Cloth colorization caused by microbial biofilm

In this study, cloth disfeaturement was investigated biologically. To clarify whether or not microbes can cause cloth disfeaturement, and to identify the microbes causing the disfeaturement, worn cloth samples were incubated on sweat-ingredient agar medium. Non-sterilized cloth samples became yellow-colored during incubation, and bacterial strains belonging to the genera Bacillus, Brevibacterium, Kocuria, Micrococcus and Staphylococcus were isolated from the yellow-colored parts. Two major isolates close to the genera Bacillus and Micrococcus were inoculated separately or together on cloth samples to examine whether or not these isolates can cause colorization. When the isolate close to Micrococcus was inoculated on its own or mixed with the isolate close to Bacillus, the samples turned yellow to a greater extent and a biofilm-like structure was observed by SEM on the colored areas. In contrast, the isolate close to Bacillus alone barely caused any colorization, and no biofilm-like structure was observed. From the yellow-colored samples, bacterial strains with the same 16S rRNA gene sequences as those of the inoculated strains were re-isolated. These results strongly suggest that the bacterial strain belonging to genus Micrococcus causes cloth colorization by forming a biofilm structure.     

On the structure of human hair melanins from an infrared spectroscopy analysis of their interactions with Cu2+ ions.

Melanins were isolated from dark and red human hair and complexed with copper ions at various pH values in a complexing medium. IR spectra of melanins and their Cu2+-complexes for pellets with KBr were obtained. The IR spectra indicate that Cu2+ ions bound to melanins are fixed by different carboxyl and hydroxyl (phenolic and/or alcoholic) groups in the macromolecule. From these results it is concluded that, generally, melanin carboxyl groups are responsible for interactions of metal ions with the melanin molecule. Complexes of melanins isolated from dark and red human hair show structural differences when analysed by IR spectroscopy. Conclusions from these investigations assist in the differentiation of structures of analysed hair melanins. IR spectral analysis of melanin samples and their complexes suggest that melanin samples obtained from red hair may contain eumelanin.     

A two-dimensional proteome map of Shigella flexneri

Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans. In this study, extracellular proteins from the culture medium and whole cell proteins in cellular extracts of S. flexneri 2a strain 2457T were examined by two-dimensional (2-D) gel electrophoresis using immobilized pH gradient (IPG) technology. Proteins were identified by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with Mascot search program. In total, among the 488 proteins spots processed, 388 proteins were identified. The identified proteins represented 169 genes. By comparing results of Mascot search against databases of Escherichia coli and genomes of S. flexneri 2a, one S. flexneri-specific protein was identified and one possible gap was found in 2457T genome sequences. Although this proteome map is still incomplete, it is already a useful reference for future studies involving pathogenicity, vaccine development, design of novel antibacterial drugs, etc. Proteome maps and a table of all identified proteins are available on the internet at www.proteomics.com.cn.     

Mass spectrometric characterization of lipid-modified peptides for the analysis of acylated proteins

The analysis of acylated proteins by mass spectrometry (MS) has largely been overshadowed in proteomics by the analysis of glycosylated and phosphorylated proteins; however, lipid modifications on proteins are proving to be of increasing importance in biomedical research. In order to identify the marker ions and/or neutral loss fragments that are produced upon collision-induced dissociation, providing a means to identify the common lipid modifications on proteins, peptides containing an N-terminally myristoylated glycine, a palmitoylated cysteine and a farnesylated cysteine were chemically synthesized. Matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF-TOF), electrospray ionization quadrupole time-of-flight (ESI Q-TOF), and electrospray ionization hybrid triple-quadrupole/linear ion trap (ESI QqQ(LIT)) mass spectrometers were used for the analysis. The peptide containing the N-terminally myristoylated glycine, upon CID, produced the characteristic fragments a1 (240.4 Th) and b1 (268.4 Th) ions as well as a low-intensity neutral loss of 210 Da (C14H26O). The peptides containing a farnesylated cysteine residue fragmented to produce a marker ion at a m/z of 205 Th (C15H25) as well as other intense farnesyl fragment ions, and a neutral loss of 204 Da (C15H24). The peptides containing a palmitoylated cysteine moiety generated neutral losses of 238 Da (C16H30O) and 272 Da (C16H32OS); however, no marker ions were produced. The neutral losses were more prominent in the MALDI-TOF-TOF spectra, whereas the marker ions were more abundant in the ESI QqQ(LIT) and Q-TOF mass spectra.     

