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1.
Ion mobility coupled to mass spectrometry has been an important tool in the fields of chemical physics and analytical chemistry for decades, but its potential for interrogating the structure of proteins and multiprotein complexes has only recently begun to be realized. Today, ion mobility–mass spectrometry is often applied to the structural elucidation of protein assemblies that have failed high-throughput crystallization or NMR spectroscopy screens. Here, we highlight the technology, approaches and data that have led to this dramatic shift in use, including emerging trends such as the integration of ion mobility–mass spectrometry data with more classical (e.g., ‘bottom-up’) proteomics approaches for the rapid structural characterization of protein networks.  相似文献   

2.
A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.  相似文献   

3.
The gas chromatographic and mass spectrometric properties of 19 catechol estrogens and catechol estrogen methyl ethers are reported. The gas chromatographic behaviour of the TMS-derivatives on the stationary phases OV-1, OV-3, OV-7, and OV-17 is examined and correlated with their molecular weight, shape, and polarity. The characteristic mass spectrometric features of the compounds result from the aromatic ring A, which is able to stabilize positive charge within the molecular ions. Consequently the molecular ions form the base peaks of the spectra. Fragmentation patterns highly specific for the catechols as well as for their monomethyl and dimethyl ethers are discussed and substantiated by determination of metastable ions and high resolution mass measurements.  相似文献   

4.
Physiological functions depend on a coordinated interplay of numerous different cell types. Proteins serve as major signaling molecules between cells; however, their comprehensive investigation in physiologically relevant settings has remained challenging. Mass spectrometry (MS)–based shotgun proteomics is emerging as a powerful technology for the systematic analysis of protein-mediated intercellular signaling and regulated post-translational modifications. Here, we discuss recent advancements in cell biological, chemical, and biochemical MS-based approaches for the profiling of cellular messengers released by sending cells, receptors expressed on the cell surface, and their interactions. We highlight methods tailored toward the mapping of dynamic signal transduction mechanisms at cellular interfaces and approaches to dissect communication cell specifically in heterocellular systems. Thereby, MS-based proteomics contributes a unique systems biology perspective for the identification of intercellular signaling pathways deregulated in disease.  相似文献   

5.
Biological imaging techniques are the most efficient way to locally measure the variation of different parameters on tissue sections. These analyses are gaining increasing interest since 20 years and allow observing extremely complex biological phenomena at lower and lower time and resolution scale. Nevertheless, most of them only target very few compounds of interest, which are chosen a priori, due to their low resolution power and sensitivity. New chemical imaging technique has to be introduced in order to overcome these limitations, leading to more informative and sensitive analyses for biologists and physicians.Two major mass spectrometry methods can be efficiently used to generate the distribution of biological compounds over a tissue section. Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) needs the co-crystallization of the sample with a matrix before to be irradiated by a laser, whereas the analyte is directly desorbed by a primary ion bombardment for Secondary Ion Mass Spectrometry (SIMS) experiments. In both cases, energy used for desorption/ionization is locally deposited -some tens of microns for the laser and some hundreds of nanometers for the ion beam- meaning that small areas over the surface sample can be separately analyzed. Step by step analysis allows spectrum acquisitions over the tissue sections and the data are treated by modern informatics software in order to create ion density maps, i.e., the intensity plot of one specific ion versus the (x,y) position.Main advantages of SIMS and MALDI compared to other chemical imaging techniques lie in the simultaneous acquisition of a large number of biological compounds in mixture with an excellent sensitivity obtained by Time-of-Flight (ToF) mass analyzer. Moreover, data treatment is done a posteriori, due to the fact that no compound is selectively marked, and let us access to the localization of different lipid classes in only one complete acquisition.  相似文献   

6.
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7.
YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.  相似文献   

8.
This paper reviews liquid chromatographic–mass spectrometric (LC–MS) procedures for the identification and/or quantification of drugs of abuse, therapeutic drugs, poisons and/or their metabolites in biosamples (whole blood, plasma, serum, urine, cerebrospinal fluid, vitreous humor, liver or hair) of humans or animals (cattle, dog, horse, mouse, pig or rat). Papers published from 1995 to early 1997, which are relevant to clinical toxicology, forensic toxicology, doping control or drug metabolism and pharmacokinetics, were taken into consideration. They cover the following analytes: amphetamines, cocaine, lysergide (LSD), opiates, anabolics, antihypertensives, benzodiazepines, cardiac glycosides, corticosteroids, immunosuppressants, neuroleptics, non-steroidal anti-inflammatory drugs (NSAID), opioids, quaternary amines, xanthins, biogenic poisons such as aconitines, aflatoxins, amanitins and nicotine, and pesticides. LC–MS interface types, mass spectral detection modes, sample preparation procedures and chromatographic systems applied in the reviewed papers are discussed. Basic information about the biosample assayed, work-up, LC column, mobile phase, interface type, mass spectral detection mode, and validation data of each procedure is summarized in tables. Examples of typical LC–MS applications are presented.  相似文献   

