Biomedical subjects

Catherine Fenselau

Publications and source records attributed to Catherine Fenselau.

At least 19 recordsLinked to original sources

PubMed2007

Analysis of a model virus using residue-specific chemical cleavage and MALDI-TOF mass spectrometry.

A nonenzymatic proteomics strategy is applied to the rapid identification of viruses. The approach provides solubilization and subsequent digestion of viral coat proteins in under 30 s. Acid digestions were carried out using a laboratory-quality microwave system equipped with temperature, pressure, and power controls, which allowed for precise optimization of experimental parameters. Under optimal conditions, this method provides an efficient alternative to traditional enzymatic digestion-based methods for virus identification. Following rapid microwave heating of a suspension of a model virus, RNA bacteriophage MS2, 13 chemical digestion products were detected in parallel with the coat protein precursor using MALDI-TOF MS. Because of the high sequence coverage obtained, the bacteriophage MS2 coat protein was identified with high confidence and the specificity of the identification allowed for the discrimination between bacteriophage MS2 and other closely related RNA bacteriophages.

Amino Acid Sequence↗

PubMed2006

A review of quantitative methods for proteomic studies.

An overview is provided of six strategies for relative or absolute quantitation of protein abundances that are widely used in proteomic studies. Strengths and limitations are discussed. Four of these involve stable isotope labeling and isotope ratio measurements by mass spectrometry. In another, mass spectra are used to deconvolute overlapping peptide HPLC peaks to provide relative quantitation based on peak areas. The sixth provides relative abundances of proteins based on 2-D gel arrays. It should be noted that these strategies measure peptide and protein abundances, and cannot directly assess changes in regulation or expression.

Amino Acid Sequence↗

PubMed2006

Rapid chemical digestion of small acid-soluble spore proteins for analysis of Bacillus spores.

A method for the rapid identification of Bacillus spores is proposed, based on the selective release and chemical digestion of small, acid-soluble spore proteins (SASPs). Microwave-assisted acid hydrolysis of SASPs from B. anthracis str. Sterne and B. subtilis str. 168 was accomplished in a single step requiring only 90 s of heating. The peptide products of the chemical digestion were identified by postsource decay sequencing with a MALDI-TOF-MS equipped with a curved-field reflectron. The specificity of the observed SASP peptides was evaluated using a cross-species sequence search. The incomplete nature of the acid digestion under these conditions allowed detection of the digest products along with the proteins from which they originated, which increased species identification confidence. The feasibility of this approach for the rapid identification of Bacillus species was further demonstrated by analyzing a mixture of B. subtilis str. 168 and B. anthracis str. Sterne spores.

Amino Acid Sequence↗

PubMed2006

Alterations in the mitochondrial proteome of adriamycin resistant MCF-7 breast cancer cells.

A 2D-gel based comparative proteomic analysis has been conducted of changes in mitochondrial protein abundances in MCF-7 human breast cancer cells selected for resistance to Adriamycin accompanied by verapamil. We identified and compared 156 unique proteins from 184 spots. Eleven mitochondrial proteins were found with abundances altered more than 2-fold. Transcription was evaluated for two of these, using quantitative RT-PCR. Implications of the changes are considered with respect to drug resistance.

Breast Neoplasms↗

PubMed2005

Characterization of Enterobacteria using MALDI-TOF mass spectrometry.

A method is proposed for the rapid classification of Gram-negative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order.

Amino Acid Sequence↗

PubMed2005

Rapid characterization of Bacillus spores targeting species-unique peptides produced with an atmospheric pressure matrix-assisted laser desorption/ionization source.

New and improved strategies are eagerly sought for the rapid identification of microorganisms, particularly in mixtures. Mass spectrometry remains a powerful tool for this purpose. Small acid-soluble proteins (SASPs), which are relatively abundant in Bacillus spores, represent potential biomarkers for species characterization. Despite sharing extensive sequence homology, these proteins differ sufficiently in sequence for discrimination between species. This work focuses on the differences in sequence between SASPs from various Bacillus species. Compilation of SASP sequences from protein database searches, followed by in silico trypsin digestion and analysis of the resulting fragments, identified several species-specific peptides that could be targeted for analysis using mass spectrometry. This strategy was tested and found to be successful in the characterization of Bacillus spores both from individual species and in mixtures. Analysis was performed using an ion trap mass spectrometer with an atmospheric pressure MALDI source. This instrumentation offers the advantage of increased speed of analysis and accurate precursor ion selection for tandem mass spectrometric analysis compared with vacuum matrix-assisted laser desorption/ionization and time-of-flight instruments. The identification and targeting of species-specific peptides using this type of instrumentation offers a rapid, efficient strategy for the identification of Bacillus spores and can potentially be applied to different microorganisms.

Amino Acid Sequence↗

PubMed2005

Painting canvases.

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Biochemistry↗

PubMed2004

Proteomic phenotyping: metastatic and invasive breast cancer.