The determination of the linear alkylbenzene sulfonate isomers in water samples by gas-chromatography/mass spectrometry

A number of 20 compounds of linear alkylbenzene sulfonates (LASs) family were identified by electron impact mass spectrometry (EI-MS) in water samples collected from wastewater treatment plants (WWTP). This paper presents the mass spectra of 20 compounds, the proposed mechanism of formation of the diagnostic ions obtained by EI-MS and the distribution of individual isomers in water samples collected from compartments of WWTP. The individual isomers from four homolog series C(10)-, C(11)-, C(12)- and C(13)-LAS were analyzed as methyl derivatives.     

Electrospray mass spectrometry of lanthanide β-diketone complexes. II-Ligand-exchange processes involving acetate ions and Ln(tfc)3 complexes

Electrospray ionization on Ln(tfc)₃ complexes (Ln = Eu, Yb; tfc = D -3-trifluoroacetylcamphorate) was performed with samples dissolved in methanol–water containing acetic acid. The spectra were obtained with a four-sector mass spectrometer. The mass spectra exhibit ions containing tfc ligand in addition to ions with acetato ligand. Electro-spray and tandem mass spectra are presented.     

Mass spectrometric evidence for different complexes of peptides and proteins with arsenic(III), arsenic(V), copper(II), and zinc(II) species

Trivalent and pentavalent arsenic were incubated with sulfur-containing amino acid, peptide and protein solutions both as organic compounds (phenylarsine oxide, phenylarsonic acid, dimethylarsinic acid, monomethylarsonic acid) and as inorganic compounds (arsenite, As(III), and arsenate, As(V)). After incubation of phenylarsine oxide solutions with cysteine and glutathione the mass spectra showed a covalent bond between arsenic and sulfur, which was stable at both acidic and neutral pH values. The mass spectra were dominated by monovalent ions at m/z 272 for cysteine samples and at m/z 458 for glutathione samples. Based on these masses the ionic structures could be ascribed to either fragment ions of the covalent arsenic-sulfur complexes or to other arsenic-bonding sites presumably at the amino group. Interestingly, under the same conditions no interactions of inorganic arsenite or arsenate could be measured. In the presence of added Cu(2+) ions all mass signals caused by a reaction of phenylarsine oxide with glutathione disappeared. In these mass spectra only the oxidised form of glutathione (GSSG) was found because of the redox activity of Cu(II). For the model protein lysozyme, no interactions with arsenic could be detected, whereas definite Cu- and Zn-lysozyme complexes with a stoichiometry of 1:1 and 2:1 for Zn(2+) ions and Cu(2+) ions, respectively, were observed. In contrast, for thioredoxin a bonding of As that depended on the concentration of the disulfide-reducing agent tris(2-carboxyethyl) phosphine was demonstrated.For three different phenylarsonic acids and for dimethylarsinic acid that all contain pentavalent arsenic, complexes with glutathione appeared in the mass spectra, which can be attributed to non-covalent interactions or to a covalent bond caused by an additive reaction.The optimisation of the experimental conditions necessary for the mass spectrometric analysis of the interactions of the arsenic species with peptides and proteins is described and the obtained mass spectra that provide information on the kinds of bonds are discussed.     

Lipid composition of membranes of Escherichia coli by liquid chromatography/tandem mass spectrometry using negative electrospray ionization

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using reversed-phase chromatography was developed for the analysis of phospholipids from bacterial extracts of a wild-type strain of Escherichia coli. Product ion mass spectra from [M--H](-) precursor ions allowed an identification of individual phospholipid species that includes both fatty acid composition and fatty acyl location on the glycerol backbone using diagnostic product ions. Thus, complete assignment, including sn-1/sn-2 fatty acyl position, was achieved for this strain of E. coli. In addition, the phospholipids were quantified relative to one another using an internal standard method.     

Phosphate effect on the content of selected elements in a lettuce variety grown at a contaminated soil

The purpose of this study was to evaluate the efficiency of superphosphate fertilizer in remediating a contaminated soil with potentially toxic elements. For this, different phosphorus doses were used in a number of lettuce plants. The element concentrations determined in their leaves were compared with those found in control lettuce plants. Instrumental neutron activation analysis was the analytical technique used to determine element concentration in lettuce leave samples. The application of 250 mg kg^?1 of P was the most effective treatment to reduce the concentrations of Br, Ca, Cd, Cl, Co, Cr, Fe, K, Mg, Mn, Sb and Zn in lettuce leaves.     

