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1.
The applicability of electrospray ionization (ESI) mass spectrometry to protein analyses has been studied. The molecular weight of hen egg lysozyme (HEL) was determined with an accuracy of +/- 2 u. The choice of solvents and additives in sample preparations was important to achieve high sensitivity as well as high precision of molecular weight measurements.  相似文献   

2.
We report here on the analysis of synthetic oligonucleotides by electrospray ionization mass spectrometry (ESI-MS). After intensive removal of salt ions (especially sodium cations), negative ion mass spectra, allowing mass measurement with an accuracy of 0.01%, were obtained on several oligonucleotides up to 80 nucleotides. In most cases, the resolution was sufficient to observe n-1 and n-2 forms due to internal deletions during automated synthesis, and to identify the missing nucleotides. A 132-mer, whose size is close to the limit of automated chemical synthesis, was also successfully mass measured. A quantitative study showed that ESI-MS can provide quantitative data on oligonucleotides of similar size and structure. The described methodology is used to characterize oligonucleotide analogues such as phosphorothioate oligonucleotides designed for antisense applications. Finally, analyses in the positive ion mode on a trimer TpTpT in the presence of different amine bases were performed and allowed a better understanding of the influence of these bases on the ions formation.  相似文献   

3.
Desorption electrospray ionization (DESI) was utilized to monitor the presence of targeted central carbon metabolites within bacterial cell extracts and the quench supernatant of Escherichia coli. The targeted metabolites were identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation in the negative ion mode. Picogram detection limits were achieved for a majority of the metabolites during MS/MS analysis of standard metabolite solutions. In a [U-(13)C]glucose pulse experiment, where uniformly labeled glucose was fed to E. coli, the corresponding fragment ions from labeled metabolites in extracts were generally observed. There was evidence of matrix effects including moderate suppression by other metabolites within the spectra of the labeled and unlabeled extracts. To improve the specificity and sensitivity of detection, optimized in situ ambient chemical reactions using DESI and extractive electrospray ionization (EESI) were carried out for targeted compounds. This study provides the first indication of the potential to perform in situ targeted metabolomics of a bacterial sample via ambient ionization mass spectrometry.  相似文献   

4.
El-Hawiet A  Kitova EN  Klassen JS 《Biochemistry》2012,51(21):4244-4253
The development of analytical methods capable of characterizing carbohydrate-protein interactions, which are critical for many biological processes, represents an active area of research. Recently, the direct electrospray ionization mass spectrometry (ESI-MS) assay has emerged as a valuable tool for identifying and quantifying carbohydrate-protein complexes in vitro. The assay boasts a number of strengths, including its simplicity, speed, low level of sample consumption, and the unique ability to directly probe binding stoichiometry and to measure multiple binding equilibria simultaneously. Here, we describe the implementation of the direct ESI-MS assay for the determination of carbohydrate-protein binding stoichiometries and affinities. Common sources of error encountered with direct ESI-MS analysis of carbohydrate-protein interactions are identified along with strategies for minimizing their effects. The application of ESI-MS and a catch-and-release strategy for carbohydrate library screening are also described. The utility of the direct ESI-MS assay can be extended by combining the technique with competitive protein or ligand binding. An overview of these "indirect" ESI-MS methods is given, as well as examples of recent applications.  相似文献   

5.
Desorption electrospray ionization (DESI) allows the direct analysis of ordinary objects or pre-processed samples under ambient conditions. Among other applications, DESI is used to identify and record spatial distributions of lipids and drug molecules in biological tissue sections. This technique does not require sample preparation other than production of microtome tissue slices and does not involve the use of ionization matrices. This greatly simplifies the procedure and prevents the redistribution of analytes during matrix deposition. Images are obtained by continuously moving the sample relative to the DESI sprayer and the inlet of the mass spectrometer. The timing of the protocol depends on the size of the surface to be analyzed and on the desired resolution. Analysis of organ tissue slices at 250 microm resolution typically takes between 30 min and 2 h.  相似文献   

