共查询到20条相似文献,搜索用时 31 毫秒
1.
John B. Briggs Rodney G. Keck Stacey Ma Wendy Lau Andrew J.S. Jones 《Analytical biochemistry》2009,389(1):40-8149
A novel system for characterizing complex N-linked oligosaccharide mixtures that uses a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) has been developed. In this study, oligosaccharides released from recombinant TNK-tPA (tissue plasminogen activator) were derivatized with 5-amino-2-naphthalenesulfonic acid (ANSA). The negative charge imparted by the ANSA label facilitated the analysis of the oligosaccharides by MALDI-TOF MS by allowing the observation of both neutral and sialylated oligosaccharides in a single negative ion mode spectrum. Labeling with ANSA was also determined to be advantageous in the characterization of oligosaccharides by both HPLC and CE. The ANSA label was demonstrated to provide superior resolution over the commonly used label 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in both the CE and HPLC analysis of oligosaccharides. To date, no other labels that enable the analysis of complex oligosaccharide mixtures in a single mass spectral mode, while also enabling high-resolution chromatographic and electrophoretic separation of the oligosaccharides, have been reported. By integrating the structural information obtained by MALDI-TOF MS analysis with the ability of CE and HPLC to discriminate between structural isomers, the complete characterization of complex oligosaccharide mixtures is possible. 相似文献
2.
Lipoarabinomannans are key molecules of the mycobacterial envelopes involved in many steps of tuberculosis immunopathogenesis. Several of the biological activities of lipoarabinomannans are mediated by their ability to bind human C-type lectins, such as the macrophage mannose receptor, the mannose-binding protein and the surfactant proteins A and D. The lipoarabinomannan mannooligosaccharide caps have been demonstrated to be involved in the binding to the lectin carbohydrate recognition domains. We report an original analytical approach, based on capillary electrophoresis monitored by laser-induced fluorescence, allowing the absolute quantification, in nanomole quantities of lipoarabinomannan, of the number of mannooligosaccharide units per lipoarabinomannan molecule. Moreover, this analytical approach was successful for the glycosidic linkage determination of the mannooligosaccharide motifs and has been applied to the comparative analysis of parietal and cellular lipoarabinomannans of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv, H37Ra and Erdman strains. Significant differences were observed in the amounts of the various mannooligosaccharide units between lipoarabinomannans of different strains and between parietal and cellular lipoarabinomannans of the same strain. Nevertheless, no relationship was found between the number of mannooligosaccharide caps and the virulence of the corresponding strain. The results of the present study should help us to gain more understanding of the molecular basis of lipoarabinomannan discrimination in the process of binding to C-type lectins. 相似文献
3.
Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis 总被引:4,自引:0,他引:4
A method for quantitative analysis of monosaccharides including N-
acetylneuraminic acid derived from sialic acid-containing oligosaccharides
and glycoproteins is presented. The analysis is based on the combination of
chemical and enzymatic methods coupled with capillary electrophoretic (CE)
separation and laser-induced fluorescence (LIF) detection. The present
method utilizes a simplified acid hydrolysis procedure consisting of mild
hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis
(2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from
strong acid hydrolysis of oligosaccharides and glycoproteins were
reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS)
along with neutral sugars in the presence of sodium cyanoborohydride to
yield quantitatively the highly stable fluorescent APTS adducts. N-
acetylneuraminic acid (Neu5Ac), a major component of most mammalian
glycoproteins, was converted in a fast specific reaction by the action of
neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to
N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with
APTS in the same manner as the other monosaccharides. This method was
demonstrated for the quantitation of pure Neu5Ac and the species derived
from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine
fetuin glycan. Quantitative recovery of the N-acetylmannosamine was
obtained from a known amount of Neu5Ac in a mixture of seven other
monosaccharides or from the sialylated oligosaccharides occurring in
glycoproteins. The sequence of procedures consists of acid hydrolysis,
enzymatic conversion and APTS derivatization which produced quantitative
recovery of APTS- monosaccharide adducts. The detection limits for sugars
derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50
pmol for the other sugars.