Bacterial Lux-Fluoro test for biological assessment of pollutants in water samples from urban and rural origin

During the 20th century, population growth and urbanization, together with changes in production and consumption, have placed unprecedented demands on the quality of water. The ongoing extraordinary economic growth, industrialization, and urbanization of many developing countries results in widespread water pollution from agricultural, industrial, and domestic sources. In consequence, people consume contaminated drinking water, thereby increasing the risk of exposure not only to infectious and parasitic disease but also to a growing volume of genotoxic and cytotoxic chemicals. In light of these trends, new, rapid and low-cost approaches are urgently needed to assess the quality of water supplies. Because of their simplicity and sensitivity, bacterial tests play an important role in the detection and screening of genotoxins or cytotoxins in water. Thus, the bacterial Lux-Fluoro test, which is a combination of two bioassays that simultaneously measure the genotoxicity (SOS-Lux test) and the cytotoxicity (LAC-Fluoro test), was used to identify polluted water from samples of rural and urban sources, collected from 10 different locations in the Punjab rivers’ basin. We identified at least three samples from rural origin having a high cytotoxic potential. The highest toxicity was found for the sample obtained from a draining canal collecting runoff water from the fields. The two other highly contaminated samples identified were taken from two ponds of different villages. The water samples obtained from the Ravi river and from the water tap in a suburb of the megacity Lahore showed no sign of genotoxicity or cytotoxicity. Seven control samples with differing genotoxic and cytotoxic potency were shown for comparison.     

Metal ion analysis in contaminated water samples using anodic stripping voltammetry and a nanocrystalline diamond thin-film electrode

Boron-doped nanocrystalline diamond thin-film electrodes were employed for the detection and quantification of Ag (I), Cu (II), Pb (II), Cd (II), and Zn (II) in several contaminated water samples using anodic stripping voltammetric (ASV). Diamond is an alternate electrode that possesses many of the same attributes as Hg and, therefore, appears to be a viable material for this electroanalytical measurement. The nanocrystalline form has been found to perform slightly better than the more conventional microcrystalline form of diamond in this application. Differential pulse voltammetry (DPASV) was used to detect these metal ions in lake water, well water, tap water, wastewater treatment sludge, and soil. The electrochemical results were compared with data from inductively coupled plasma mass spectrometric (ICP-MS) and or atomic absorption spectrometric (AAS) measurements of the same samples. Diamond is shown to function well in this electroanalytical application, providing a wide linear dynamic range, a low limit of quantitation, excellent response precision, and good response accuracy. For the analysis of Pb (II), bare diamond provided a response nearly identical to that obtained with a Hg-coated glassy carbon electrode.     

Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification

Flow-injection electrospray ionization mass spectrometry (FI-ESI-MS) of unfractionated cell-free extracts obtained from bacterial cells suspended in a solvent mixture was investigated as a rapid analytical method for reproducible, high-throughput bacterial identification. Five bacterial strains (two Escherichia coli, two Bacillus spp. and one Brevibacillus laterosporus) were studied in this investigation. Axenically grown bacterial cells were suspended in an acidic organic solvent and the cell-free extract was sequentially injected into a solvent flow stream that was sprayed into the ionization chamber of the ESI-MS. The spectra produced contained reproducible information, which was useful for discriminating between the bacteria. Tandem mass spectrometry was used to characterize further the peaks, and at least three classes of macromolecules, namely phospholipids, glycolipids, and proteins, were found to contribute most to the spectral information. Bacterial extracts stored under different conditions gave very similar mass spectra for each of the five bacterial strains, indicating that the extracts were stable even at room temperature for up to 24 h, with no loss of information content, which has obvious implications for automated high-throughput analysis. An analysis of the components of the extracting solvent mixture and their effects on the spectral information showed that acetonitrile contributes most significantly to the extraction process and hence to the information content of the spectra.     

