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PubMed2004

A rapid method for analyzing recombinant protein inclusion bodies by mass spectrometry.

The identification and characterization of a protein overexpressed in insoluble inclusion bodies in Escherichia coli are the first crucial and time-limiting steps in recombinant protein expression. Here, a straightforward approach to the analysis of recombinant proteins in inclusion bodies is presented. Inclusion bodies were dissolved in 8M urea and analyzed by matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry without prior desalting. Mass determination was achieved by direct spotting of the samples onto the MALDI target and serial dilution in the matrix. The masses of four different proteins, expressed in inclusion bodies, were determined with a mass accuracy better than 0.1%. Furthermore, protein modifications, such as N-terminal processing of single amino acids or artificial cyanylation caused by incubation of the inclusion bodies with urea at elevated temperatures, could be detected. Similarly, tryptic digests were directly analyzed in 2M urea to obtain peptide mass fingerprints for identification and more detailed information on the primary protein structure and secondary modifications. Due to the presence of ammonia in the urea-containing buffers, no Na(+) adducts were observed in the peptide mass fingerprint analysis. Taken together, the rapid and robust procedures presented here greatly facilitate the analysis of recombinant proteins.

Antibodies, Monoclonal↗

PubMed2005

Multi-component HPLC fingerprinting of Radix Salviae Miltiorrhizae and its LC-MS-MS identification.

The multi-component fingerprinting method of Radix Salviae Miltiorrhizae (Root of Salvia miltiorrhiza BGE.), an important and popular medicinal herb in traditional Chinese medicine, was studied using reverse-phase HPLC and LC-MS-MS. Extract containing both the water-soluble phenolic compounds and nonpolar diterpenoid compounds known to be the herb's main bioactive components was prepared by a two-step extractive procedure. An HPLC fingerprinting method which can simultaneously separate these two types of compounds was established with gradient elution mode and photodiode array detection at 280 nm. Eighteen peaks in this HPLC fingerprint were structurally identified by employing LC-MS-MS techniques. The electrospray ionization (ESI) MS-MS spectra of most salvianolic acids displayed a characteristic behavior of loss of danshensu and caffeic acid moieties, while those of tanshinones showed a particular behavior of loss of H2O which is quite different from the fragmentation pattern in electron ionization mass spectrometry (EI-MS). The HPLC fingerprints of 7 batches of crude drugs showed similar separation pattern and provided much chemical information of the pharmacologically-active compounds in the crude drugs, which is useful for the authentication and quality evaluation of this medicinal herb.

Chromatography, High Pressure Liquid↗

PubMed1998

Allergy to Aspergillus-derived enzymes in the baking industry: identification of beta-xylosidase from Aspergillus niger as a new allergen (Asp n 14).

BACKGROUND: Aspergillus-derived enzymes are used in dough improvers in bakeries. Some of these enzymes are identified as causing IgE-mediated sensitization in up to 25% of bakers with workplace-related symptoms. OBJECTIVE: The aim of this study was to compare the frequency of sensitization to Aspergillus xylanase, cellulase, and glucoamylase with the sensitization to alpha-amylase (Asp o 2) and to identify IgE-reactive proteins in enzyme preparations. METHODS: Sensitization to Aspergillus-derived enzymes and cross-reactivity were retrospectively studied by enzyme allergosorbent test (EAST) and EAST-inhibition experiments. IgE-reactive proteins were detected by electrophoretic separation and immunoblotting. Liquid chromatography with electrospray ionization mass spectrometry and Edman degradation of tryptic protein fragments were used for the biochemical identification of an unknown IgE-binding protein. RESULTS: Twenty-three percent of 171 tested bakers had specific IgE to alpha-amylase, 8% reacted to glucoamylase, 13% reacted to cellulase, and 11% reacted to xylanase. Xylanase and cellulase preparations, each containing at least 6 different proteins, showed cross-reactivity in the range of 80%. The main IgE-binding protein in the xylanase preparation recognized in 7 of 8 xylanase-positive subjects was a protein of about 105 kd. This protein was identified as beta-xylosidase by peptide mass spectrometric fingerprinting. The identification was confirmed by matching 12 peptide sequences obtained by N-terminal and mass spectrometric sequencing to this protein. CONCLUSIONS: Beta-Xylosidase from Aspergillus niger is an occupational allergen present in currently used baking additives, which causes sensitization in at least 4% of symptomatic bakers. According to the International Union of Immunological Societies nomenclature, we suggest the term Asp n 14 for this allergen.

Adult↗

PubMed1979

Studies on the specificity of preribosomal RNA transcription in nucleoli after selective deproteinization.

