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1.
Amino acid analysis has been an integral part of analytical biochemistry for more than 50 years. However, its experimental design, which includes derivatization of amino acids followed by some kind of chromatographic separation, has not changed over the years. We have developed a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based method for the quantitative analysis of amino acids. This method does not require any amino acid modification, derivatization, or chromatographic separation. The data acquisition time is decreased to several seconds for a single sample. No significant ion suppression effects were observed with the developed sample deposition technique, and the method was found to be reproducible. Linear responses between the amino acid concentration and the peak intensities ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 microM, and correlation coefficients were between 0.983 (for arginine) and 0.999 (for phenylalanine). Limits of quantitation were between 0.03 microM (for arginine) and 3.7 microM (for histidine and homocysteine). This method was applicable to the mixtures of free amino acids as well as to HCl hydrolysates of proteins. Furthermore, we have shown that this method can be applied to other biologically important low-molecular weight compounds such as glucose. 相似文献
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Succinoglycan monomers (M1, M2, and M3) are octasaccharides with acetyl, pyruvyl, and/or succinyl groups as substituents derived from Sinorhizobium meliloti 1021. The dissociation patterns of the octasaccharides caused by low-energy collision-activated dissociation (CAD) were investigated using triple quadrupole tandem mass spectrometry (MS) equipped with an electrospray ionization (ESI) source with increasing collision energy (CE) in negative ion mode. None of the succinoglycan monomers were fragmented at a CE of −25 eV. When the CE was applied to −50 or −70 eV, the loss of the terminal Gal residue and/or the succinyl group of the monomers was observed in the product ion scan mode. Interestingly, the acetyl and the pyruvyl groups in the succinoglycan monomers were not lost even when a CE of −70 eV was applied, indicating that the substituents are more stable than the succinyl group in the octasaccharides. 相似文献
4.
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
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与传统的微生物鉴定技术相比,基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS)是一种准确、可靠和快速的鉴定和分型的技术。本文通过检索近年来国内外相关研究论文,总结最新的研究进展,发现MALDI-TOF MS在临床病原微生物、食源性微生物以及环境微生物等鉴定中有较大的优势,加快了微生物鉴定的进程,同时探索该技术在新领域的最新进展和面临的挑战,以期为我国基质辅助激光解吸电离飞行时间质谱技术的发展提供参考。 相似文献
6.
Ogawa Y Traina J Zimmermann E Yu T Schneider DW Pungor E 《Analytical biochemistry》2007,368(2):214-221
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
7.
Fernández LE Sørensen HR Jørgensen C Pedersen S Meyer AS Roepstorff P 《Molecular biotechnology》2007,35(2):149-160
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
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M.Kaniz Fatema Diogo R.B. Ducatti M. Eug?nia R. Duarte Alberto S. Cerezo María C. Matulewicz 《Carbohydrate research》2010,345(2):275-2314
MALDI-TOF mass spectrometry analyses of several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kappa- and iota-carrageenans using different matrices (2,5-dihydroxybenzoic acid, nor-harmane, ferulic acid, and the ionic liquid matrices 2,5-dihydroxybenzoic acid-n-butylamine and ferulic acid-n-butylamine) were conducted. These carbohydrates were selected as model compounds to study the MALDI prompt and post-source decay (PSD) fragmentation processes of both families of oligosaccharides. Sulfated alditols showed in the negative-ion mode the molecular ion as [M−Na]− together with the species yielded by their prompt fragmentation (mainly desulfation) while the sulfated oligosaccharides (aldoses) showed mainly glycosidic prompt fragmentation (glycosidic C-cleavages and desulfation). Non-sulfated aldoses and alditols, which could only be analyzed in positive-ion mode ([M+Na]+), did not suffer any prompt fragmentation. The former yielded cross-ring fragmentation in the PSD mode. Best results were obtained by using 2,5-dihydroxybenzoic acid and/or nor-harmane as matrices for all the compounds studied. 相似文献
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The utilization of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the analytical detection and quantification of phosphoinositides and other lipids in lipid extracts from biological samples was explored. Since phosphatidylcholine species in crude extracts have been shown to cause ion suppression of the MS signals for other lipids, a minicolumn of a silica gel cation exchanger was used to adsorb the cationic lipids including the phosphatidylcholine species from the chloroform phase of fetal and adult murine brain extracts. In positive ion mode, lipid peaks that had been completely suppressed in the crude extract became readily detectable and quantifiable in the flow-through fraction from the column. In negative ion mode, improved sensitivity made it possible to readily detect and measure phosphatidylinositol-4,5-bisphosphate (PIP(2)) which had been only marginally detectable before the fractionation. By incorporating an internal standard into the samples, the relative MALDI-TOF MS signals obtained for increasing concentrations of mammalian phosphatidylinositol (PtdIns) increased linearly with correlation coefficients >0.95. Using strong cation exchange minicolumn treated extracts, the levels of PtdIns and PIP(2) in adult and fetal murine brains were measured and compared. The removal of cationic lipids from the chloroform-methanol murine brain extracts resulted in improved overall detection of neutral and anionic lipids and quantification of phosphoinositides by MALDI-TOF MS. 相似文献
10.