9.
Rappsilber J  Mann M 《Genome biology》2002,3(8):comment2008.1-comment20085
Recent advances in mass spectrometry will soon allow routine analysis of protein expression levels. How close are we to true quantitative proteomics?  相似文献   

10.
The modern phenotypic and genetic methods except for Multi Locus Sequence Typing do not allow the reliable differentiation within Mitis group of α-hemolytic streptococci. During this study the MALDI mass spectra were acquired for 28 clinical isolates initially identified as S. pneumoniae by routine bacteriological tests. Due to Multi Locus Sequence Typing these isolates were found to belong to two closely related species - S. pneumoniae (n = 22) and S. mitis (n = 6). Distribution of those isolates in accordance with cluster analysis of collected mass spectra matched to Multi Locus Sequence Typing data. The diagnostic model based on Genetic Algorithm classifier demonstrated the differentiation of α-hemolytic streptococci with 100% sensitivity and 94.6% accuracy. Statistical analysis of MS peak areas revealed 2 peaks which are different for S. mitis and S. pneumoniae groups.  相似文献   

11.
Cysteinyl leukotrienes (LT) C4, LTD4, and LTE4 are potent mediators of anaphylaxis and inflammatin. LTE4 is extensively metabolized in man mainly by ω-oxidation followed by subsequent β-oxidation to more polar and biologically inactive metabolites. This paper describes a method for the synthesis of [1,20−18O2]-carboxy-LTE4, [1,18−18O2]-carboxy-dinor-LTE4, and [1,16−18O2]-carboxy-14,15-dihydro-tetranor-LTE4 starting from the unlabelled dimethyl esters of 20-carboxy-LTA4, 18-carboxy-dinor-LTA4 and 16-carboxy-14,15-dihydro-tetranor-LTA4, respectively, by separate chemical conjugation with cysteine hydrochloride in H218O-methanol followed by alkaline hydrolysis with Li18OH. The isotopic purity of the isolated reaction products was 94% at 18O for all three preparations while only 0.3% remained unlabelled as confirmed by negative-ion chemical-ionization gas chromatography-mass spectrometry (GC-NICI-MS) after their catalytical reduction/desulphurization and derivation. The 18O2-labelled compounds are demonstrated to be suitable internal standards for quantification by GC-NICI-MS and GC-NICI-tandem MS. We found by GC-NICI-tandem MS that the excretion rate of 20-carboxy-LTE4 is comparable to that of LTE4 (both in nmol/mol creatinine, mean ± S.E.) in healthy children (26.7 ± 4.7 vs. 32.0 ± 6.0, n = 9) and adults (13.9 ± 1.1 vs. 27.2 ± 5.4, n = 3).  相似文献   

12.
Periplasmic, cyclic β-glucans isolated from Bradyrhizobium elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium yuanmingense strains have been investigated by means of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), 1D and 2D nuclear magnetic resonance (NMR), as well as standard chemical methods. These compounds are built of 10–13 d-glucose residues. The main fractions contain molecules assembled of 12 hexose units (Mw = 1945.363 Da). Glucose monomers are linked by β-(1→3) or β-(1→6) glycosidic bonds. The ratio of β-(1→3) to β-(1→6) linked glucose is approximately 1:2. Moreover, methylation analysis demonstrated the presence of terminal, non-reducing, as well as branched (i.e., 3- and 6-substituted) glucoses. Thus, the basic structure of the investigated compounds is similar to that of periplasmic oligosaccharides from Bradyrhizobium japonicum and Azorhizobium caulinodans strains. The analyzed cyclic β-glucans are substituted by phosphocholine (PC) (one or two residues per ring) and highly decorated with acetate and succinate. The substituents are arranged diversely in the population of cyclic β-glucan molecules. The concentrations of cyclic β-glucans in Bradyrhizobium periplasmic space are osmotically regulated and increase in response to a decrease of medium osmolarity.  相似文献   

13.
Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography–mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid–histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.  相似文献   

14.