Fidelity of preribosomal RNA transcription in vitro was studied after selective deproteinization of nucleoli using either sequential salt extraction or sodium deoxycholate treatment. Homochromatography fingerprinting and identification of marker oligonucleotides from a T1 ribonuclease digest of the transcripts were used to evaluate the RNA products. These studies indicated that: (1) nucleoli retained their endogenous RNA polymerase I activity and the specificity of transcription up to 0.6 M NaCl extraction; (2) exogenous RNA polymerase I transcribed nucleolar chromatin only after 1.0 M NaCl extraction and the transcription pattern, like that of totally deproteinized DNA, was completely random; (3) extraction of nucleoli with deoxycholate resulted in a DNP complex in which the endogenous RNA polymerase I transcribed pre-rRNA specifically; however, it also initiated random transcription, producing a "mixed" fingerprint pattern on the homochromatogram. The random transcription was selectively inhibited either by deoxycholate or rifampicin AF/013. These studies indicate that the selectivity of pre-rRNA transcription is due both to the endogenous RNA polymerase I molecules that were involved in transcription in vivo and are tightly bound to the template and to factors in intact nucleoli which prevent random transcription by the released RNA polymerase I molecules.

Animals↗

PubMed1988

Radiologic evaluation of mass casualty victims: lessons from the Gander, Newfoundland, accident.

This study describes the use of radiologic methods in the identification of 256 bodies after the crash of an airliner in Gander, Newfoundland. Two hundred thirty-one (90%) of the victims were identified positively with dental and/or fingerprint comparisons. Radiologic data confirmed identification in 29 of these victims. Seventeen bodies without dental or fingerprint identification were presumptively identified with a variety of data, which included radiologic characteristics in four cases. Eight bodies were identified with an exclusion matrix. Radiologic input was critical in two of these. The procedures described provide practical information for radiologists in a mass casualty disaster investigation.

Accidents, Aviation↗

PubMed2004

[Study on the fingerprints of Belamcanda chinensis (L.) DC by capillary electrophoresis].

A capillary electrophoresis fingerprints (CEFP) method was established to evaluate the quality of Belamcanda chinensis (L.) DC. The background electrolyte (BGE) was a 80 mmol/L boric acid containing 15 mmol/L borate solution (adjusted pH 9.7 with KOH solution). The detection wavelength was 228 nm and 12.1 kV was applied. The Belamcanda chinensis (L.) DC was extracted by water to obtain a CEFP containing 21 peaks. The CEFP has good precision, reproducibility and stability with the relative standard deviations (RSDs) of the relative migration time less than 3.5% and the RSDs of the relative peak area about 5.0%. There were good similarities between the standard CEFP and each CEFP of samples collected from ten different places. The coefficients were from 0.913 to 0.993. Meanwhile, fundamental studies related to the methods in peak identification of fingerprints and content evaluation of different sample constituents were carried out.

Boric Acids↗

PubMedDate not supplied

Comparison of genomic profiles of Escherichia coli isolates from urinary tract infections.

Formally included in the larger category of extraintestinal pathogenic Escherichia coli (ExPEC), the uropathogenic E. coli remains the most frequent cause of urinary tract infection (UTI), an important endemic health problem. The genomic DNA of E. coli urinary isolates from adults diagnosed with urinary tract infections and of E. coli fecal isolates from healthy subjects was analysed by PCR for the presence of virulence factor encoding genes pap, sfa/foc, afa, hly and cnf and by field inversion gel electrophoresis (FIGE) fingerprinting of XbaI DNA macrorestriction fragments. The aim was to obtain more detailed microbiological data regarding the community circulating strains in respect of their virulence potential and genetic relatedness. Almost 70% of the urinary strains carried at least one of the target virulence genes, and only 35.5% of the fecal E. coli strains were positive in the PCR screening. Taking into account the virulence genotypes exhibited, a part of the strains isolated from the urinary tract could be defined as belonging to the ExPEC pathotype. A unique FIGE profile was obtained for each of the selected isolates and the dendrogram generated by Taxotron software package analysis suggested a polyclonal population of potential uropathogenic strains clustered into 14 groups of only 60% similarity. For better understanding the epidemiology of UTIs, diseases commonly caused by such a heterogeneous species like E. coli, molecular analysis methods could be essential due to their increased power of identification and fingerprinting.

Adult↗

PubMed2001

The proteome of maize leaves: use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints.

