共查询到20条相似文献,搜索用时 31 毫秒
1.
A hybrid mass spectrometer operated in low-resolution selected decomposition monitoring (SDM) mode was used for the analysis of whole fish samples for the 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and poly-chlorinated dibenzofurans (PCDD/Fs). These fish samples were previously analyzed for PCDD/Fs using a high-resolution mass spectrometer following EPA protocols. The hybrid tandem mass spectrometric method using loss of COCl gave similar quantification results to those obtained by high-resolution mass spectrometry and eliminated the interferences attributed to polychlorinated biphenyls that were encountered in the high-resolution mass spectrometric analysis. Comparison of the two methods shows that the high-resolution mass spectrometric method surpasses the tandem mass spectrometric method in other analytical figures of merit, such as detection limit, linearity and reproducibility. 相似文献
2.
Electrospray ionization mass spectrometric peptide mapping: a rapid, sensitive technique for protein structure analysis 总被引:4,自引:0,他引:4
S K Chowdhury V Katta B T Chait 《Biochemical and biophysical research communications》1990,167(2):686-692
Electrospray ionization mass spectrometric peptide mapping is demonstrated to be a useful new technique for protein structure analysis. The procedure involves the digestion of the protein with trypsin and subsequent analysis of the total unfractionated digest by electrospray ionization mass spectrometry. The utility of the technique for investigating protein structure is illustrated by a peptide mapping analysis of human apolipoprotein AI (Mr = 28 kDa). The technique is rapid, sensitive, and requires no prior separation of the peptides. The discrimination effects observed in other mass spectrometric methods are less important in the present procedure. 相似文献
3.
Advanced analytical methods for hemoglobin variants 总被引:3,自引:0,他引:3
Wada Y 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,781(1-2):291-301
Hemoglobin variants are the protein mutations most often encountered in the clinical scene. They have been useful for developing methods to analyze mutant proteins because of their size and ease of collection in large amounts. Improvements in analytical methods have been directed toward higher resolution in electrophoresis and shorter elution times in chromatography. More importantly, in the last 20 years, hemoglobin variants have been used in the development of mass spectrometric strategies for analyzing protein mutations. This approach consists of a series of steps: measurement of the molecular mass of globins to detect or confirm the presence of mutations, peptide mass mapping or peptide mass fingerprinting of an enzymatic digest to identify mutated peptides, and tandem mass spectrometry to determine or confirm the site and type of mutation. The mass spectrometric strategy has enabled rapid analysis and demonstrated a superb ability to detect a number of hemoglobin variants, particularly those without a change in electrophoretic or chromatographic properties. Even with the recent advances in DNA analysis, protein analysis is still essential, because post-translational modifications following amino acid substitutions can occur including N-terminal acetylation, deamidation and oxidation-mediated processes. 相似文献
4.
Application of mass spectrometry for identification and structural studies of flavonoid glycosides 总被引:2,自引:0,他引:2
Stobiecki M 《Phytochemistry》2000,54(3):237-256
Mass spectrometry is an important tool for the identification and structural determination of flavonoid glycosides. The advantages of mass spectrometry are high sensitivity and possibilities of hyphenation with liquid chromatographic methods for the analysis of mixtures of compounds. Different desorption ionization methods allow the analysis of underivatized glycosides. A review of mass spectrometric techniques applied to the identification and structural studies of flavonoid glycosides is presented. 相似文献
5.
Vollmer S Astorga-Wells J Alvelius G Bergman T Jörnvall H 《Analytical biochemistry》2008,374(1):154-162
Identification of peptides from a complex mixture can be difficult because of the wide concentration range and the different ionization efficiencies of peptides during analysis by electrospray ionization (ESI) mass spectrometry (MS). Preconcentration methods are necessary to allow low-abundance and low-intensity peptides to reach the ionization threshold of the mass spectrometer. Here we demonstrate peptide enrichment based on electroimmobilization. Peptides are immobilized without the use of solid support or chemical binding by application of an electric field along a microflow stream in an electrocapture cell. Once enriched/preconcentrated inside the cell, they are released by removal of the electric field and via an interface with an electrospray emitter are submitted to online mass spectrometric analysis. Tandem mass spectrometric analysis of a peptide mixture containing hemoglobin, myoglobin, bovine serum albumin (BSA), and cytochrome c was successful. Amplification factors up to 16-fold were achieved with improvement of the signal-to-noise values for the preconcentrated sample. The limit of detection for one of the preconcentrated peptides was 3.6 fmol. 相似文献
6.
