Proteomic strategies for rapid characterization of micro-organisms.

Bioinformatic considerations are offered to illustrate strengths and limitations of the characterization of Bacillus spores based on proteomic interpretation of matrix-assisted laser desorption/ionization spectra. In particular, species-specific biomarkers are evaluated in the context of both experimental access and uniqueness in silico.

Characterization of protein biomarkers desorbed by MALDI from whole fungal cells.

In this publication, the use of ultraviolet (UV) matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS) for rapid identification and characterization of Saccharomyces cerevisiae, a fungus, is reported. S. cerevisiae is a unicellular eukaroyte that can serve as a model to study more complex organisms. We have determined that the best technique for cell wall lyses for MALDI involves the use of high concentrations of formic acid solutions. We also have shown that different fungal species exhibit different mass spectra, which can be used to distinguish them readily. Protein peaks from S. cerevisiae spectra have been tentatively identified using bioinformatics and are mainly assigned to ribosomal and mitochondrion-related proteins.

Factors contributing to peak broadening and mass accuracy in the characterization of intact spores using matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry.

Factors contributing to peak broadening, accuracy and precision in mass assignment in the matrix-assisted laser desorption/ionization characterization of a lipopeptide desorbed from intact Bacillus spores were investigated. These spores were studied as an example of a thick, topologically irregular sample, which present a more difficult target than a pure peptide or protein. The type of matrix, matrix:sample ratio, laser fluence, and localized repetitive laser irradiation were all found to affect the full-width at half maximum of the biomarker. Both in-source and post-source phenomena were shown to contribute. Sample thickness had less effect. Precision and accuracy of mass assignment were also affected by matrix:sample ratio and laser fluence. In general, this sample was responsive to the same experimental variables as pure peptides, and the use of an internal standard produced significant improvements in precision and accuracy.

Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus.

A new method for proteolytic stable isotope labeling is introduced to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools or their subfractions. Two 18O atoms are incorporated universally into the carboxyl termini of all tryptic peptides during the proteolytic cleavage of all proteins in the first pool. Proteins in the second pool are cleaved analogously with the carboxyl termini of the resulting peptides containing two 16O atoms (i.e., no labeling). The two peptide mixtures are pooled for fractionation and separation, and the masses and isotope ratios of each peptide pair (differing by 4 Da) are measured by high-resolution mass spectrometry. Short sequences and/or accurate mass measurements combined with proteomics software tools allow the peptides to be related to the precursor proteins from which they are derived. Relative signal intensities of paired peptides quantify the expression levels of their precursor proteins from proteome pools to be compared, using an equation described in the paper. Observation of individual (unpaired) peptides is mainly interpreted as differential modification or sequence variation for the protein from the respective proteome pool. The method is evaluated here in a comparison of virion proteins for two serotypes (Ad5 and Ad2) of adenovirus, taking advantage of information already available about protein sequences and concentrations. In general, proteolytic 18O labeling enables a shotgun approach for proteomic studies with quantitation capability and is proposed as a useful tool for comparative proteomic studies of very complex protein mixtures.

Analysis of viral glycoproteins by MALDI-TOF mass spectrometry.

Membrane glycoproteins were shown to be useful biomarkers of enveloped viruses using on-target deglycosylation and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Sindbis virus, the prototype alpha-virus, was used as a model system. The glycoproteins and the capsid protein of the Sindbis virus were successfully detected by MALDI-TOF MS using two solvent systems. One of them is 0.5% n-octyl glucoside/0.5% trifluoroacetic acid. The two components of this solvent acted synergistically on the virus to help release and solubilize the structural proteins. The other is 70% acetonitrile/30% formic acid. This solvent solubilized the integral membrane glycoproteins very effectively even after serious aggregation. On-target deglycosylation was performed to confirm the detection of the glycoprotein peak and to produce protein moieties that can be used as biomarkers. After a simple and fast incubation using peptide-N-glycosidase F on target, sequential mass shifts were observed, which proved that the proteins detected at 51 000 Da have N-linked carbohydrate moieties at two sites. Observation of this mass shift could provide confirmatory evidence for viral identification.

Characterization of the protein subset desorbed by MALDI from whole bacterial cells.

This study characterizes various features of the proteins that are detected in MALDI mass spectra when whole bacteria cells are analyzed, in an effort to understand why some proteins are successfully detected and many others are not. Forty peaks observed in the mass range 4,000-20,000 Da in the spectra of Escherichia coli K-12 and 11775 are tentatively assigned to proteins in a protein database, and these proteins are characterized by cell location, copy number, pI, and hydropathicity. Those detected originate in the cytosol and generally share the traits of high abundance within the cell, strong bacisity, and medium hydrophilicity.