相似文献
4.
With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE-mass spectrometry (CE-MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE-MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE-laser-induced fluorescence (CE-LIF). For each study, CE-MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE-MS for profiling biomolecules. 相似文献
5.
A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined. 相似文献
6.
毛细管电泳分离寡糖衍生物及其电泳行为研究 总被引:6,自引:0,他引:6
将葡聚糖部分酸水解成寡糖混合物,经ANTS胺化还原衍生,在pH2.5、50mmol/L磷酸缓冲液(含或不含10mmol/L TEA)中,以及在pH9.3、100mmol/L硼砂缓冲液中用毛细管电泳分离衍生物,分别得到1至21和1至18个聚合度的衍生物电泳梯度图谱。作出了准确定位的毛细管电泳双向电泳图。对电泳行为研究发现,在低pH磷酸电泳缓冲液中,衍生物相对电迁移率(μep)r与(Mr^-2/3)、 相似文献
7.
N-Glycosidically linked oligosaccharides were released from glycoproteins by digestion with trypsin followed by hydrazinolysis and subsequently re-N-acetylated and reductively pyridylaminated. Derivatives of sialic acid-containing oligosaccharides were further desialylated with neuraminidase. The final derivatives of asialo-oligosaccharides were analyzed by capillary zone electrophoresis in two carriers, an acidic phosphate buffer and an alkaline borate buffer. The former carrier allowed direct zone electrophoresis as cationic immonium ions, accordingly size-dependent separation, whereas the latter realized indirect electrophoresis as anionic borate complexes, i.e., separation based on the structural variation in outermost monosaccharide residues. Two-dimensional plots of relative mobilities of the derivatives in these dual separation modes to reductively pyridylaminated glucose provided a good tool for identification of oligosaccharides. 相似文献
8.
Mauri PL Basilico F Pietta PG Pasini E Monti D Sauerwein W 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,788(1):9-16
Boron neutron capture therapy is a promising binary treatment for cancer. It is based on the nuclear fission that occurs when non-radioactive 10B absorbs thermal neutrons. One of the two boron compounds currently used in clinical trials for this therapy is BSH. To ensure differentiated retention in the tumour versus normal tissue prior to treatment, routine analytical methods to determine pharmacokinetics must be available. For this purpose we have developed a new, easy and time saving approach, in which the separation of boron derivatives is performed by means of capillary electrophoresis (CE). The CE method allows analyses to be performed in short times (less than 18 min), sensitively (LOD 8 pg loaded on the capillary) quantitatively (LOQ 5 microg/ml) and with a high efficiency of separation. Moreover it is simpler than HPLC and more reproducible (intra- and inter-day values were +/-1% and +/-3%, respectively), and does not require a specific column of derivatization. Mass spectrometry analysis of boron derivatives in different samples was also performed to ensure correct attribution of the CE peaks. 相似文献
9.
Eugene C. Rickard Ronald J. Bopp David J. Skanchy Kerri L. Chetwyn Bettina Pahlen John F. Stobaugh 《Chirality》1996,8(1):108-121
In the past several years, capillary electrophoresis (CE) has generated considerable interest from pharmaceutical companies for control of both the chiral and achiral purity of bulk drugs and drug products. This paper evaluates the use of CE as: (1) a technique complementary to HPLC for the determination of peak homogeneity of a drug, (2) for determination of chiral purity, and (3) for determination of achiral purity. It would be greatly advantageous if CE could be used to determine both the chiral and achiral purity in a single assay. This investigation compares the results obtained for the separation of the enantiomers of duloxetine using several neutral cyclodextrins to those obtained using anionic cyclodextrins (sulfobutyl ether derivatives) as chiral selectors added to the separation buffer. In addition, it reports chiral separations obtained by using neutral cyclodextrins in a sulfonic acid-coated capillary column, which give a negatively charged capillary surface and electro-osmotic flow even in low pH buffers. The possible mechanism of separation is discussed. © 1996 Wiley-Liss, Inc. 相似文献
10.