Origin and removal of adducts (molecular mass = 98 u) attached to peptide and protein ions in electrospray ionization mass spectra

Electrospray ionization of peptides and proteins often produces intense adduct ions resulting from the attachment of a moeity with mass 98 u. The formation of these adduct ions results in a substantial reduction in the mass spectrometric sensitivity and an undesirable increase in the complexity of the mass spectra. In the present study it was shown that the removal of the attached adducts from peptide and protein ions can be affected by collisional activation and that the adducts arise from the attachment of sulfuric acid or phosphoric acid to peptide and protein ions. When sulfate and phosphate ions are removed from the samples by chemical means, adduct free ions are obtained from proteins yielding spectra with improved quality and sensitivity.     

Identification of microbial mixtures by LC-selective proteotypic-peptide analysis (SPA)

This paper describes a method--using a combination of LC-MS/MS of selected bacteria-specific peptides and database search--for determining the species of bacteria present in a mixture. We identified the proteotypic peptides that were associated with specific bacteria by searching protein databases for the LC-MS/MS data. The retention time windows for specific peptide markers were used as an extra constraint so that the peptide markers of many bacterial species could be analyzed in a single LC-selective proteotypic-peptide analysis (SPA). We performed LC-MS/MS analyses on the proteolytic digest of cell extracts and monitored only the selected marker peptide ions at given elution time windows. The corresponding bacterial species could be characterized when the selected peptides that eluted at expected elution windows were identified correctly from the database. We managed to identify up to eight bacterial species simultaneously during a single LC-MS/MS analysis, as well as bacteria mixed in various abundances. Two marker ions having similar values of m/z, but obtained from two different bacterial samples, which would otherwise be selected as precursors within mass tolerance and would complicate the MS/MS data, were time-resolved using LC and then used to correctly identify their bacterial sources. The coupling of selective MS/MS monitoring with separation methods, such as LC, provides a highly selective and accurate analytical method for characterizing complex mixtures of bacterial species.     

In-source fragmentation and analysis of polysaccharides by capillary electrophoresis/mass spectrometry

In order to develop a robust and easy-to-use technique for characterization of bacterial polysaccharides, a pseudo-hydrolysis strategy was investigated. Based on in-source collision-induced dissociation, polysaccharide molecular ions were fragmented within the orifice-skimmer region of an electrospray ionization (ESI) mass spectrometer. The fragment ions thus generated were then analyzed similarly to the conventional ESI mass spectrometry approach. MS/MS scanning was applied to obtain product-ion spectra of the primary fragments for sequencing. To further improve the sensitivity and separation of polysaccharides from other components in the samples, a pressure-assisted capillary electrophoresis/mass spectrometry (CE/MS) system was employed. Using bacterial polysaccharides as model compounds, the mass spectra obtained for polysaccharide repeating units generated through chemical hydrolysis and in-source fragmentation were directly compared, both in positive and negative ion modes. With the additional separation of impurities provided by CE, the success of this technique has been demonstrated for structural analysis of O-chain polysaccharides (O-PS) and capsular polysaccharides (CPS). In-source fragmentation was applied to promote the formation of structurally relevant repeating units of heterogeneous CPS that would remain undetected using conventional ESI conditions. This approach was proven to be particularly useful for probing the subtle structural differences in monosaccharide composition and functionalities arising across bacterial serotypes.     

Species identification of Oetzi's clothing with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on peptide pattern similarities of hair digests

Identification of ancient biological samples from the 1991-discovered and more than 5300-year-old Tyrolean mummy, also called iceman or Oetzi, is very difficult. The species of origins of four animal-hair-bearing samples of the accoutrement of the mummy not yet diagnosed were identified by a special proteomics method. Ha 43/91/130 and Ha 6/91, two samples from his coat, and Ha 5/91, a sample from his leggings, were assigned to sheep. The upper leather of his moccasins, Ha 2/91, was made from cattle. Despite the enormous age of these samples with partial (bio)chemical alterations, reliable identification was possible using a recently developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometric ((MALDI-TOF MS)-based analytical method. The method is exclusively based on the analysis of proteins and uses minute amounts of peptides directly derived from tryptic hair digests without any separation or enrichment steps. Unknown species are identified by comparison of their peptide ion patterns with known spectra stored in existing databases. Hereby, the correlation distance, a form of Euclidean distance, and deduced parameters are used to measure similarities.If more than one potential hit remains, specific diagnostic peptide ions are used to stepwise exclude incorrect matches. These ions are specific for orders, families, subfamilies/genera and/or even species. Peptide mass fingerprinting data combined with those from collision-induced dissociation spectra (combined MS & MS/MS) were used for interpretation with the MASCOT search engine and the NCBI database to find the potential parentage of hair proteins. For this technique, selected precursor ions were identified as specific diagnostic peptide ions. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Application of combined high-performance thin-layer chromatography immunostaining and nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry to the structural characterization of high- and low-affinity binding ligands of Shiga toxin 1