Peer Stehling Martin Gohlke Rudolf Fitzner Werner Reutter 《Glycoconjugate journal》1998,15(4):339-344
N-Acetylneuraminic acid (a sialic acid) occurs mainly as a terminal substituent of oligosaccharides of glycoconjugates. Derivatives of neuraminic acid occur widely, substituted in the amino and hydroxy side chains, as well in the C-9 carbon skeleton. These derivatives are responsible for specific functions of sialic acids during cell-cell, cell-substrate, or cell-virus interactions. The study of O-acetylated neuraminic acids is difficult, because only small amounts are extractable from natural sources and they are generally unstable to acids and bases. We report a new method for the rapid analysis of O-acetylated neuraminic acids, using a combination of reversed phase HPLC and MALDI-TOF mass spectrometry. A mixture of neuraminic acids from bovine submaxillary gland mucins was analysed, as well as neuraminic acids variously substituted in the amino and hydroxy side chains with acetyl and glycolyl groups, respectively. © 1998 Rapid Science Ltd 相似文献
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Delphine Martiny Alain Visscher Boudewijn Catry Sonia Chatellier Olivier Vandenberg 《Journal of microbiological methods》2013
Growth conditions – including growth medium and incubation temperature – may influence the identification of Campylobacter by MALDI-TOF MS. 相似文献
13.
Sato Y Fukuyama Y Nonami H Erra-Balsells R Stortz CA Cerezo AS Matulewicz MC 《Carbohydrate research》2007,342(17):2567-2574
Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) has shown to be a very useful technique for the study of the non-volatile and thermally non-stable N-acylated glycopyranosyl- and glycofuranosyl-amines. Of the several matrices tested, 2,5-dihydroxybenzoic acid (DHB) was the most effective giving good spectra in the positive-ion mode. In the linear and reflectron modes, the [M+Na](+) ions appeared with high intensity. Their fragmentation patterns were investigated by post-source decay (PSD) UV-MALDI-TOF-MS showing mainly cross-ring cleavages. In addition, N,O-acylated glycopyranosyl- and glycofuranosyl-amines were also analyzed by this technique. PSD UV-MALDI-TOF-MS gave significant signals for several primary fragment ions, which were proposed but not detected, or observed with very low abundance, in electron ionization mass spectrometry (EI-MS) experiments. 相似文献
14.
Elena Urso Sabrina Bossio Teresa Sprovieri Antonio Qualtieri 《Analytical biochemistry》2010,402(1):13-19
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been applied to the analysis of a wide range of biomolecules. To date, there are two specific areas of application where MALDI-TOF-MS is viewed as impractical: analysis of low-mass analytes and relative quantitative applications. However, these limitations can be overcome and quantification can be routine. Increased levels of thymosin β4 (TB4) have been recently found in cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients. Our objective was to apply a label-free quantitative application of MALDI-TOF-MS to measure TB4 levels in human CSF by adding the oxidized form of TB4 as an internal standard. The relative peak area or peak height ratios of the native TB4 to the added oxidized form were evaluated. Considering the relative peak area ratios, healthy individuals showed a mean value of 40.8 ± 21.27 ng/ml, whereas CJD patients showed high values with a mean of 154 ± 59.07 ng/ml, in agreement with the previous observation found in CJD patients. Similar results were obtained considering peak height ratios. The proposed method may provide a simple and rapid screening method for quantification on CSF of TB4 levels suitable for diagnostic purposes. 相似文献
15.
Karine Guitot Silvia Scarabelli Thierry Drujon Gérard Bolbach Mehdi Amoura Fabienne Burlina Albert Jeltsch Sandrine Sagan Dominique Guianvarc’h 《Analytical biochemistry》2014
Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors. 相似文献
16.