6.
Lipoproteins are of fundamental importance for the lipid transport and cardiovascular disease. The function and metabolism of lipoproteins is intimately linked to the biophysical properties of their surface lipids. Although a number of disease associations were found for lipid species in plasma, only a few studies reported lipid profiles of lipoproteins. Here, we provide an overview of techniques for lipoprotein separation, methods for lipid species analysis based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) as well as data from recent lipidomic studies on lipoprotein fractions. We also discuss the different analytical strategies and how lipid profiling can expand our understanding of the biology and structures of lipoproteins.  相似文献   

7.
A novel hemoglobin variant was detected by electrospray ionization mass spectrometry. Hb Zurich-Hottingen is characterized by an Asn --> Ser replacement in the alpha-chain at position 9 as confirmed by DNA analysis. This hemoglobin variant is silent in isoelectric focusing, reversed-phase chromatography, and cation-exchange chromatography. The mutant alpha-chain was detectable only with electrospray mass spectrometry by its mass shift of -27 Da. The carrier was found to be heterozygous for the new hemoglobin variant. These results illustrate the power of ESI mass spectrometry for hemoglobin analysis.  相似文献   

8.
Nanoelectrospray ionization mass spectrometry has been used to measure the binding of ATP and ADP to the active site of rabbit skeletal myosin-S1. Increases in the molecular mass of myosin-S1 of 425 +/- 10 Da were obtained with the binding of ADP to the active site and by 530 +/- 10 Da with either ATP or hydrolysis products ADP and phosphate. Active site titrations of myosin-S1 with ADP gave a stoichiometry of approximately 1 ADP/S1 with an affinity in the micromolar range. The binding of ATP to myosin-S1 could be observed in the presence of up to 60 muM of excess MgATP without nonspecific binding of MgATP to the myosin. Conversion of the nucleotide complex containing an equilibrium mixture of ATP and ADP-Pi bound to myosin-S1 to one containing only bound ADP occurs at a rate consistent with that of the known steady-state rate of ATP hydrolysis. We expect this method to be of considerable use in the analysis of ligand binding and hydrolysis by the active sites of expressed myosin and myosin subfragments, which are not available in sufficient quantities for conventional methods of measurement of ligand binding.  相似文献   

9.
Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.  相似文献   

10.
A fast and direct method for the monitoring of enzymatic DNA hydrolysis was developed using electrospray ionization mass spectrometry. We incorporated the use of a robotic chip-based electrospray ionization source for increased reproducibility and throughput. The mass spectrometry method allows the detection of DNA fragments and intact non-covalent protein–DNA complexes in a single experiment. We used the method to monitor in real-time single-stranded (ss) DNA hydrolysis by colicin E9 DNase and to characterize transient non-covalent E9 DNase–DNA complexes present during the hydrolysis reaction. The mass spectra showed that E9 DNase interacts with ssDNA in the absence of a divalent metal ion, but is strictly dependent on Ni2+ or Co2+ for ssDNA hydrolysis. We demonstrated that the sequence selectivity of E9 DNase is dependent on the ratio protein:ssDNA or the ssDNA concentration and that only 3′-hydroxy and 5′-phosphate termini are produced. It was also shown that the homologous E7 DNase is reactive with Zn2+ as transition metal ion and that this DNase displays a different sequence selectivity. The method described is of general use to analyze the reactivity and specificity of nucleases.  相似文献   

11.
Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.  相似文献   

12.
Neutral oligosaccharides were fluorescently conjugated with 7-amino-1, 3-naphthalenedisulfonic acid. A mixture of fluorescently labeled chitobiose, chitotriose, and chitotetrose were successfully separated by preparative capillary electrophoresis (CE) and the individual components characterized by electrospray ionization-mass spectrometry (ESI-MS). By combining fluorescent labeling with CE, the use of highly specific exoglycosidases and ESI-MS, a more structurally complex N-linked glycan was analyzed.  相似文献   

