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1.
The analysis of chlorophylls and their derivatives by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry is described. Four matrices—sinapinic acid, a-cyano-4-hydroxycinnnamic acid, terthiophene, and 3-aminoquinoline—were examined to determine optimal conditions for analysis of the molecular mass and structure of chlorophyll a as a representative chlorophyll. Among them, terthiophene was the most efficient without releasing metal ions, although it caused fragmentation of the phytol-ester linkage. Terthiophene was useful for the analyses of chlorophyll derivatives as well as porphyrin products such as 8-deethyl-8-vinyl-chlorophyll a, pheophorbide a, pyropheophorbide a, bacteriochlorophyll a esterified phytol, and protoporphyrin IX. The current method is suitable for rapid and accurate determination of the molecular mass and structure of chlorophylls and porphyrins.  相似文献   

2.
We applied the metabolomic analysis of comprehensive small-molecular metabolites using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) and principal component analysis to identify uremic toxins accumulated in the serum of chronic renal failure (CRF) rats. CRF rats were produced by 5/6-nephrectomy. Indoxyl sulfate was demonstrated to be the first principal serum metabolite which differentiates CRF from normal, followed by phenyl sulfate, hippuric acid and p-cresyl sulfate. Then, we measured the serum levels of indoxyl sulfate, phenyl sulfate, hippuric acid and p-cresyl sulfate by the selected reaction monitoring (SRM) of LC/ESI-MS/MS, and demonstrated that these serum levels were markedly increased in CRF rats as compared with normal rats. As creatinine clearance decreased, the serum levels of the metabolites increased.  相似文献   

3.
Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) was evaluated as a technique to characterize strains of the nitrogen-fixing actinomycete Frankia. MALDI-TOF MS reliably distinguished 37 isolates within the genus Frankia and assigned them to their respective host infection groups, i.e., the Alnus/Casuarina and the Elaeagnus host infection groups. The assignment of individual strains to sub-groups within the respective host infection groups was consistent with classification based on comparative sequence analysis of nifH gene fragments, confirming the usefulness of MALDI-TOF MS as a rapid and reliable tool for the characterization of Frankia strains.  相似文献   

4.
Chemerin is a chemoattractive protein acting as a ligand for the G-protein-coupled receptor ChemR23/CMKLR1 and plays an important role in the innate and adaptive immunity. Proteolytic processing of its C terminus is essential for receptor binding and physiological activity. Therefore, we investigated the plasma stability of the decapeptide chemerin 145-154 (P(145)-F(154)) corresponding to the C terminus of the physiologically active chemerin variant E(21)-F(154) from human hemofiltrate. For monitoring concentration-time profiles and degradation products we developed a novel matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry procedure using an internal peptide standard (hemorphin LVV-H7) for quantification. The linear range covers 2.5 orders of magnitude in the lower micromolar concentration range (lower limit of quantification 0.312 microg/ml, 0.25 microM) characterized by satisfactory reproducibility (CV < or =9%), accuracy (< or =10%), ruggedness, and recovery (98%). We found that chemerin 145-154 is C-terminally truncated in human citrate plasma by the cleavage of the penultimate dipeptidyl residue. N-terminal truncation was not observed. In contrast to citrate plasma, no degradation was detected in ethylenediammetetraacetate (EDTA) plasma. We identified angiotensin-converting-enzyme (ACE) to be responsible for C-terminal truncation, which could be completely inhibited by EDTA and captopril. These results are relevant to clarify the natural processing of chemerin and the potential involvement of ACE in mediating the immune response.  相似文献   

5.
We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.  相似文献   

6.
A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW ∼ 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galβ1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galβ1-4GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc) as the common core structure, and most of them contained Fucα1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galβ1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galβ1-4GlcNAcβ1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position.  相似文献   