As a first step in establishing a proteome database for maize, we have embarked on the identification of the leaf proteins resolved on two-dimensional (2-D) gels. We detected nearly 900 spots on the gels with a pH 4-7 gradient and over 200 spots on the gels with a pH 6-11 gradient when the proteins were visualized with colloidal Coomassie blue. Peptide mass fingerprints for 300 protein spots were obtained with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer and 149 protein spots were identified using the protein databases. We also searched the pdbEST databases to identify the leaf proteins and verified 66% of the protein spots that had been identified using the protein databases. Sixty-seven additional protein spots were identified from expressed sequence tags (ESTs). Many abundant leaf proteins are present in multiple spots. Functions of over 50% of the abundant leaf proteins are either unknown or hypothetical. Our results show that EST databases in conjunction with peptide mass fingerprints can be used for identifying proteins from organisms with incomplete genome sequence information.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2003

Identification of disease- and nutrient-related metabolic fingerprints in osteoarthritic Guinea pigs.

Osteoarthritis (OA), one of the most common diseases among the elderly, is characterized by the progressive destruction of joint tissues. Its etiology is largely unclear and no effective disease-modifying treatment is currently available. Metabolic fingerprinting provides a novel tool for the identification of biomarkers. A metabolic fingerprint consists of a typical combination of metabolites in a biological fluid and is identified by a combination of (1)H NMR spectroscopy and multivariate data analysis (MVDA). The current feasibility study was aimed at identifying a metabolic fingerprint for OA and applying this in a nutritional intervention study. Urine samples were collected from osteoarthritic male Hartley guinea pigs (n = 44) at 10 and 12 mo of age, treated from 4 mo onward with variable vitamin C doses (2.5-3, 30 and 150 mg/d) and from healthy male Strain 13 guinea pigs (n = 8) at 12 mo of age, treated with 30 mg vitamin C/d. NMR measurements were performed on all urine samples. Subsequently, MVDA was carried out on the data obtained using NMR. An NMR fingerprint was identified that reflected the osteoarthritic changes in guinea pigs. The metabolites that comprised the fingerprint indicate that energy and purine metabolism are of major importance in OA. Metabolic fingerprinting also allowed detection of differences in OA-specific metabolites induced by different dietary vitamin C intakes. This study demonstrates the feasibility of metabolic fingerprinting to identify disease-specific profiles of urinary metabolites. NMR fingerprinting is a promising means of identifying new disease markers and of gaining fresh insights into the pathophysiology of disease.

Animal Nutritional Physiological Phenomena↗

PubMedDate not supplied

Use of phenotypic and genotypic fingerprinting methods in the strain identification of Aspergillus fumigatus.

Aspergillus fumigatus isolates (n = 6) from a lung transplant recipient, one isolate from a patient who had been on the same ward and a reference strain (NCPF 2140) were compared using three typing methods: SDS-PAGE, immunoblotting with serum from the transplant patient and random amplified polymorphic DNA (RAPD) assay. Neither the SDS-PAGE, immunoblot nor RAPD assay with single primers revealed differences between the eight isolates. Digestion of one primer product with the endonuclease EcoRI discriminated between the six patient isolates and the other two strains. The RAPD assay using pairwise combined primers showed identical patterns for the patient's strains but differentiated between the two other strains. It is concluded that any single technique may fail to detect strain differences and that a spectrum of typing methods is necessary in order to reveal or to exclude cross-infections with Aspergillus fumigatus.

Aspergillus fumigatus↗

PubMed1995

Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting.

A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3' terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3'-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially in murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes.

Animals↗

PubMed2002

Peptide mass fingerprinting peak intensity prediction: extracting knowledge from spectra.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry has become a valuable tool in proteomics. With the increasing acquisition rate of mass spectrometers, one of the major issues is the development of accurate, efficient and automatic peptide mass fingerprinting (PMF) identification tools. Current tools are mostly based on counting the number of experimental peptide masses matching with theoretical masses. Almost all of them use additional criteria such as isoelectric point, molecular weight, PTMs, taxonomy or enzymatic cleavage rules to enhance prediction performance. However, these identification tools seldom use peak intensities as parameter as there is currently no model predicting the intensities based on the physicochemical properties of peptides. In this work, we used standard datamining methods such as classification and regression methods to find correlations between peak intensities and the properties of the peptides composing a PMF spectrum. These methods were applied on a dataset comprising a series of PMF experiments involving 157 proteins. We found that the C4.5 method gave the more informative results for the classification task (prediction of the presence or absence of a peptide in a spectra) and M5' for the regression methods (prediction of the normalized intensity of a peptide peak). The C4.5 result correctly classified 88% of the theoretical peaks; whereas the M5' peak intensities had a correlation coefficient of 0.6743 with the experimental peak intensities. These methods enabled us to obtain decision and model trees that can be directly used for prediction and identification of PMF results. The work performed permitted to lay the foundations of a method to analyze factors influencing the peak intensity of PMF spectra. A simple extension of this analysis could lead to improve the accuracy of the results by using a larger dataset. Additional peptide characteristics or even PMF experimental parameters can also be taken into account in the datamining process to analyze their influence on the peak intensity. Furthermore, this datamining approach can certainly be extended to the tandem mass spectrometry domain or other mass spectrometry derived methods.