The adriamycin resistant breast cancer cell line (MCF-7/ADR) is a subject of ongoing debate concerning its origin and or source. Previous studies in our laboratory showed that MCF-7/ADR has a unique cytosolic protein expression pattern when compared to that of the parental MCF-7 cell line and other drug resistant MCF-7 cell lines. Protein expression patterns obtained using two-dimensional gel electrophoresis and mass spectrometry indicated that this MCF-7/ADR cell line shares some similarities with the metastatic breast cancer cell lines MDA-MB. Further comparisons with available two-dimensional gel electrophoresis maps in the literature indicate that MCF-7/ADR has a protein expression signature even closer to of the ductal infiltrating breast carcinoma cell line 8701. These observations suggest that MCF-7/ADR cells might have originated in a selection of ductal infiltrating carcinoma cells, which were present among the original MCF-7 cell population. These ductal infiltrating carcinoma cells may possess an intrinsic adriamycin resistance phenotype.

Antibiotics, Antineoplastic↗

PubMed2004

Complete sequences of small acid-soluble proteins from Bacillus globigii.

Three abundant small acid-soluble proteins (SASPs) from spores of Bacillus globigii were sequenced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with post-source decay and nanoelectrospray collision-induced dissociation tandem mass spectrometry. The proteins were extracted from spores with 1 M HCl. Scanning electron micrographs of spores before and after acid extraction show that the spores retain their overall structure but have a shriveled texture following the acid treatment. Extracted SASPs were purified by high-performance liquid chromatography and molecular masses of the SASPs were identified at 7068 (SASP-1), 7332 (SASP-2), and 8889 (gamma-SASP). De novo peptide sequencing was used to determine the protein sequences. The correct ordering of peptide sequences was aided by mapping overlapping enzymatic digests and by comparison with homologous SASPs from Bacillus stearothermophilus. B. globigii is used in many field tests as a surrogate for B. anthracis. Thus complete SASP sequences from B. globigii will facilitate the development of methods for rapid identification of bacteria based on mass spectrometry and the examination of taxonomic relationships between Bacillus species.

Acids↗

PubMed2004

A targeted proteomics approach to the rapid identification of bacterial cell mixtures by matrix-assisted laser desorption/ionization mass spectrometry.

A proteomic approach to the rapid identification of bacteria is presented, which relies on the solubilization of a limited number of proteins from intact cells combined with on-probe tryptic digestion. Within 20 min, complete cleavage products of a limited set of bacterial proteins with molecular masses of about 4-125 kDa were obtained by on-probe digestion with immobilized trypsin. Bacterial peptides suitable for unimolecular decomposition analysis were generated within 5 min, and the sequence information obtained allowed identification of abundant proteins, and accordingly, their bacterial sources via searches in the NCBI database. Analysis of fragmentation products was also shown to allow for identification of bacterial peptides identical in mass but differing slightly in amino acid sequence by manual data analysis. In this work, Bacillus subtilis 168, B. globigii, B. sphaericus 14577, B. cereus T, and B. anthracis Sterne were examined, and various cold shock proteins were identified in all species. In addition, DNA-binding, 60 kDa-heat shock, surface-related and other stress-protective proteins were identified in the bacterial cell digests, and species-specific tryptic peptides could be generated from each of the Bacillus species studied. Bacterial peptides could be analyzed with greater sensitivity and mass accuracy than the parent proteins. The applicability of this targeted proteomics approach to the rapid identification of Bacillus species was further established by analyzing binary cell mixtures.

Amino Acid Sequence↗

PubMed2004

Analysis of Bacillus anthracis spores in milk using mass spectrometry.

New approaches for identifying biological threat agents in raw milk using spectroscopy were tested using Bacillus anthracis (BA) Sterne strain spores seeded into unpasteurized bulk tank milk. Direct filtration onto Tyvek membranes provided the optimal filtration approach from raw milk, but detection limits were not ideal. When beads coated with anti-BA antibodies were mixed with spores in raw milk, the beads were capable of concentrating the spores that could be later detected and characterized by MALDI spectroscopy based on presence of previously characterized small acid-soluble proteins (SASP's). This approach could provide a very rapid assessment of whether milk or milk products have been purposefully contaminated with BA spores. This work was fundamentally a proof-of-concept study demonstrating feasibility of the approach in milk. Other parameters could be changed to potentially lower detection limits, and additional studies are currently underway to improve the approach.

Animals↗

PubMed2004

Contact regions in the dimer of Alzheimer beta-amyloid domain [1-28] studied by mass spectrometry.

Information is provided about the amino acid residues in the [1-28] domain of the Alzheimer b- amyloid protein, which participate in interstrand pairing and initiate fibillogenesis. The study was carried out using electrospray ionization on a four sector mass spectrometer, measuring kinetic energy release for a fragmentation process, and modeling the transition state with molecular dynamics calculations. The results eliminate the sequence [11-24] proposed earlier as the central core, and are consistent with, but do not distinguish between, residues [17-28] and [17-23] proposed by others based on biochemical studies.