7.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used for many years to rapidly identify whole bacteria. However, no consistent methodology exists for the rapid identification of bacteria cultured in liquid media. Thus, in this study we explored the use of MALDI-TOF MS analysis for rapid identification of cells cultured in liquid media. We determined that 2,5-dihydroxybenzoic acid (50 mg mL?1, 50% acetonitrile, 0.1% trifluoroacetic acid) was the best matrix solution for MALDI-TOF MS for this type of study. Moreover, the tested strains were successfully differentiated by principal component analysis, and the main characteristics of the mass peaks for each species were found in mixed culture samples. In addition, we found that the minimum number of cells for detection was 1.8×103. In conclusion, our findings suggest that MS-based techniques can be developed as an auxiliary method for rapidly and accurately identifying bacteria cultured in liquid media.  相似文献   

8.
The physiological response to small molecules (secondary messengers) is the outcome of a delicate equilibrium between biosynthesis and degradation of the signal. Cyclic diguanosine monophosphate (c-di-GMP) is a novel secondary messenger present in many bacteria. It has a complex cellular metabolism whereby usually more than one enzyme synthesizing and degrading c-di-GMP is encoded by a bacterial genome. To assess the in vivo conditions of c-di-GMP signaling, we developed a high-performance liquid chromatography (HPLC)-mass spectrometry-based method to detect c-di-GMP with high sensitivity and to quantify the c-di-GMP concentration in the bacterial cell as described here in detail. We successfully used the methodology to determine and compare the c-di-GMP concentrations in bacterial species such as Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae. We describe the use of the methodology to assess the change in c-di-GMP concentration during the growth phase and the contribution of a point mutation in S. typhimurium to the overall cellular c-di-GMP concentration.  相似文献   

9.
We report here the preliminary characterization of oligosaccharides present in an enzyme-treated industrial fermentation residue using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ion trap mass spectrometry (ESI-ITMS), and gas chromatography mass spectrometry (GC-MS). After sample cleaning with carbon graphite columns, analysis of oligosaccharides present in the sample using MALDI-TOF-MS resulted in identification of molecular ions representing sodiated hexose and pentose oligo/polysaccharides. The GC-MS analyses revealed that the signals observed in the mass spectrum for hexose oligomers represent linear structures, whereas the pentose oligomers were identified as arabinoxylans with a (1-->4) linked Xylp backbone where the Xylp residues were either not substituted or singly substituted with Araf branching residues at positions C-2 or C-3 of the Xylp ring. Analyses by ESI-ITMS of the signals corresponding to arabinoxylan oligosaccharides with four and five monosaccharide residues showed the presence of isomeric structures differing in degree of branching and localization of the branched residue along the Xylp backbone.  相似文献   

10.
A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.  相似文献   

11.
We report a method to detect the presence of dimethylarginines on proteins. Peptides with dimethylarginines were hydrolyzed in acid. The hydrolysates were subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis using a mixture of alpha-cyano-4-hydroxycinnamic acid and nitrocellulose as matrix. Both asymmetric omega-N(G),N(G)-dimethylarginine and symmetric omega-N(G),N(G')-dimethylarginine give a clear signal at m/z 203. Recombinant Sbp1p modified by Hmt1p in vivo were isolated by affinity chromatography followed by electrophoresis on a polyacrylamide gel and subjected to acid hydrolysis. MALDI-TOF analysis of the acid hydrolysates confirmed the presence of dimethylarginines. The detection limit of the method is estimated at approximately 1pmol of protein.  相似文献   

12.
Chemical analysis for the characterisation of micro-organisms is rapidly evolving, after the recent advent of new ionisation methods in mass spectrometry (MS): electrospray (ES) and matrix-assisted laser desorption/ionisation (MALDI). These methods allow quick characterisation of micro-organisms, either directly or after minimum sample preparation. This review provides a brief introduction to ES and MALDI MS and a discussion of micro-organism characterisation capabilities. Some attention is devoted to the analysis of mixtures of proteins, lipids and other compounds, to the combination of polymerase chain reaction technology and MS, and to the analysis of whole bacteria and their lysates. The review of results produced hitherto is concluded with an outlook on future developments.  相似文献   