Acrylamide↗

PubMed1981

Polymorphic variants in human chromosome 15.

We found eight polymorphic variants in human chromosome 15 using Q, C, Q-C and Ag-NOR staining methods. These variants included brightly or dully fluorescent pericentric segments and satellites, giant satellites, increased amounts of short arm hetrochromatin (ph+) and darkly (C band-positive) or lightly (C band-negative) Giemsa-stained pericentric Q-negative segments. These staining properties indicated that the entire short arm of 15 contained at least four distinct chromatin segments: Q-negative centromeric heterochromatin, a Q-variable distal segment, a Q-negative satellite stalk, and Q-variable satellites, in that order, from proximal to distal ends. The BrdU-Hoechst 33258-stained R bands (RBH) and high resolution G subbands were also studied for karyologic characterization of chromosome 15. Most of these variants were reported also in 13, but insufficiently documented in other D and G chromosomes. Together with polymorpic pericentric fluorochromes seen in 3 and 4, Yq, and nonpathogenic t(D;Yq), the pattern of these variants can be used as karyologic fingerprints for identification of each individual and his or her cell explants both in vivo and in vitro.

Azure Stains↗

PubMed2005

Fast fingerprinting by MALDI-TOF mass spectrometry of urinary sediment glycosphingolipids in Fabry disease.

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid (GSL) metabolism, caused by a deficiency of the lysosomal alpha-galactosidase A, which results in high levels in lysosomes and biological fluids of globotriaosylceramide (Gb3) and digalactosylceramide (Ga2), also known as galabiosylceramide. We report here a detailed study of the molecular species of GSLs in urinary samples obtained from hemizygous and heterozygous patients by use of matrix-assisted laser desorption ionisation and tandem mass spectrometry (MALDI-MS-MS). Twenty-two and fifteen molecular species were identified in the globotriaosylceramide and digalabiosylceramide series, respectively. The major sphingoid base was sphingosine (d18:1), and dihydrosphingosine (C18:0) and sphingadienine (d18:2) were also present. The molecular profiles obtained by MALDI-TOF-MS enabled us to show significant differences between GSLs composition for young, adult or atypic hemizygote and heterozygote patients. Thus, MALDI-TOF-MS and MS-MS proved a powerful tool for screening a population of patients with clinical signs suggestive of FD by direct and rapid GSL fingerprinting and identification, and for study of the biological processes occurring in glycosphingolipid accumulation.

Adult↗

PubMed2006

Capillary electrophoresis fingerprinting, quantification and mass-identification of various 9-aminopyrene-1,4,6-trisulfonate-derivatized oligomers derived from plant polysaccharides.

Various plant polysaccharide derived mono- and oligosaccharides were derivatized with the fluorescent 9-aminopyrene-1,4,6-trisulfonate (APTS) and subjected to capillary electrophoresis (CE) in combination with laser induced fluorescence (LIF) detection. CE-LIF was suitable for mol-based quantification of various APTS-monosaccharides. CE-LIF of APTS-oligosaccharides showed high resolutions, while analysis times were at maximum 15 min. The coupling of CE to electrospray-iontrap mass spectrometery (MS) with online UV detection showed to be a powerful technique in the identification of APTS-oligosaccharides. For the first time, various APTS-xylo-oligosaccharides, having either no, O-acetyl, arabinosyl or xylosyl substitutions at varying positions, were identified by using CE-LIF and CE-MS(n).

Arabinose↗

PubMed2005

Proteomics analysis of human astrocytes expressing the HIV protein Tat.

