Does MS have a future after the Nobel Prize?
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Magnetic Resonance Spectroscopy↗
Biomedical subjects
Catherine Fenselau
Publications and source records attributed to Catherine Fenselau.
Mass spectrometry-based proteolytic mapping for rapid virus identification.
A novel method is proposed for rapid identification of viruses and other organisms that show a low number of biomarkers, based on the construction of databases of organism-specific tryptic peptide masses. The peptide products of any protease that cuts at specific residues can be accommodated. Experimentally, a sample of intact virus, e.g., one collected from the atmosphere, is digested with a selective protease for a short time, and the digestion products are analyzed by MALDI-TOF mass spectrometry without fractionation or purification. In the present proof of concept, the Sindbis virus AR 339 was identified by using the masses of observed tryptic peptide products to query a database composed of tryptic peptide masses generated in silico for six viruses whose genomes have been sequenced. Two algorithms were tested for identification--a direct score-ranking algorithm and an algorithm that evaluates the probability of random matching. The Sindbis virus was unambiguously identified by either approach. The influence of factors such as experimental mass accuracy, number of missed cleavages, and database size on the identification algorithms has also been evaluated, with the objective of extending the approach to other microorganisms.
Databases, Factual↗
Metallothionein dimers studied by nano-spray mass spectrometry.
Both transient and stable dimers of metallothionein have been characterized, based on earlier studies using NMR, circular dichroism and size-exclusion chromatography. Here additional characterization is provided by nanospray mass spectrometry. Rapid redistribution of metal ions between monomeric Cd7- and Zn7-metallothionein 2a is monitored by nanospray. An experiment in which theses two forms of the monomeric protein are separated by a dialysis membrane, which will pass metal ions but not proteins, confirms that a transient dimer must form for metal ions to be redistributed. On the other hand, size-exclusion chromatography of reconstituted Zn7- or Cd7-metallothionein revealed the presence of monomeric and dimeric species. These dimers do not equilibrate readily to form monomers and they are shown to be covalent.
Animals↗
Structural characterization of lipopeptide biomarkers isolated from Bacillus globigii.
Spectra obtained using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of Bacillus globigii (Bacillus subtilis niger) spores, vegetative cells and the culture supernatant show a cluster of biomarkers centered at a molecular mass of 1478 Da. Three biomarkers were isolated from the cell-free culture supernatant by solid-phase extraction and reversed-phase high-performance liquid chromatography, and characterized using various kinds of mass spectrometry. A Fourier transform mass spectrometer with a MALDI source was used to determine the monoisotopic protonated masses at 1463.8, 1477.8, and 1505.8 Da in order of elution. The mass differences of 14 and 28 Da suggest that they are homologous molecules. Alkaline hydrolysis of each species showed that it contained a lactone linkage. Strong acid hydrolysis released a fatty acid from an amide bond, consistent with a lipopeptide. A quadrupole time-of-flight instrument with a nanospray source was used to sequence the hydrolyzed forms of the three biomarkers. The cyclic lipopeptides were found to have amino acid sequences identical with those in fengycins and plipastatins, antimicrobial compounds with phospholipase inhibitor activity, previously identified in related species of Bacillus subtilis and Bacillus cereus.
Bacillus↗
Qualitative characterization of biomolecular zinc complexes by collisionally induced dissociation.
Nanospray and collisionally induced dissociation (CID) on a quadrupole/time-of-flight mass spectrometer were used to examine the complexes formed between the zinc ion binding protein metallothionein and a series of peptides related to glutathione. The objective of the study was to determine if CID could be used to distinguish complexes that are stabilized by co-chelation of a zinc ion from non-covalent complexes that were formed in some other way. Differences in the collision energy required for dissociation and, more importantly, differences in the distribution of zinc ions between the pairs of dissociation products suggest that mass spectrometry can provide qualitative information about the bimolecular chelation of metal ions. The potential application to zinc chelates is particularly important, since biological chelates do not provide signals directly detectable by NMR, Mössbauer or other spectroscopies. The observations reported here also allowed a molecular mechanism to be proposed to explain the differences observed by others in the physiological interactions of reduced and oxidized glutathione with metallothionein.
Animals↗
Rapid microorganism identification with on-slide proteolytic digestion followed by matrix-assisted laser desorption/ionization tandem mass spectrometry and database searching.
A simple method for microorganism identification is proposed in this paper. In this method, no isolation or fractionation of microorganisms and no special search algorithm or database are needed. Experimentally, a sample of the unfractionated intact microorganism is subjected to brief on-slide proteolytic digestion and the digestion product ions detected in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) are selected for tandem mass spectrometry (MS/MS). Database searching using the MS/MS spectrum identifies the microorganism protein, and thereby, according to the source of the protein, the microorganism. The method was demonstrated to be feasible with enterobacteriophage MS2 as the model microorganism. The novel method is simple, rapid, and requires a small quantity of sample.