Proteomics studies of pathogenic bacteria are an important basis for biomarker discovery and for the development of antimicrobial drugs and vaccines. Especially where vaccines are concerned, it is of great interest to explore which bacterial factors are exposed on the bacterial cell surface and thus can be directly accessed by the immune system. One crucial step in proteomics studies of bacteria is an efficient subfractionation of their cellular compartments. We set out to compare and improve different protocols for the fractionation of proteins from Gram-negative bacteria into outer membrane, cytoplasmic membrane, periplasmic, and cytosolic fractions, with a focus on the outer membrane. Overall, five methods were compared, three methods for the fast isolation of outer membrane proteins and two methods for the fractionation of each cellular compartment, using Escherichia coli BL21 as a model organism. Proteins from the different fractions were prepared for further mass spectrometric analysis by SDS gel electrophoresis and consecutive in-gel tryptic digestion. Most published subfractionation protocols were not explicitly developed for proteomics applications. Thus, we evaluated not only the separation quality of the five methods but also the suitability of the samples for mass spectrometric analysis. We could obtain high quality mass spectrometry data from one-dimensional SDS-PAGE, which greatly reduces experimental time and sample amount compared to two-dimensional electrophoresis methods. We then applied the most specific fractionation technique to different Gram-negative pathogens, showing that it is efficient in separating the subcellular proteomes independent of the species and that it is capable of producing high-quality proteomics data in electrospray ionization mass spectrometry. 相似文献
7.
8.
A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder. 相似文献
9.
Thingholm TE Jensen ON Robinson PJ Larsen MR 《Molecular & cellular proteomics : MCP》2008,7(4):661-671
The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 mug of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a 3-fold increase in recovery of multiply phosphorylated peptides. 相似文献
10.
Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques.
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Z. Yu G. Friso J. J. Miranda M. J. Patel T. Lo-Tseng E. G. Moore A. L. Burlingame 《Protein science : a publication of the Protein Society》1997,6(12):2568-2577
Diaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric-based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5-dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size-exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP-HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin beta-chain, and two hemoglobin alpha-chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb. 相似文献
11.
Dorothy R. Ahlf Wheatcraft Xin Liu Amanda B. Hummon 《Journal of visualized experiments : JoVE》2014,(94)
Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI. 相似文献
12.
Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes
The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples. 相似文献
13.
The analysis of antimalarials by high-performance liquid chromatography (HPLC)/mass spectrometry demonstrates a new dimension in specificity along with increased sensitivity compared to conventional HPLC detection methods. Both direct liquid introduction and thermospray HPLC/mass spectrometry interfaces provided molecular weight information as well as characteristic fragment ions for antimalarials not normally amenable to direct probe or gas chromatographic/mass spectrometric techniques. The direct liquid introduction interface, which incorporated a 1/100 split, showed a detection limit of 30 ng using selected ion monitoring. The thermospray technique showed less than 1 ng detection limits using selected ion monitoring. 相似文献
14.
Hasegawa Y Iga M Kimura M Shigematsu Y Yamaguchi S 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,823(1):13-17
We performed prenatal diagnosis of organic acid disorders using two mass spectrometric methods; gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS). Of 28 cases whose amniotic fluid was tested, 11 cases were diagnosed as "affected". All cases whose samples were diagnosed as "unaffected" were confirmed to have no symptoms or abnormalities in urinary organic acid analysis after birth. Of the 11 "affected" cases, two cases were missed by ESI/MS/MS but not by GC/MS. When the stability of metabolites in amniotic fluid was checked, it was found that acylcarnitines degraded in one week at room temperature, whereas organic acids such as methylmalonate or methylcitrate were stable for at least 14 days. Prenatal diagnosis by analysis using simultaneous two or more methods may be more reliable, though attention should be paid to sample transportation conditions. 相似文献
15.