Astrocyte infection in HIV has been associated with rapid progression of dementia in a subset of HIV/AIDS patients. Astrogliosis and microglial activation are observed in areas of axonal and dendritic damage in HIVD. In HIV-infected astrocytes, the regulatory gene tat is over expressed and mRNA levels for Tat are elevated in brain extracts from individuals with HIV-1 dementia. Tat can be detected in HIV-infected astrocytes in vivo. The HIV-1 protein Tat transactivates viral and cellular gene expression, is actively secreted mainly from astrocytes, microglia and macrophages, into the extracellular environment, and is taken up by neighboring uninfected cells such as neurons. The HIV-1 protein Tat released from astrocytes reportedly produces trimming of neurites, mitochondrial dysfunction and cell death in neurons, while protecting its host, the astrocyte. We utilized proteomics to investigate protein expression changes in human astrocytes intracellularly expressing Tat (SVGA-Tat). By coupling 2D fingerprinting and identification of proteins by mass spectrometry, we identified phosphatase 2A, isocitrate dehydrogenase, nuclear ribonucleoprotein A1, Rho GDP dissociation inhibitor alpha, beta-tubulin, crocalbin like protein/calumenin, and vimentin/alpha-tubulin to have decreased protein expression levels in SVGA-Tat cells compared to the SVGA-pcDNA cells. Heat shock protein 70, heme oxygenase-1, and inducible nitric oxide synthase were found to have increased protein expression in SVGA-Tat cells compared to controls by slotblot technique. These findings are discussed with reference to astrocytes serving as a reservoir for the HIV virus and how Tat promotes survival of the astrocytic host.

Analysis of Variance↗

PubMed2005

Proteomic analysis of oxidatively modified proteins induced by the mitochondrial toxin 3-nitropropionic acid in human astrocytes expressing the HIV protein tat.

The human immunodeficiency virus (HIV)-Tat protein has been implicated in the neuropathogenesis of HIV infection. However, its role in modulating astroglial function is poorly understood. Astrocyte infection with HIV has been associated with rapid progression of dementia. Intracellularly expressed Tat is not toxic to astrocytes. In fact, intracellularly expressed Tat offers protection against oxidative stress-related toxins such as the mitochondrial toxin 3-nitroproprionic acid (3-NP). In the current study, human astrocytes expressing Tat (SVGA-Tat) and vector controls (SVGA-pcDNA) were each treated with the irreversible mitochondrial complex II inhibitor 3-NP. Proteomics analysis was utilized to identify changes in protein expression levels. By coupling 2D fingerprinting and identification of proteins by mass spectrometry, actin, heat shock protein 90, and mitochondrial single-stranded DNA binding protein were identified as proteins with increased expression, while lactate dehydrogenase had decreased protein expression levels in SVGA-Tat cells treated with 3-NP compared to SVGA-pcDNA cells treated with 3-NP. Oxidative damage can lead to several events including loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, ultimately leading to neuronal death. Identification of specific proteins protected from oxidation is a crucial step in understanding the interaction of Tat with astrocytes. In the current study, proteomics also was used to identify proteins that were specifically oxidized in SVGA-pcDNA cells treated with 3-NP compared to SVGA-Tat cells treated with 3-NP. We found beta-actin, calreticulin precursor protein, and synovial sarcoma X breakpoint 5 isoform A to have increased oxidation in control SVGA-pcDNA cells treated with 3-NP compared to SVGA-Tat cells treated with 3-NP. These results are discussed with reference to potential involvement of these proteins in HIV dementia and protection of astrocytes against oxidative stress by the HIV virus, a prerequisite for survival of a viral host cell.

Astrocytes↗

PubMed2002

Intact cell mass spectrometry (ICMS) used to type methicillin-resistant Staphylococcus aureus: media effects and inter-laboratory reproducibility.

Intact cell mass spectrometry (ICMS) rapidly analyses the surface composition of microorganisms providing rapid, discriminatory fingerprints for identification and subtyping of important nosocomial pathogens such as methicillin resistant Staphylocccus aureus (MRSA). In this study, ICMS using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI TOF/MS) was assessed for the identification and subtyping of MRSA. An intra- and inter-laboratory reproducibility study was carried out and the effects of culture media (an important source of variation for ICMS) were also studied. Several media used for the cultural identification of MRSA were examined using a panel of well-characterised staphylococcal isolates (n=26). Six MRSA isolates were analysed over a 1-month period for intra-laboratory reproducibility on the same instrument and three different culture media. Spectra were consistent for each isolate between the four experiments on the same culture medium. Individual isolates produced different spectral profiles on different culture media. Spectra from organisms grown on Columbia blood agar contained more peaks (approximately 120) compared to Columbia agar (approximately 50) and methicillin mannitol salt agar (approximately 25). All 26 staphylococcal isolates were subjected to an inter-laboratory study on two MALDI instruments. For each isolate, the overall spectral profile was the same for each of the two instruments but the baseline threshold values was adjusted due to instrument differences in detector sensitivities. Differences between certain regions of the spectra reproducibly identified isolates belonging to the two major MRSA strains (EMRSA phage group 15 and 16). These results demonstrate ICMS with appropriate media selection is a rapid and reproducible technique for identification and discrimination of MRSA.

Culture Media↗