Amino Acid Sequence↗
Proteolytic 18O labeling for comparative proteomics: evaluation of endoprotease Glu-C as the catalytic agent.
Recently, proteolytic 18O labeling has been demonstrated as a promising strategy for comparative proteomic studies (Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-42). In this approach, protein mixtures are digested in parallel in H216O and H218O and the ratios of isotopically distinct peptide products are measured by mass spectrometry. In the initial report from this laboratory, trypsin was shown to catalyze incorporation of two 18O atoms into the carboxyl terminus of each new peptide formed by cleavage of the adenovirus proteome. In the present study, a second enzyme, endoprotease Glu-C, is evaluated as an agent for cleavage and labeling. Proteolytic 18O labeling by Glu-C is shown to occur readily with phosphorylated and glycosylated proteins and with cysteinealkylated and disulfide-linked proteins. A sequential double-labeling strategy is used to characterize N-linked glycopeptides. Labeled and unlabeled peptide pairs are found to coelute chromatographically, and measurements of isotope ratios by nanospray and capillary LC-MS are found to be accurate and precise.
Adenoviridae↗
Differential protein expression in the cytosol fraction of an MCF-7 breast cancer cell line selected for resistance toward melphalan.
Analysis of differential protein expression in the cytosol of melphalan-resistant and -susceptible MCF-7 cell lines has been carried out using a combination of two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics. Comparison of multiple digitized gel arrays detected several spots as candidates for differentially expressed proteins in melphalan-resistant MCF-7 cells. The up-regulated proteins included retinoic acid binding protein II, an isoform of the macrophage migration inhibition factor, and other unidentified proteins. The down-regulated proteins included calreticulin, cyclophin A, and an isoform of the 27 kD heat shock protein. Correlation of the differential expression of some of the proteins with acquired resistance of MCF7 cells to melphalan is discussed.
Antineoplastic Agents, Alkylating↗
Dissection of proteolytic 18O labeling: endoprotease-catalyzed 16O-to-18O exchange of truncated peptide substrates.
Proteolytic labeling in H2(18)O has been recently revived as a versatile method for proteomics research. To understand the molecular basis of the labeling process, we have dissected the process into two separate events: cleavage of the peptide amide bonds and exchange of the terminal carboxyl oxygens. It was demonstrated that both carboxyl oxygens can be catalytically labeled, independent of the cleavage step. Reaction kinetics of the tryptic 16O-to-18O exchange of YGGFMR, YGGFMK, and the tryptic digest of apomyoglobin were studied by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. A larger KM for the Lys-peptide (4400 +/- 700 microM), when compared to that of the Arg-peptide (KM 1300 +/- 300 microM), was mainly responsible for the slower reaction with YGGFMK (kcat/KM 0.64 +/- 0.14 microM(-1)min(-1)) compared to YGGFMR (kcat/KM 2.6 +/- 0.9 microM(-1)min(-1)). Multiplexed kinetic studies showed that endoprotease-catalyzed oxygen exchange is a general phenomenon, allowing homogeneous 18O2-coding of a variety of peptides. It was demonstrated for the first time that chymotrypsin 18O2-codes peptides during proteolysis. On the basis of the analyses reported here, we propose that proteolytic 18O labeling can be advantageously decoupled from protein digestion, and endoproteases can be used in a separate step to 18O2-code peptides for comparative studies after proteolysis has taken place.
Animals↗
Highly altered protein expression profile in the adriamycin resistant MCF-7 cell line.
The protein expression pattern in the cytosol fraction of the adriamycin resistant MCF-7 cell line (MCF-7/ADR) was compared to that of the parental MCF-7 cell line using two-dimensional gel electrophoresis and mass spectrometry. Twenty proteins with altered abundances were identified and studied in MCF-7/ADR. Both up regulation and down regulation are characterized. The most striking differences were found for proteins that were uniquely expressed in this cell line and not detectable in the parental MCF-7 cell line. These proteins include annexin I, the neuronal ubiquitin carboxyl hydrolase isoenzyme L-1 (also known as PGP9.5), glutathione-S-transferase pi class, nicotinamide N-methyltransferase, and interleukin-18 precursor. On the other hand, catechol-O-methyltransferase was expressed in the parental cell line, but was not detected in the adriamycin resistant cell line. This protein expression pattern was unique to MCF-7/ADR and not observed in MCF-7 cell lines selected for resistant to etoposide, mitoxantrone or melphalan.
Breast Neoplasms↗
Investigation of doxorubicin resistance in MCF-7 breast cancer cells using shot-gun comparative proteomics with proteolytic 18O labeling.