Archaea and a number of groups of environmentally important bacteria, e.g., sulfate-reducing bacteria, anoxygenic phototrophs, and some thermophiles, are difficult to characterize using current methods developed for phenotypically differentiating heterotrophic bacteria. We have evaluated matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) as a rapid method for identifying different groups of extremophilic prokaryotes using a linear mass spectrometer (Micromass, UK). The instrument is designed to acquire mass-spectral patterns from prokaryotic cell-wall components between masses of 500 and 10,000 Da in a statistically robust manner and create a database that can be used for identification. We have tested 28 archaea (10 genera, 20 spp.) and 42 bacteria (25 genera, 37 spp.) and found that all species yield reproducible, unique mass-spectral profiles. As a whole, the profiles for the archaea had fewer peaks and showed less differentiation compared to the bacteria, perhaps reflecting fundamental differences in cell-wall structure. The halophilic archaea all had consistent patterns that showed little differentiation; however, the software was able to consistently distinguish Halobacterium salinarium, Halococcus dombrowski, and Haloarcula marismortui from one another, although it could not always correctly distinguish four strains of Hb. salinarium
from one another. The method was able to reliably identify 105 cells of either
Albidovulum inexpectatum or Thermococcus litoralis and could detect as low as 103
cells. We found that the matrix, alpha-cyano-4-hydroxy-cinnamic acid yielded better spectra for archaea than 5-chloro-2-mercapto-benzothiazole. Overall, the method was rapid, required a minimum of sample processing, and was capable of distinguishing and identifying a very diverse group of prokaryotes.Communicated by F. Robb 相似文献
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Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of chemical damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this article, we describe the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with nonstandard synthesis, deprotection, or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and use. 相似文献
18.
Agus Darwanto Alvin Farrel Daniel K. Rogstad Lawrence C. Sowers 《Analytical biochemistry》2009,394(1):13-23
The DNA of all organisms is persistently damaged by endogenous reactive molecules. Most of the single-base endogenous damage is repaired through the base excision repair (BER) pathway that is initiated by members of the DNA glycosylase family. Although the BER pathway is often considered to proceed through a common abasic site intermediate, emerging evidence indicates that there are likely distinct branches reflected by the multitude of chemically different 3′ and 5′ ends generated at the repair site. In this study, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS) to the analysis of model DNA substrates acted on by recombinant glycosylases. We examine the chemical identity of several possible abasic site and nicked intermediates generated by monofunctional and bifunctional glycosylases. Our results suggest that the intermediate from endoIII/Nth might not be a simple β-elimination product as described previously. On the basis of 18O incorporation experiments, we propose a new mechanism for the endoIII/Nth family of glycosylases that may resolve several of the previous controversies. We further demonstrate that the use of an array of lesion-containing oligonucleotides can be used to rapidly examine the substrate preferences of a given glycosylase. Some of the lesions examined here can be acted on by more than one glycosylase, resulting in a spectrum of damaged intermediates for each lesion, suggesting that the sequence and coordination of repair activities that act on these lesions may influence the biological outcome of damage repair. 相似文献
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The discrimination of onychomycoses from endogenous diseases showing macroscopically similar symptoms is difficult. Long-lasting but ineffective antifungal therapies using systemic medicaments with often severe adverse reactions may be the consequence. We introduce a novel mass spectrometric method for the discrimination of fungal infections and nonfungal affections. Horn samples from patients infected by Trichophyton rubrum, from patients with psoriasis affecting nails, and from healthy persons were investigated. Onychomycoses are basically associated with proteolytic attacks of the virulent fungi-secreting proteases partly hydrolyzing the horn material. Endogenous diseases lack these proteolytic activities, conserving intact structural proteins. Tryptical digestion of horn material produced cleavage peptides detectable by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mass spectra of horn material infected by T. rubrum were clearly different from those originating from healthy test persons and from patients with psoriasis. Two methods were successfully applied to quantify the differences between groups of samples. One is based on the Euclidean match factor, and the other is based on the identification of specific peptide peaks occurring exclusively within one group of persons. The Euclidean match factor distributions and the occurrence of specific peptide peaks allowed a clear differentiation of T. rubrum infections from psoriasis patients and healthy test persons. No differences were found between healthy test persons and psoriasis patients. The method is rapid and does not require any cultivation. 相似文献
20.
Florence Gaucher-Wieczorek Vincent GuérineauDavid Touboul Sophie Thétiot-LaurentFranck Pelissier Marie-Ange Badet-DenisotBernard Badet Philippe Durand 《Analytical biochemistry》2014
Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5′-diphospho-N-acetyl-d-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts d-fructose-6-phosphate (Fru-6P) and l-glutamine (Gln) into d-glucosamine-6-phosphate (GlcN-6P) and l-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. 相似文献