Lectin and carbohydrate affinity capture surfaces for mass spectrometric analysis of microorganisms.

In a preliminary report (Bundy, J. L.; Fenselau, C. Anal. Chem 1999, 71, 1460-1463), we demonstrated the use of lectin-derivatized surfaces to capture and concentrate complex carbohydrates as well as microorganisms from sample matrixes unamenable to direct MALDI mass spectrometry. Here, we extend the work to include samples representative of a wider variety of microorganisms of importance to human health and of enveloped viruses. In this study, lectins were immobilized directly to a membrane surface via primary amines. A complementary approach was also explored, using immobilized carbohydrates to capture bacteria via microbial lectins expressed on their surfaces. The carbohydrate-based surfaces were constructed by first immobilizing streptavidin to the membrane, followed by attachment of a commercially produced biotin/carbohydrate polymer. Acid treatment of the sample prior to mass spectrometric analysis permits the observation of protein biomarkers from the captured microbial samples in the 5-20 kDa mass range. Bacteria samples were detected from physiological buffers, urine, milk, and processed chicken samples using the biocapture probes. Viral samples were detected from culture based on glycoprotein moieties desorbed directly from the surface. The carbohydrate-based system provided greater sensitivity than the lectin system, possibly due to the larger number of accessible saccharide ligands on the polymer.

Tandem mass spectrometry of intact proteins for characterization of biomarkers from Bacillus cereus T spores.

Intact protein biomarkers from Bacillus cereus T spores have been analyzed by high-resolution tandem Fourier transform ion cyclotron resonance mass spectrometry. Two techniques have been applied for excitation of the isolated multiply charged precursor ion species: sustained off-resonance irradiation/collisionally activated dissociation and electron capture dissociation. Fragmentation-derived sequence tags and BLAST sequence similarity proteome database searches allow unequivocal identification of the major biomarker protein with unprecedented specificity. Sequence-specific fragmentation patterns further confirm protein identification. Moreover, methodology combining accurate mass measurements of intact proteins with additional information contained in a proteome database permits tentative assignment of several other protein biomarkers isolated from the B. cereus T spores. We argue that approaches involving tandem MS of protein biomarkers, combined with bioinformatics, can drastically improve the specificity of individual microorganism identification, particularly in complex environments.

Characterization of intact microorganisms by MALDI mass spectrometry.

The application of MALDI mass spectrometry to desorb protein biomarkers from intact viruses, bacteria, fungus, and spores is the focus of this review. Instrumentation, sample collection, sample preparation, and algorithms for data analysis are summarized. Optimally these analyses should be carried out in less than five minutes. Successful applications are discussed from biotechnology, cell biology, and the pharmaceutical industry.

Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis.

A novel class of lipopeptides was isolated from Bacillus thuringiensis kurstaki HD-1. Four compounds (1-4) were separated by high-performance liquid chromatography and their primary structures determined using a combination of chemical reactions and mass spectrometry. The four lipopeptides were found to have the same amino acid sequence, Thr-Gly-Ala-Ser-His-Gln-Gln, but different fatty acids. The fatty acyl chain is linked to the N-terminal amino acid residue via an amide bond. Each lipopeptide has a lactone linkage between the carboxyl terminal amino acid and the hydroxyl group in the side chain of the serine residue. Antifungal activity was demonstrated against Stachybotrys charatum.

Matrix-assisted laser desorption/ionization time-of-flight analysis of Bacillus spores using a 2.94 microm infrared laser.

The performance of infrared (2.94 microm) and ultraviolet (337 nm) lasers were compared for analysis of purified spores of B. subtilis, B. cereus and B. globigii on a four-inch end-cap reflectron time-of-flight instrument. Infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectra of these microorganisms displayed a larger number of biomarker peaks above m/z 4000, compared with UV-MALDI. Biomarker peaks were observed at higher m/z values with the IR laser.

Testing the significance of microorganism identification by mass spectrometry and proteome database search.