Background  

Mass Spectrometry coupled to Liquid Chromatography (LC-MS) is commonly used to analyze the protein content of biological samples in large scale studies. The data resulting from an LC-MS experiment is huge, highly complex and noisy. Accordingly, it has sparked new developments in Bioinformatics, especially in the fields of algorithm development, statistics and software engineering. In a quantitative label-free mass spectrometry experiment, crucial steps are the detection of peptide features in the mass spectra and the alignment of samples by correcting for shifts in retention time. At the moment, it is difficult to compare the plethora of algorithms for these tasks. So far, curated benchmark data exists only for peptide identification algorithms but no data that represents a ground truth for the evaluation of feature detection, alignment and filtering algorithms.  相似文献   

15.
Gas chromatography–mass spectrometry (GC–MS) was compared with gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for measurements of cholesterol 13C enrichment after infusion of labeled precursor ([13C1,2]acetate). Paired results were significantly correlated, although GC–MS was less accurate than GC–C–IRMS for higher enrichments. Nevertheless, only GC–MS was able to provide information on isotopologue distribution, bringing new insights to lipid metabolism. Therefore, we assessed the isotopologue distribution of cholesterol in humans and dogs known to present contrasted cholesterol metabolic pathways. The labeled tracer incorporation was different in both species, highlighting the subsidiarity of GC–MS and GC–C–IRMS to analyze in vivo stable isotope studies.  相似文献   

16.
17.
《Journal of Proteomics》2008,71(6):601-608
For relative protein quantitation by mass spectrometry we metabolically labeled E. coli bacteria with 15N-enriched diets. Proteins extracted from 15N-labeled and unlabeled E. coli bacteria were mixed, separated by two-dimensional gel electrophoresis and enzymatically digested. The resulting tryptic peptides were analyzed by MALDI mass spectrometry. For the relative protein quantitation we developed fully automated software, QuantiSpec (Quantitative Mass Spectrometry Analysis Software), which uses data from MALDI TOF mass spectrometry and the Mascot database search engine. QuantiSpec detects natural as well as partially or fully labeled peptide isotope distributions. For each identified peptide the 15N incorporation rate is determined by comparing the experimental to a set of theoretical isotope patterns based on the peptide sequence. Relative quantitation is accomplished by calculating the signal intensity ratios for each 14N/15N peptide pair.  相似文献   

18.
In an effort to develop DS02, a new radiation dosimetry system for the atomic bomb survivors of Hiroshima and Nagasaki, measurements of neutron-induced activities have provided valuable information to reconstruct the radiation situation at the time of the bombings. In Hiroshima, the depth profile of (152)Eu activity measured in a granite pillar of the Motoyasu Bridge (128 m from the hypocenter) was compared with that calculated using the DS02 methodology. For calculation of the (152)Eu production due to the thermal-neutron activation reaction, (151)Eu(n,gamma)(152)Eu, information on the hydrogen content in granite is important because the transport and slowing-down process of neutrons penetrating into the pillar is strongly affected by collisions with the protons of hydrogen. In this study, proton-proton elastic recoil coincidence spectrometry has been used to deduce the proton density in the Motoyasu pillar granite. Slices of granite samples were irradiated by a 20 MeV proton beam, and the energies of scattered and recoil protons were measured with a coincidence method. The water concentration in the pillar granite was evaluated to be 0.30 +/- 0.07%wt. This result is consistent with earlier data on adsorptive water (II) and bound water obtained by the Karl Fisher method.  相似文献   

19.
Screening of newborns for inborn errors of metabolism (IEM) in China is both a challenging and undeveloped area for gynecologists and pediatricians. Since 1999, the Capital Institute of Pediatrics has been studied as regards screening for IEM using advanced gas chromatography–mass spectrometry (GC–MS) method in collaboration with the Matsumoto Institute of Life Science (MILS), Japan, and has successfully diagnosed 51 cases of IEM in a total of 393 patients. Galactosemia, phenylketonuria and methylmalonic acidemia were the most frequent disorders among 51 cases of IEM. Treatment by suitable drugs and/or diet therapy was very effective in the most cases.  相似文献   

20.
A liquid chromatography–mass spectrometry (LC–MS) method for the analysis of corticosteroids in equine urine was developed. Corticosteroid conjugates were hydrolysed with β-glucuronidase; free and enzyme-released corticosteroids were then extracted from the samples with ethyl acetate followed by a base wash. The isolated corticosteroids were detected by LC–MS and confirmed by LC–MS–MS in the positive atmospheric pressure chemical ionisation mode. Twenty-three corticosteroids (comprising hydrocortisone, deoxycorticosterone and 21 synthetic corticosteroids), each at 5 ng/ml in urine, could easily be analysed in 10 min.  相似文献   

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