Shiga toxin 1 (Stx1) represents an AB5 toxin produced by enterohemorrhagic Escherichia coli, which cause gastrointestinal diseases in humans that are often followed by potentially fatal systemic complications, such as acute encephalopathy and hemolytic uremic syndrome. The expression of the preferential Stx1 receptor, Gb3Cer/CD77 (Gal alpha1-4Gal beta1-4Glc beta1-1Cer), is one of the primary determinants of susceptibility to tissue injury. Due to the clinical importance of this life-threatening toxin, a combined strategy of preparative high-performance thin-layer chromatography (HPTLC) overlay assay and mass spectrometry was developed for the detection and structural characterization of Stx1-binding glycosphingolipids (GSLs). A preparation of neutral GSLs from human erythrocytes, comprising 21.4% and 59.1% of the high- and low-affinity Stx1-binding ligands Gb3Cer/CD77 and Gb4Cer, respectively, was separated on silica gel precoated HPTLC plates and probed for the presence of Stx1 receptors. Stx1 positive on the one hand and anti-Gb3Cer/CD77 and anti-Gb4Cer antibody positive bands from parallel reference runs on the other hand were extracted with chloroform/methanol/water (30/60/8, v/v/v). These crude extracts were used without any further purification for a detailed structural analysis by nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) in the negative ion mode. In all extracts investigated, neutral GSLs were detected as singly charged deprotonated molecular ions, [M-H]-, and neither buffer-derived salt adducts nor coextracted contaminants from the overlay assay procedure or the silica gel layer were observed. For the structural characterization of Stx1- and antibody-binding GSLs low-energy collision-induced dissociation (CID) was applied to high and low abundant receptor species of the crude extracts. All MS/MS spectra obtained contained full series of Y-type ions, B-type ions and additional ions generated by ring cleavages of the sugar moiety. Only analytical quantities in the microgram scale of a single GSL species within the complex GSL mixture were required for the structural MS characterization of Stx1 ligands as Gb3Cer/CD77 and Gb4Cer. This effective combined HPTLC/MS procedure offers a broad range of applications, not only for toxins of bacterial origin, but also for any GSL-binding agents such as plant-derived lectins or human proteins with yet unknown binding specificities.     

天麻中对羟基苯类化合物的高效液相色谱-质谱研究

天麻中对羟基苯类化合物的(EsI(-))一级质谱图中除分子离子峰[M-1]外，还可见明显的[M-1 20] m[2m-1]峰，由此规律可方便确证未知同类样品的分子离子峰。由二级质谱(ESI/MS/MS)的特征碎片离子93、105和107等，结合紫外光谱及核磁结果确定了3个不纯样品中主要成分的结构，同时初步确定了两个同类杂质的结构。     

Complementary analysis of lipids in whole bacteria cells by thermally assisted hydrolysis and methylation-GC and MALDI-MS combined with on-probe sample pretreatment

The molecular structure of lipids in whole bacteria cells was characterized in detail by using two different and complementarily direct analyses; thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with on-probe sample pretreatment. First, THM-GC in the presence of tetramethylammonium hydroxide (TMAH) was applied to compositional analysis of the fatty acid components of lipids in whole bacterial cells. On the resulting chromatograms, a series of fatty acid components in bacterial lipids were clearly observed as resolved peaks of their methyl esters. The fatty acid compositions determined on the basis of the peak intensities were in good agreement with those obtained by the conventional technique involving solvent extraction of the lipids in the samples. Furthermore, MALDI-MS combined with the on-probe sample treatment, using 2,5-dihydroxybenzoic acid (DHB) and sodium iodide (NaI) as matrix and cationization reagents, respectively, was used to detect intact phospholipids directly from whole bacterial cells. The MALDI spectra thus obtained showed an array of ions generated from bacterial phospholipids, such as phosphatidylethanolamines (PEs) and phosphatidylglycerols (PGs). Finally, the bacterial lipids were comprehensively characterized in terms of the acyl groups and the molecular structures by taking both of the results obtained by THM-GC and MALDI-MS into consideration.