13.
An on-line reversed-phase (RP) high-performance liquid chromatography/mass spectrometry (MS) method has been developed for profiling and characterizing N-glycans from recombinant immunoglobulin G antibodies. In this method, released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2AB) and separated on a RP column with on-line fluorescence and MS detection. The method achieves good resolution of all major glycans and segregates glycan types (high-mannose, hybrid, and complex) to different regions of the chromatogram, thus allowing accurate quantification of N-glycans from the fluorescent signal alone. Moreover, the mobile phase used allows high quality on-line MS detection. The 2AB-labeled N-glycans demonstrate good ionization efficiency in electrospray and generate primarily doubly charged [M+2H](2+) ions. The mass and structural information can be readily obtained from the on-line MS and tandem MS data. As little as 70 fmol glycan species can be detected and identified.  相似文献   

14.
Eight pyridylamino (PA) derivatives of fucose-containing oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by high-performance liquid chromatography (HPLC) on normal-phase and reversed-phase columns, and by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Six out of eight PA-oligosaccharides were clearly separated by both normal- and reversed-phase HPLC at a column temperature of 40 degrees C, but two PA-oligosaccharides, lacto-N-fucopentaose II [Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-3Gal beta1-4GIcPA] and lacto-N-fucopentaose III [Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-3Gal beta1-4GIcPA], were not separated. The two unresolved PA-oligosaccharides were finally separated by reversed-phase HPLC at a column temperature of 11 degrees C. MALDI-TOF mass spectra of PA-oligosaccharides demonstrated pseudo-molecular ions as the predominant signals, therefore information about the molecular mass of each PA-oligosaccharide was easily obtained. Post-source decay (PSD) MALDI-TOF mass spectra of PA-oligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PA-oligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PA-oligosaccharides by HPLC, MALDI-TOF mass spectrometry, and PSD MALDI-TOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDI-TOF mass spectrometry or HPLC and PSD MALDI-TOF mass spectrometry is quite useful for the structural characterization of sub-pmol or pmol levels of fucose-containing oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.  相似文献   

15.
Nanoscale liquid chromatography coupled to electrospray ionization mass spectrometry was used to identify the nature of the ligand that binds noncovalently to siderocalin (lipocalin 2). The folded state siderocalin-ligand complex was separated from free, unfolded siderocalin using reversed phase chromatography, and the molecular weight of the siderocalin ligand was then determined from the deconvoluted molecular weights of the complex and of the free protein. The ligand was identified as dihydroxybenzoyl-serine, a breakdown product of enterobactin, an iron-chelating compound ("siderophore") synthesized in bacteria. These results demonstrate that, in some cases, electrostatic noncovalent protein complexes can survive the denaturing conditions of reversed phase liquid chromatography and the gas phase transfer occurring during electrospray ionization.  相似文献   

16.
A new strategy has been employed for the identification of the covalent modification sites (mainly acetylation and methylation) of histone H3 from chicken erythrocytes using low enzyme/substrate ratios and short digestion times (trypsin used as the protease) with analysis by HPLC separation, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), matrix-assisted laser desorption ionization-postsource decay, and tandem mass spectrometric techniques. High-accuracy MALDI-TOF mass measurements with representative immonium ions (126 for acetylated lysine, 98 for monomethylated lysine, and 84 for di-, tri-, and unmethylated lysine) have been effectively used for differentiating methylated peptides from acetylated peptides. Our results demonstrate that lysines 4, 9, 14, 27, and 36 of the N-terminal of H3 are methylated, while lysines 14, 18, and 23 are acetylated. Surprisingly, a non-N-terminal residue, lysine 79, in the loop region hooking up to the bound DNA, was newly found to be methylated (un-, mono-, and dimethylated isoforms coexist). The reported mass spectrometric method has the advantages of speed, directness, sensitivity, and ease over protein sequencing and Western-blotting methods and holds the promise of an improved method for determining the status of histone modifications in the field of chromosome research.  相似文献   