13.
The complexation of calcium and zinc cations by pyrroglutamate analogs has been studied in the gas phase by means of electrospray ionization mass spectrometry (ESI–MS). Complexes were obtained from the solutions of calcium perchlorate and zinc perchlorate in acetonitrile. The complexes with calcium are singly and doubly charged with various stoichiometries while zinc complexes are singly charged except for one ligand. Solvation with acetonitrile and presence of perchlorate counter-ions are observed when the complexes are in the gas phase. The complexes formed with both metals are mainly L2M and LM species. All tested compounds are better complexing agents for calcium than for zinc.  相似文献   

14.
Electrospray ionization mass spectrometry (ESI-MS) has been used to determine the dissociation constants (K(D)s) and binding stoichiometry for tobramycin and paromomycin with a 27-nucleotide RNA construct representing the A-site of the 16S ribosomal RNA. K(D) values determined by holding the ligand concentration fixed are compared with K(D) values derived by holding the RNA target concentration fixed. Additionally, the effect of solution conditions such as the amount of organic solvent present and the amount of salt present in the solution on the K(D) measurement is investigated. It is shown that the preferred method for determining dissociation constants using ESI-MS is holding the RNA target concentration fixed below the expected K(D) and titrating the ligand. K(D) measurements should also be carried out at as high as possible salt concentration to minimize nonspecific binding due primarily to electrostatic interactions. For tobramycin, two nonequivalent binding sites were found with K(D1) = 352 nM and K(D2) = 9 microM. For paromomycin, there is only one binding site with K(D) = 52 nM.  相似文献   

15.
We developed and characterized 6 new cysteine mass tags for high-sensitivity peptide analysis. The structural features are: (1) iodoacetyl group for thiol tagging, (2) hydrophilic character for reducing sample loss, (3) tertiary amino, quaternary ammonium, or guanidino group for high proton affinity, and (4) no amide bonding for minimizing fragmentation of tag moiety in collision-induced dissociation. By using these tags, 2- to 200-fold MS sensitivity was achieved, compared to control peptide with carbamydomethylation.  相似文献   

16.
Carbohydrate Deficient Glycoprotein Syndrome (CDGS) is an inherited metabolic disease affecting all parts of the body. The biochemical diagnosis of this syndrome is based on the presence of a special marker in blood, Carbohydrate Deficient Transferrin (CDT), which is also a marker of chronic alcohol abuse. CDT is characterized by abnormal glycoforms of serum transferrin (Tf). In the present study, electrophoretic separation of human serum transferrin glycoforms was carried out using a bare fused-silica capillary and the glycoforms present in commercial Tf were baseline separated. The limit of detection (LOD) of human Tf was around the nmol concentration range. The LOD of the trisialo- and disialo-Tf, expressed as percentages of the tetrasialo-Tf peak area, were 0.5% for trisialo-Tf and 0.4% for disialo-Tf, and these values were appropriate for CDGS diagnosis. Moreover, Tf glycoforms were characterized using mass spectrometry (MS). The method was applied to the analysis of normal and pathological serum samples, after dilution. The results obtained suggest a way of making a rapid and simple CDGS diagnosis.  相似文献   

17.
Low molecular weight glutenin subunits (LMW-GS) are typically subdivided into three groups, according to their molecular weights and isoelectric points, namely the B-, C-, and D groups. Enriched B- and C-type LMW-GS fractions extracted from the bread wheat cultivar Chinese Spring were characterized using high performance liquid chromatography (HPLC) directly interfaced with electrospray ionization mass spectrometry and HPLC coupled off-line with matrix-assisted laser desorption/ionization mass spectrometry, in order to ascertain the number and relative molecular masses of the components present in each fraction and determine the number of cysteine residues. About 70 components were detected in each of the fractions examined by the combined use of these two techniques, with 18 components common to both fractions. Analysis of the fractions after alkylation with 4-vinylpyridine allowed determination of the number of the cysteines present in about 40 subunits. The proteins detected were tentatively classified based on the relative molecular masses and number of cysteine residues. Cross-contamination was found in both B- and C- fractions, along with the presence of D-type LMW-GS. The two fractions also contained unexpected components, probably lipid transfer proteins and omega-gliadins. The presence of extensive microheterogeneity was suggested by the detection of several co-eluting proteins with minor differences in their molecular masses.  相似文献   