Sergey P. Radko Andreas Chrambach 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,722(1-2)
Utilization of capillary electrophoresis (CE) for characterization and analytical separation of submicron- and micron-sized organic and inorganic particles as well as biological vesicles is reviewed. CE has been applied to charged polystyrene size standards, inorganic and organic colloidal particles, lipoprotein particles, liposomes, microsomes and viruses. These particle separations generally occur in a size-dependent manner and provide values of electrophoretic mobility which are in good agreement with those obtained by other electrophoretic techniques. 相似文献
11.
Petr Bartk Petr Bedn David Friedecký Antonn Haviger Juraj ev
k 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,758(2):311
Some technical aspects influencing the total time of CE analysis are discussed. A high throughput electrophoretic system based on micellar electrokinetic chromatography (MEKC) is demonstrated as an example. A short capillary, strong electric field, alkaline buffer (pH 9.5) generating strong electroosmotic flow, and parallel hydrodynamic pressure allow the separation of two uncharged isothiazolone derivatives within 45 s. 相似文献
12.
We report the formation, detection, quantitation and structural characterization of products resulting from the adduction of deoxynucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine and 5-methyldeoxycytidine) to the catechol estrogens (CE) of estrone, estradiol-17beta and estradiol-17 alpha. The crude products are obtained in a one-pot synthesis through oxidation of catechols to quinones and subsequent Michael-type reaction with the deoxynucleosides in acidic medium.In all experiments, adducts are detected by electrospray ionization mass spectrometry analysis after HPLC separation (LC/ESI/MS(n)). The two pyrimidines deoxycytidine and 5-methyldeoxycytidine yield only CE adducts to deoxynucleosides, which correspond to stable adducts on DNA. For purines, the results depend on the CE (2,3- or 3,4-catechols) used, the function and configuration on carbon 17 (ketone for estrone, alcohol for alpha and beta isomers of estradiol), and on the purine itself (deoxyadenosine or deoxyguanosine). Both stable adducts and deglycosylated adducts are formed, and therefore formation of stable adducts on DNA as well as the loss of purines from the DNA strands could be possible. MS(2) and MS(3) experiments prove to be relevant for further structural determinations, enabling in some cases the elucidation of the regiochemistry of adduction on the A and B rings of the steroid moiety. 相似文献
13.
Feng HT Wong N Wee S Lee MM 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,870(1):131-134
Twelve nucleotides and seven nucleotide sugars in Chinese Hamster ovary (CHO) cells were determined by capillary electrophoresis (CE). The CE operating conditions of buffer pH value, ion strength, capillary temperature, polymer additive and cell extraction method were investigated. Optimum separation was achieved with 40 mM sodium tetraborate buffer (pH 9.5) containing 1% (w/v) polyethylene glycol (PEG) at a capillary temperature of 22 degrees C. Acetonitrile and chloroform were used for intracellular extraction. This method can be used to monitor intracellular carbohydrate metabolism. 相似文献
14.
Cholesteryl ester (CE) and diacylglycerol (DAG) molecular species are important lipid storage and signaling molecules. Mass spectrometric analyses of these lipids are complicated by the presence of isobaric molecular ions shared by these lipid classes and by relatively poor electrospray ionization, which is a consequence of an inherently weak dipole moment in these lipid classes. The current study demonstrates that lithiated adducts of CE and DAG molecular ions have enhanced ionization and lipid class-specific fragmentation in tandem mass spectrometry (MS/MS) scan modes, thereby allowing the implementation of strategies capable of lipid class-specific detection. Using neutral loss (NL) mode for the loss of cholestane from cholesterol esters (NL 368.5) and specific selected reaction monitoring for DAG molecular species, the response of specific molecular species to that of internal standards was determined. CE and DAG molecular species were quantified in human coronary artery endothelial cells (HCAECs) incubated with both palmitic acid and oleic acid. Furthermore, NL 368.5 spectra revealed the oxidation of the aliphatic fatty acid residues of CE molecular species. Taken together, these studies demonstrate a new analytical approach to assessing CE and DAG molecular species that exploits the utility of lithiated adducts in conjunction with MS/MS approaches. 相似文献
15.