17.
Phosphoamino acid modifications on substrate proteins are critical components of protein kinase signaling pathways. Thus, diverse methodologies have been developed and applied to identify the sites of phosphorylated amino acids within proteins. Despite significant progress in the field, even the determination of phosphorylated residues in a given highly purified protein is not a matter of routine and can be difficult and time-consuming. Here we present a practicable approach that integrates into a liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry (LC–MALDI MS) workflow and allows localization and quantification of phosphorylated peptides on the MALDI target plate prior to MS analysis. Tryptic digests of radiolabeled proteins are fractionated by reversed-phase LC directly onto disposable MALDI target plates, followed by autoradiographic imaging. Visualization of the radiolabel enables focused analysis of selected spots, thereby accelerating the process of phosphorylation site mapping by decreasing the number of spectra to be acquired. Moreover, absolute quantification of the phosphorylated peptides is permitted by the use of appropriate standards. Finally, the manual sample handling is minimal, and consequently the risk of adsorptive sample loss is very low. Application of the procedure allowed the targeted identification of six novel autophosphorylation sites of AMP-activated protein kinase (AMPK) and displayed additional unknown phosphorylated peptide species not amenable to detection by MS. Furthermore, autoradiography revealed topologically inhomogeneous distribution of phosphorylated peptides within individual spots. However, accurate analysis of defined areas within single spots suggests that, rather than such quantitative differences, mainly the manner of matrix crystallization significantly affects ionization of phosphopeptides.  相似文献   

18.
Polymeric tannins, extracted from grape berries (Gamay variety), were fractionated according to their mean degree of polymerisation (mDP) on a styrene-divinylbenzene phase eluted with a gradient of methanol:chloroform. Increasing the percentage of methanol led to the solubilisation of higher molecular weight tannins. The mean mDP of each collected fraction was determined by acid-catalysed degradation in the presence of a nucleophilic reagent. The fractionation method produced a linear gradient of mDP varying between 1.84 and 19.34. The fractions were partially characterised by matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). The spectra showed a complex mixture of proanthocyanidins and galloylated proanthocyanidins up to 4000 amu.  相似文献   

19.
The results of the characterization of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based method that was developed to establish the stoichiometry of CHX-A'-diethylenetriaminepentaacetic acid (DTPA) or benzyl-DTPA conjugated to a recombinant immunoglobulin G (IgG) are reported. This simple method does not require an accurate measurement of the sample protein concentration to accurately quantify the number of DTPA conjugated. It is also not necessary to thoroughly remove nonconjugated DTPA from the sample. The average number of moles of DTPA attached per mole of IgG was calculated from the difference in the observed masses of DTPA-IgG and nonconjugated IgG divided by the molecular weight of the DTPA derivative. As more DTPA is attached, the [M+H](+) peak of DTPA-IgG becomes broader and noisier. Also, the signal intensity in the mass spectrum decreases, apparently due to the increase in the heterogeneity in the number of DTPA attached to each molecule of IgG. The standard deviation of the measured mass and that of the stoichiometry of the DTPA attached per IgG increased as more DTPA was attached. The standard deviation, expressed as coefficient of variation for samples with 2 to 4 mol of DTPA attached per mole of IgG, was 8 to 9%.  相似文献   

20.
Immunoglobulin M is an especially important product of the immune system because it plays a critical role in early protection against infections. In this report, the glycosylation pattern of the protective murine monoclonal IgM 12A1 to Cryptococcus neoformans polysaccharide was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Peptide mapping studies covering 88% of the deduced amino acid sequence indicated that of the six potential N-glycosylation sites in this antibody only five were utilized, as the tryptic peptide derived from monoclonal IgM 12A1 containing Asn-260 was recovered without carbohydrates. The oligosaccharide side chains of monoclonal IgM 12A1 were characterized at each of the N-glycosylation sites. Asn-166 possessed 20 monosialylated and nonsialylated, and fucosylated and nonfucosylated complex- and hybrid-type oligosaccharides and one high-mannose-type oligosaccharide. Thirteen oligosaccharides were attached to the site at Asn-401, including six complex-type, four hybrid-type, and three high-mannose-type oligosaccharides. Twelve hybrid-type oligosaccharides were attached to Asn-378, three of which had terminal sialic acids. Eleven hybrid-type oligosaccharides were attached to Asn-331, seven of which had terminal sialic acids. Only two high-mannose type oligosaccharides were attached to Asn-363. These results indicated great complexity in the structure and composition of oligosaccharides attached to individual IgM glycosylation sites.  相似文献   

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