18.
Li C  Takazaki S  Jin X  Kang D  Abe Y  Hamasaki N 《Biochemistry》2006,45(39):12117-12124
In this study, we used peptide mapping combined with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) to examine the methionine oxidation of band 3 of erythrocyte membrane protein. Initially, we identified the methionine sites oxidized by chloramine T (N-chloro-p-toluenesulfoamide), a hydrophilic reagent. There were three oxidized methionines (Met 559, Met 741, and Met 909) in band 3, and these methionines were located in a hydrophilic region determined by previous topological studies of band 3. In addition, we found that C12E8, a polyoxyethylene detergent, leads to the oxidation of methionines in a transmembrane segment in band 3, and this oxidation occurs in a C12E8 preincubation time-dependent manner. In a previous study, it was found that peroxides accumulate in a polyoxyethylene detergent. Thus, our method enabled the direct and quantitative detection of protein damage due to detergent peroxides. Furthermore, we examined methionine oxidation in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethyl pyrocarbonate (DEPC), which induced either an outward or an inward conformation in band 3, respectively. Our results indicated that the location of Met 741 was associated with the band 3 conformation induced by band 3-mediated anion transport. In conclusion, we found that methionine oxidation can be applied to examine membrane protein structures as follows: (1) for topological studies of membrane proteins, (2) for assessing the quality of proteins in detergent solubilization studies, and (3) for the detection of conformational changes in membrane proteins.  相似文献   

19.
Molybdenum cofactor deficiency is a fatal neurological disorder, which follows an autosomal-recessive trait and is characterized by combined deficiency of the enzyme, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Early detection of molybdenum cofactor-deficient patients is essential for their proper care and genetic counseling of families at risk. We demonstrate the use of S-sulfonated transthyretin (TTR) as a marker for molybdenum cofactor deficiency. Plasma or sera obtained from 4 patients with molybdenum cofactor deficiency and 57 controls were studied by electrospray ionization mass spectrometry (ESIMS) following selective enrichment of TTR by immunoprecipitation using protein G/A agarose. The data obtained from molybdenum cofactor deficiency samples indicated a strong increase in the peak height of S-sulfonated TTR. A more significant difference was revealed if the peak height ratio of S-sulfonated TTR and the sum of the other oxidized TTR were determined. By accurate determination of the ratio, the samples of molybdenum cofactor deficiency patients could clearly be distinguished from controls without molybdenum cofactor deficiency.  相似文献   

20.
Mass spectrometry (MS) has been a major driver for metabolomics, and gas chromatography (GC)-MS has been one of the primary techniques used for microbial metabolomics. The use of liquid chromatography (LC)-MS has however been limited, but electrospray ionization (ESI) is very well suited for ionization of microbial metabolites without any previous derivatization needed. To address the capabilities of ESI-MS in detecting the metabolome of Saccharomyces cerevisiae, the in silico metabolome of this organism was used as a template to present a theoretical metabolome. This showed that in combination with the specificity of MS up to 84% of the metabolites can be identified in a high mass accuracy ESI-spectrum. A total of 66 metabolites were systematically analyzed by positive and negative ESI-MS/MS with the aim of initiating a spectral library for ESI of microbial metabolites. This systematic analysis gave insight into the ionization and fragmentation characteristics of the different metabolites. With this insight, a small study of metabolic footprinting with ESI-MS demonstrated that biological information can be extracted from footprinting spectra. Statistical analysis of the footprinting data revealed discriminating ions, which could be assigned using the in silico metabolome. By this approach metabolic footprinting can advance from a classification method that is used to derive biological information based on guilt-by-association, to a tool for extraction of metabolic differences, which can guide new targeted biological experiments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

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