Alzheimer Disease↗

PubMed2003

Bacillus spore identification via proteolytic peptide mapping with a miniaturized MALDI TOF mass spectrometer.

An approach is tested here as a rapid screening method for Bacillus spore species employing bacterial peptide analysis with a miniaturized MALDI TOF mass spectrometer. A limited set of tryptic peptides was generated in situ following selective solubilization of the small, acid-soluble protein family (SASP) from spore samples on the MALDI sample holder. To facilitate species identification, a compact database was created comprising masses of the tryptic cleavage products generated in silico from all Bacillus and Clostridium SASPs whose sequences are available in public databases. Experimental measurements were matched against the custom-made database, and a published statistical model was then used to evaluate the probability of false identifications.

Bacillus↗

PubMed2003

Characterization of Bacillus spore species and their mixtures using postsource decay with a curved-field reflectron.

A strategy is proposed for the rapid identification of Bacillus spores, which relies on the selective release of a family of proteins, referred to as small, acid-soluble spore proteins (SASPs). In this work, SASPs were selectively solubilized from Bacillus spores on the MALDI sample plate by using 10% TFA. Proteolytic digests of SASPs generated in situ from spores of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki HD-1, B. cereus T, and the nonpathogenic strain B. anthracis Sterne were prepared in 5-25 min by using trypsin immobilized on Agarose beads and subsequently analyzed by MALDI-TOFMS using a curved-field reflectron. Protein identification was obtained by partial sequencing of distinctive tryptic peptides from Bacillus spores via post-source decay analysis combined with genome-based database searches by Mascot Sequence Query. Various unique SASPs were identified, allowing the characterization of Bacillus species by obtaining sequence-specific information on single peptides. The applicability of this approach for the rapid identification of Bacillus species was further established by analyzing spore mixtures.

Amino Acid Sequence↗

PubMed2003

MALDI analysis of Bacilli in spore mixtures by applying a quadrupole ion trap time-of-flight tandem mass spectrometer.

A novel ion trap time-of-flight hybrid mass spectrometer (qIT-TOF MS) has been applied for peptide sequencing in proteolytic digests generated from spore mixtures of Bacilli. The method of on-probe solubilization and in situ proteolytic digestion of small, acid-soluble spore proteins has been recently developed in our laboratory, and microorganism identification in less than 20 min was accomplished. In this study, tryptic peptides were generated in situ from complex spore mixtures of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki, and B. cereus T, respectively. MALDI analysis of bacterial peptides generated was performed with an average mass resolving power of 6200 and a mass accuracy of up to 10 ppm using a trap-TOF tandem configuration. Precursor ions of interest were usually selected and stored in the quadrupole ion trap with their complete isotope distribution by choosing a window of +/- 2 Da. Sequence-specific information on isolated protonated peptides was gained via tandem MS experiments with an average mass resolving power of 4450 for product ion analysis, and protein and bacterial sources were identified by database searching.

Amino Acid Sequence↗

PubMed2003

Fractionation of cytosolic proteins on an immobilized heparin column.

Currently there is great interest in the development of methods to simplify complex protein mixtures for analysis by proteomic strategies. The objective of this study was to develop and evaluate immobilized heparin chromatography to simplify such mixtures and to enrich minor proteins. The method is evaluated with cytosol from human breast cancer MCF-7 cells. This protein mixture was fractionated into three portions and eluted with a stepwise salt gradient. These were characterized by protein analysis, two-dimensional gel electrophoresis, and mass spectrometry, with attention to reproducibility, overlap between fractions, simplification of protein mixtures, and enrichment of low-abundance proteins. It was possible to identify proteins enriched in the fractionated mixtures that were not even detectable in gel arrays of the total cytosol. The method was shown to be suitable for integration with other proteomics strategies.

Animals↗

PubMed2003

Use of bioactive glass slides for matrix-assisted laser desorption/ionization analysis: application to microorganisms.

Glass slides are widely used in high-throughput analysis and are available commercially with surfaces activated, etched, and channeled. Thin glass microscope slides are shown here to be suitable sample supports for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. As a demonstration, lectins immobilized on glass slides with activated surfaces are used to concentrate and purify agglutinated Bacillis spores. It is expected that such slides will provide a rapid, inexpensive way to evaluate and implement new strategies involving MALDI MS readout.

Bacillus cereus↗

PubMed2003

Small, acid-soluble proteins as biomarkers in mass spectrometry analysis of Bacillus spores.

The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different Bacillus spore species was examined. The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol). Both major and minor alpha/beta- and gamma-type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases. For all but 1 pair (B. cereus T and B. thuringiensis subsp. Kurstaki) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry.

Amino Acid Sequence↗