William E. Bronner
Allan S. Xu
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1992,580(1-2):63-75Gas chromatographic—mass spectrometric methods of analysis for the detection of 11-nor-Δ9-tetrahydrocinnabinol-9-carboxylic acid, a major metabolite of Δ9-tetrahydrocannabinol, are reviewed. Emphasis is on analytical methodology including numerous derivatization techniques developed specifically for this analyte. The majority of procedures cited in the literature were developed to detect this metabolite in the blood and urine of man. 相似文献
16.
A technique for the detection of biotransformation products of xenobiotics in crude urine extracts by field desorption mass spectrometric profile analysis is described. The method comprises determination of peak profiles of a series of blank and test samples using low resolution field desorption mass spectrometry, comparison of averaged peak profiles and noise reduction by means of Fisher and ratio weighting of peak intensities. Application of the technique to 3,5-dinitro-2-hydroxytoluene has resulted in the detection of two hitherto unknown metabolites in rat urine. By thin-layer co-chromatography, high resolution electron impact mass spectrometry and thin-layer chromatographic/field desorption mass spectrometric analysis they could be identified as 3,5-dinitro-2-hydroxybenzenemethanol and 3,5-diacetamido-2-hydroxytoluene. 相似文献
17.
Mirgorodskaya E Hassan H Wandall HH Clausen H Roepstorff P 《Analytical biochemistry》1999,269(1):54-65
In this study we present a method for determination of O-glycosylation sites in glycopeptides, based on partial vapor-phase acid hydrolysis in combination with mass spectrometric analysis. Pentafluoropropionic acid and hydrochloric acid were used for the hydrolysis of glycosylated peptides. The reaction conditions were optimized for efficient polypeptide backbone cleavages with minimal cleavage of glycosidic bonds. The glycosylated residues were identified by mass spectrometric analysis of the hydrolytic cleavage products. Although glycosidic bonds are partially cleaved under acid hydrolysis, the resulting mass spectra allowed unambiguous determination of the glycosylation sites. Examples are shown with mannosyl- and mucin-type glycopeptides. Performing the hydrolysis in vapor eliminates the risk for contamination of the sample with impurities from the reagents, thus allowing analysis of the reaction products without further purification both by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. 相似文献
18.
Schrotz-King P Wilm M Andersen JS Ashman K Podtelejnikov AV Bachi A King A Mann M 《Journal of receptor and signal transduction research》1999,19(1-4):659-672
In recent years, mass spectrometry has become the method of choice for identifying small amounts of gel separated proteins. Using high mass accuracy peptide mass mapping followed if necessary by nanoelectrospray sequencing, most mammalian proteins can now be identified quickly and sensitively either in amino acid or in EST sequence databases. These methods are illustrated here using an ongoing project in the author's laboratory, a mass spectrometric screen for new mouse brain receptors and their interaction partners. 相似文献
19.
Christina Ludwig George Rosenberger Sabine Amon Ben C Collins Ruedi Aebersold 《Molecular systems biology》2018,14(8)
Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent and quantitatively accurate proteomics data from many samples. SWATH‐MS is a specific variant of data‐independent acquisition (DIA) methods and is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy. In a SWATH‐MS measurement, all ionized peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows. To analyse SWATH‐MS data, a strategy based on peptide‐centric scoring has been established, which typically requires prior knowledge about the chromatographic and mass spectrometric behaviour of peptides of interest in the form of spectral libraries and peptide query parameters. This tutorial provides guidelines on how to set up and plan a SWATH‐MS experiment, how to perform the mass spectrometric measurement and how to analyse SWATH‐MS data using peptide‐centric scoring. Furthermore, concepts on how to improve SWATH‐MS data acquisition, potential trade‐offs of parameter settings and alternative data analysis strategies are discussed. 相似文献
20.
Markillie LM Lin CT Adkins JN Auberry DL Hill EA Hooker BS Moore PA Moore RJ Shi L Wiley HS Kery V 《Journal of proteome research》2005,4(2):268-274
Most current methods for purification and identification of protein complexes use endogenous expression of affinity-tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, and gel separation and in-gel digestion prior to mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pull-down assay with denaturing elution, trypsin digestion in organic solvent, and LC-ESI MS/MS protein identification using SEQUEST analysis. Our method is simple and easy to scale-up and automate, making it suitable for high-throughput mapping of protein interaction networks and functional proteomics. 相似文献