A shot-gun comparative proteomic investigation utilizing proteolytic 18O labeling has been carried out on a drug susceptible MCF-7 human breast cancer cell line and a related cell line that is resistant to doxorubicin. The proteolytic 18O labeling method has been further refined and optimized for application to a protein fraction stemming from the cytosol of the breast cancer cells. The comparative investigation revealed several proteins with altered expression levels in the doxorubicin resistant line. These altered proteins are considered for a possible role in doxorubicin resistance.
Breast Neoplasms↗
Integration of Jacobson's pellicle method into proteomic strategies for plasma membrane proteins.
A modified form of the cationic colloidal silica technique for plasma membrane isolation has been combined with SDS-PAGE, mass spectrometry, and bioinformatics for evaluation as a proteomics strategy with human multiple myeloma cells and human breast cancer cells. On the basis of Western blots, half of the protein isolated is estimated to come from the plasma membrane. Forty-three percent of the 366 proteins identified by mass spectrometry had been previously classified as plasma membrane proteins. Thirty proteins previously categorized as hypothetical membrane proteins are now reported to be expressed.
Breast Neoplasms↗
Evaluation of metabolic labeling for comparative proteomics in breast cancer cells.
Protein expression patterns in the cytosol of MCF-7 cells resistant to adriamycin and to adriamycin/verapamil were compared to that of the parental MCF-7 cell line and to each other using metabolic labeling and two-dimensional gel electrophoresis. Growing the parental MCF-7 cell line in 13C6-arginine- and 13C6-lysine-enriched medium resulted in C-terminal labeling of all tryptic peptides. The culture media was optimized for the incorporation of these labeled amino acids under conditions that also supported cell growth. Protein abundances were found to be distinctive in MCF-7 cells resistant to adriamycin and those selected for resistance to both adriamycin and verapamil.
Annexins↗
Proteomic evidence for roles for nucleolin and poly[ADP-ribosyl] transferase in drug resistance.
One-hundred twenty-four proteins have been identified in the soluble nuclear protein mixture from MCF-7 human breast cancer cells, of which more than 90% are classically categorized as nuclear proteins. Proteins were also studied from three drug resistant MDF-7 lines, selected previously from the same parent line by exposure to etoposide, to mitoxantrone, or to adriamycin in the presence of verapamil. Both quantitative gel comparisons and stable isotope labeling were used to identify a total of fourteen proteins whose abundances are altered by more than 2-fold in the three resistant lines. Several cytoskeleton proteins, cytokeratin 8, cytokeratin 19, septin 2, and alpha tropomyosin, are decreased in common across the three resistant cell lines. PARP-l (poly[ADP-ribosyl]transefrase or connexion) is found to be less abundant in all three resistant lines. Nucleolin is more abundant in lines resistant to etoposide and mitoxantrone, while the mitotic checkpoint protein BUB 3 is more abundant in the line resistant to adriamycin/verapamil.
Antibiotics, Antineoplastic↗
Solution isoelectric focusing for peptide analysis: comparative investigation of an insoluble nuclear protein fraction.
In this study, a solution isoelectric focusing apparatus was modified and built into a two-dimensional separation method for peptides. Newly commercialized isoelectric membranes, which carry immobilized ampholytes, were integrated to establish the pH boundaries in this apparatus. High-performance liquid chromatography was employed as the second dimension, interfaced with mass spectrometry. An insoluble nuclear protein fraction was used to evaluate and optimize this method. This two-dimensional separation method dramatically improves peptide detection and identification compared with a single dimension LC-MS analysis. Off-line reversed-phase HPLC was used to ascertain reproducibility. The two-dimensional separation method was combined with (18)O labeling for comparative analysis of protein expression in two cell lines. Separation of peptides by solution isoelectric focusing (sIEF) offers the advantage that it can be accomplished after the (18)O labels are introduced. The labeled peptides can be mixed with unlabeled ones before fractionation by sIEF. The relative abundances of nuclear proteins from a drug resistant MCF-7 cancer cell line were compared to those from the drug susceptible parent cell line using this combined strategy. The abundances of several heterogeneous nuclear ribonucleoproteins were found to be increased in the mitoxantrone-resistant line.
Cell Line, Tumor↗
Unbiased examination of changes in plasma membrane proteins in drug resistant cancer cells.
In this study, an unbiased examination is made of the abundance changes between proteins found in the basolateral plasma membranes of a drug susceptible parental MCF-7 breast cancer cell line and a cell line selected from the parent line for resistance to the anticancer drug mitoxantrone. Plasma membrane proteins were differentially labeled metabolically, enriched using the colloidal silica pellicle method, and characterized by tandem mass spectrometry. Fifteen proteins were identified with significant (>2) changes, including receptors, adhesion proteins, proteins involved in amino acid uptake, and proteins involved in glucose uptake. From 40 mug of membrane proteins, 3227 unique peptides and 540 proteins were identified.
Antineoplastic Agents↗