Semenov SN 《Journal of molecular recognition : JMR》1998,11(1-6):157-162
The possibility was considered to use the transverse thermophoresis of analytes in the capillary for capillary electrophoresis (CE) to control the separation process, decrease the peak width due to thermal effects and provide new separation parameters in CE. As the examination has shown, in non-aqueous buffers the Joule heating in the capillary for CE can provide transverse temperature gradients comparable with the temperature gradients in conventional devices for thermal field flow Fractionation (ThFFF). It was proposed to use the non-uniform velocity profile of analytes caused by the transverse temperature gradient and the temperature dependence of the buffer viscosity for the FFF-like separation of analytes besides CE separation. The expressions for the peak parameters have been derived, where the non-uniform transverse analyte concentration distribution due to the thermophoresis is taken into account, and the possibilities based on FFF-CE principles are discussed. As possible objects of this hyphenated technique, macromolecules and particles are considered. 相似文献
16.
Normal phase-high performance liquid chromatography (NP-HPLC) coupled to matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry is evaluated for the detailed structural characterization of various isomers of arabinoxylan (AX) oligosaccharides produced from endo-beta-(1-->4)-xylanase (endoxylanase) digestion of wheat AX. The fragmentation characteristics of these oligosaccharides upon MALDI-TOF/TOF high-energy collision induced dissociation (CID) were investigated using purified AX oligosaccharide standards labeled at the reducing end with 2-aminobenzoic acid (2-AA). A variety of cross-ring cleavages and 'elimination' ions in the fragment ion spectra provided extensive structural information, including Araf substitution patterns along the xylan backbone and comprehensive linkage assignment. The off-line coupling of this MALDI-CID technique to capillary normal phase HPLC enabled the separation and identification of isomeric oligosaccharides (DP 4-8) produced by endoxylanase digestion of AX. Furthermore, this technique was used to characterize structurally different isomeric AX oligosaccharides produced by endoxylanase enzymes with different substrate specificities. 相似文献
17.
《Expert review of proteomics》2013,10(2):287-298
This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise. 相似文献
18.
This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise. 相似文献
19.
Iris Rethfeld Gottfried Blaschke 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,700(1-2):249-253
This study describes the derivatization of the pseudooligosaccharide acarbose and its main metabolite, component 2, with 7-aminonaphthalene-1,3-disulfonic acid (ANDS) in human urine. Their efficient separation was possible by means of capillary zone electrophoresis, using a capillary tube of fused-silica containing 100 mM triethylammonium phosphate buffer, pH 1.5. On column laser-induced fluorescence allowed the detection of the pseudooligosaccharides in human urine in the nanomolar range. With this method, acarbose and component 2 were quantified in human urine after application of 300 mg of acarbose. 相似文献
20.
A. K. Lalloo S. C. Chattaraj I. Kanfer 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,704(1-2)
Capillary electrophoresis (CE) provides high separation efficiency and thus is suitable for the analysis of complex mixtures of structurally similar compounds. The versatile nature of CE can be realised by controlling the chemistry of the inner capillary wall, by modifying the electrolyte composition and by altering the physicochemical properties of the analyte. A CE method has been developed for the separation of three macrolide antibiotics, erythromycin, oleandomycin and josamycin. A systematic approach was used to maximise analyte differential electrophoretic mobility by manipulating electrolyte pH, molarity and composition. In addition, some instrumental parameters such as capillary length and diameter and applied voltage were varied. The effect of the sample solvent and on-capillary concentrating techniques such as field amplified sample injection were investigated. Also, the influence of the injection of a water plug on the quantity of sample injected was demonstrated. The macrolides were completely resolved in less than 30 min in a 100 cm×75 μm I.D. fused-silica uncoated capillary with a Z-shaped flow cell of path-length 3 mm. The analysis was performed in a 75 mM phosphate buffer (pH 7.5) with 50% (v/v) methanol and an applied voltage of 25 kV was selected to effect the separation. 相似文献