blood group compounds and bacterial receptor structures

Non-acid glycosphingolipid expression was studied in the large intestines from four individuals with the A1Le(a-b+), BLe(a-b+), and OLe(a-b+) blood group phenotypes. In the A1Le(a-b+) case, specimens were taken from the ascending and sigmoid parts of the large intestine in order to compare the expression of glycolipids in the proximal and distal regions of the intestine. In one blood group OLe(a-b+) individual, epithelial cells were isolated from the residual stroma to compare the glycolipid compositions in these two tissue compartments. GlcCer, GalCer, LacCer, Gb3Cer, and Gb4Cer were the major compounds in all three individuals, as shown by mass spectrometry, proton NMR spectroscopy, and degradation studies. The Lea-5 glycolipid was the major complex blood group glycolipid in all individuals, except in the proximal ascending part of the large intestine of the A1Le(a-b+) case, in which the Leb-6 glycolipid was predominant. There were trace amounts of blood group ABH glycolipids, in agreement with the ABO blood group phenotypes of the donors, Lewis antigens with more than six sugar residues in the carbohydrate chain, and blood group X and Y glycolipid antigens. The epithelial cells were dominated by monoglycosylceramides and the Lea-5 glycolipid, while only trace amounts of di-, tri-, and tetraglycosylceramide structures were present. No reactivity was seen in the epithelial cell fraction with Gal alpha 1-4Gal specific Escherichia coli, anti-Pk, or anti-P antibodies, indicating the absence of the glycolipid-borne Gal alpha 1-4Gal sequence in human large intestinal epithelial cells.     

Evidence for a role of glycosphingolipids in CXCR4-dependent cell migration

Chemotaxis induction is a major effect evoked by stimulation of the chemokine receptor CXCR4 with its sole ligand CXCL12. We now report that treatment of CHP-100 human neuroepithelioma cells with the glucosylceramide synthase (GCS) inhibitor DL-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol inhibits CXCR4-dependent chemotaxis. We provide evidence that the phenomenon is not due to unspecific effects of the inhibitor employed and that inhibition of GCS neither affects total or plasmamembrane CXCR4 expression, nor CXCL12-induced Ca(2+) mobilization. The effects of the GCS inhibitor on impairment of CXCL12-induced cell migration temporally correlated with a pronounced downregulation of neutral glycosphingolipids, particularly glucosylceramide, and with a delayed and more moderate downregulation of gangliosides; moreover, exogenously administered glycosphingolipids allowed resumption of CXCR4-dependent chemotaxis. Altogether our results provide evidence, for the first time, for a role glycosphingolipids in sustaining CXCL12-induced cell migration.     

Analysis of silicon in human tissues with special reference to silicone breast implants

The increase, in the last two decades, in the application of silicones (polysiloxanes) and inorganic silicon compounds in medicine and the food industry, has exposed the human body to extensive contacts with these substances. Most silicone breast implants contain a gel consisting of a crosslinked silicone elastomer swollen by silicone oil (PDMS). Diffusion of PDMS through the silicone elastomer envelope and rupture of the envelope with release of the gel contents both occur clinically. The amount and distribution of silicone compounds in various tissues are key issues in the assessment of health problems connected with silicone implants. We have measured by GFAAS the Si content of tissues from normal and implant patients and the organic solvent extractable Si levels (assumed to be silicone), using careful control of sample collection and preparation. Whole blood levels were: implant patients mean 38.8 (SD 25.6) (microg/kg), controls mean 24.2 (SD 26.7) (microg/kg) in one study and subsequently 103.8 (SD 112.1) and 74.3 (SD 86.5) (microg/kg) in another study. Capsular tissue levels were: gel implants 25047 (SD 39313) (mg/kg of dry tissue), saline implants 20.0 (SD 27.3) (mg/kg of dry tissue) and controls 0.24 (SD 0.39) (mg/kg of dry tissue). Breast milk levels were: implant patients mean 58.7 (SD 33.8) (microg/kg), controls mean 51.1 (SD 31.0) (microg/kg); infant formula mean was 4.40 (mg/kg). Various precautions were undertaken to avoid Si contamination in this work, the most important being a) the use of a Class 100 laboratory for sample preparation and b) application of strict and elaborate washing procedure for specimen collection tools and laboratory plasticware. This data demonstrated that to properly interpret the importance of these numbers for human health, a larger study of "normal" levels of Si in human tissues should be undertaken and factors such as diet, water, race and geographical location should be considered.     

Chromosomal aberrations in arsenic-exposed human populations: a review with special reference to a comprehensive study in West Bengal, India

For centuries arsenic has played an important role in science, technology, and medicine. Arsenic for its environmental pervasiveness has gained unexpected entrance to the human body through food, water and air, thereby posing a great threat to public health due to its toxic effect and carcinogenicity. Thus, in modern scenario arsenic is synonymous with "toxic" and is documented as a paradoxical human carcinogen, although its mechanism of induction of neoplasia remains elusive. To assess the risk from environmental and occupational exposure of arsenic, in vivo cytogenetic assays have been conducted in arseniasis-endemic areas of the world using chromosomal aberrations (CA) and sister chromatid exchanges (SCE) as biomarkers in peripheral blood lymphocytes. The primary aim of this report is to critically review and update the existing in vivo cytogenetic studies performed on arsenic-exposed populations around the world and compare the results on CA and SCE from our own study, conducted in arsenic-endemic villages of North 24 Parganas (district) of West Bengal, India from 1999 to 2003. Based on a structured questionnaire, 165 symptomatic (having arsenic induced skin lesions) subjects were selected as the exposed cases consuming water having a mean arsenic content of 214.96 microg/l. For comparison 155 age-sex matched control subjects from an unaffected district (Midnapur) of West Bengal were recruited. Similar to other arsenic exposed populations our population also showed a significant difference (P < 0.01) in the frequencies of CA and SCE between the cases and control group. Presence of substantial chromosome damage in lymphocytes in the exposed population predicts an increased future carcinogenic risk by this metalloid.     

Quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spots using liquid chromatography-tandem mass spectrometry

A high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spot (DBS) samples, which were produced by spotting 20 μl whole blood onto FTA cards. A 3mm disc was cut from the DBS samples and extracted using methanol, followed by liquid-liquid extraction with MTBE. The reconstituted extracts were chromatographed using a Halo C(18) column and gradient elution for MS/MS detection. The possible impact of hematocrit, blood sample volume and punching location on DBS sampling was investigated. The results showed that blood sample volume or punching location has no impact on assay performance, but the presence of a high hematocrit resulted in significantly increased analyte concentrations measured from the high QC samples. The current method was fully validated over the range of 10.0-5000 ng/ml with correlation coefficients (r(2)) for three validation batches equal to or better than 0.991. The accuracy and precision (CV) at the LLOQ were -0.7 to 6.0% bias of the nominal value (10.0 ng/ml) and 10.2-2.3%, respectively. For the balance of QC samples (20.0, 50.0, 750, 1500 and 3750 ng/ml), the precision (CV) ranged from 3.2 to 11.7% and from 5.6 to 10.2%, respectively, for the intra-day and inter-day evaluations. The accuracy ranged from -6.8 to 8.5% and -0.2% to 2.7% bias, respectively, for the intra-day and inter-day batches. NIM811 is stable in the DBS samples for at least 24h at room temperature and 4h at 60°C. Interestingly, the long term stability (LTS) assessment showed that the stability of the analyte is better when the DBS samples were stored at a lower storage temperature (e.g. ≤ -60°C) compared to storage at room temperature. This is probably due to the interaction of the additives and/or other materials (e.g. cellulose, etc) on the DBS card with NIM811, a cyclic peptide. The current methodology has been applied to determine the NIM811 levels in DBS samples prepared from a clinical study.     

Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis

The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.     

Progestin and estrogen receptors: characterization and localization in rat submandibular glands, with special reference to epidermal growth factor

By using progestin (P) and estrogen (E), the localization and characterization of both steroid receptors were examined in the submandibular gland (SMG) of 6-week-old immature castrated rats, with special reference to localization of epidermal growth factor (EGF). In the castrated male and female rats, both 3H-estradiol-17 beta (3H-E2 beta) and 3H-promegestone (3H-R5020) bound to SMG cytosol with high affinity and low capacity. These values were similar to those reported for other tissues. However, E-treatment after castration inhibited the specific binding. In sucrose density gradient ultracentrifugation, it was found that P receptors in both castrated males and females had a sedimentation coefficient of 7S, whereas E receptors had sedimentation coefficients of 4S and 7S. A histochemical study of the SMG of castrated male and female rats showed that the E-peroxidase complex (EPC)- and P-peroxidase complex (PPC)-stained cells were predominantly located in the epithelium of the duct system including the excretory duct and the granular convoluted tubules. Few cells were located in the intercalated duct, and none were found in the acinus. EGF-immunoreactive cells were also located in the epithelium of the same tissue region as in PPC- and EPC-stained sections. Moreover, E-treatment after castration inhibited the intensity of staining and immunoreactivity. These results clearly suggest that rat SMG contains specific P and E receptors which are mainly located in the epithelial cells of the duct system in which EGF-containing cells are identified. We discussed the possibility that P and E might affect EGF immunoreactivity, which reflects EGF production, through their receptors in the epithelium of the duct system.     

Culture-independent analysis of fecal microbiota in infants, with special reference to Bifidobacterium species

Fecal microbiota of 31 breast-fed, 26 mix-fed, and 11 bottle-fed infants were analyzed by using terminal restriction fragment length polymorphism (T-RFLP), and culture method. We first determined the total and cultivated bacterial counts in infant fecal microbiota. Only approximately 30% of bacteria present in fecal microbiota were cultivable while the remainder was yet-to-be cultured bacteria. Sixty-eight fecal samples were divided into two clusters (I and II) by T-RFLP analysis, and then subdivided into five subclusters (Ia, Ib, IIa, IIb and IIc). There was no clear relationship between clusters and feeding method. A proportion of bifidobacteria was detected in the fecal material by PCR method using species-specific primers. The predominant Bifidobacterium spp. was Bifidobacterium longum longum type (43 samples (63.2%)), followed by B. longum infantis type (23 samples (33.8%)) and B. breve (16 samples (23.5%)). The distribution of Bifidobacterium spp. was similar in the three feeding groups. In contrast, the high incidence of B. breve in cluster I, especially subcluster Ia and B. longum longum type in cluster II, especially subcluster IIa and IIc were characterized by T-RFLP method. Our results showed that the colonization of Bifidobacterium spp. in infant feces correlated with the T-RFLP clusters.