We derive and validate a simple statistical model that predicts the distribution of false matches between peaks in matrix-assisted laser desorption/ionization mass spectrometry data and proteins in proteome databases. The model allows us to calculate the significance of previously reported microorganism identification results. In particular, for deltam = +/-1.5 Da, we find that the computed significance levels are sufficient to demonstrate the ability to identify microorganisms, provided the number of candidate microorganisms is limited to roughly three Escherichia coli-like or roughly 10 Bacillus subtilis-like microorganisms (in the sense of having roughly the same number of proteins per unit-mass interval). We conclude that, given the cluttered and incomplete nature of the data, it is likely that neither simple ranking nor simple hypothesis testing will be sufficient for truly robust microorganism identification over a large number of candidate microorganisms.

Rapid characterization of spores of Bacillus cereus group bacteria by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry was used to characterize the spores of 14 microorganisms of the Bacillus cereus group. This group includes the four Bacillus species B. anthracis, B. cereus, B. mycoides, and B. thuringiensis. MALDI mass spectra obtained from whole bacterial spores showed many similarities between the species, except for B. mycoides. At the same time, unique mass spectra could be obtained for the different B. cereus and B. thuringiensis strains, allowing for differentiation at the strain level. To increase the number of detectable biomarkers in the usually peak-poor MALDI spectra of spores, the spores were treated by corona plasma discharge (CPD) or sonicated prior to MALDI analysis. Spectra of sonicated or CPD-treated spores displayed an ensemble of biomarkers common for B. cereus group bacteria. Based on the spectra available, these biomarkers differentiate B. cereus group spores from those of Bacillus subtilis and Bacillus globigii. The effect of growth medium on MALDI spectra of spores was also explored.

Intracellular sequestration of anti-tumor drugs by metallothionein.

Acquired drug resistance is one of the most important problems in cancer chemotherapy. One of the mechanisms proposed to contribute to this phenomenon is the sequestration of alkylating agents by metallothionein (MT) in vivo. In this study cadmium-induced human bladder tumor T24 cells were exposed to the therapeutic agents chlorambucil and melphalan. MT-2a, was shown by capillary electrophoresis to comprise 56% of the MT isoforms in induced cells, and drug adducts of MT-2a were isolated and characterized by HPLC and electrospray ionization mass spectrometry. One to four equivalents of drug were found to be covalently adducted. Major binding sites on metallothionein were located in the C-terminal domain by peptide mapping, consistent with previous studies in vitro.

Metastable decay of peptide ions on a Fourier transform mass spectrometer equipped with an external ion source.

Metastable decay rates of two peptides, RPPGFSPF and PKPQQFFGLM, were determined from ions produced in an external matrix-assisted laser desorption/ionization source with a Fourier transform mass spectrometer. An isolation and subtraction method that gives difference spectra was employed to monitor the product formation and metastable decays. The dependence of metastable decay rates on laser fluences and matrixes was demonstrated.

Molecular dynamics simulation of metallothionein-drug complexes.

The intermolecular interactions of metallothionein with nitrogen mustard drugs were studied by molecular dynamics simulations. Previous laboratory experiments have defined selective alkylation of two cysteine residues, and selective binding was proposed to precede alkylation. The present study provides information about accessibility to cysteines based on evaluating the intermolecular energies and distances in the first few ps of dynamics simulations. A series of dynamics simulations was performed with three drug molecules positioned at the eight most solvent accessible cysteine residues of the dimeric form of the protein. Sites proximal to the sulfhydryl groups of Cys-33 and Cys-48 were found to be the most favorable for complexing the aziridinium forms of chlorambucil, melphalan, and mechlorethamine. The sites for preferential binding are in qualitative agreement with the sites of selective alkylation defined experimentally.

Identification of Bacillus spores by matrix-assisted laser desorption ionization-mass spectrometry.

Unique patterns of biomarkers were reproducibly characterized by matrix-assisted laser desorption ionization (MALDI)-mass spectrometry and were used to distinguish Bacillus species members from one another. Discrimination at the strain level was demonstrated for Bacillus cereus spores. Lipophilic biomarkers were invariant in Bacillus globigii spores produced in three different media and in B. globigii spores stored for more than 30 years. The sensitivity was less than 5,000 cells deposited for analysis. Protein biomarkers were also characterized by MALDI analysis by using spores treated briefly with corona plasma discharge. Protein biomarkers were readily desorbed following this treatment. The effect of corona plasma discharge on the spores was examined.

Microorganism identification by mass spectrometry and protein database searches.

A method for rapid identification of microorganisms is presented, which exploits the wealth of information contained in prokaryotic genome and protein sequence databases. The method is based on determining the masses of a set of ions by MALDI TOF mass spectrometry of intact or treated cells. Subsequent correlation of each ion in the set to a protein, along with the organismic source of the protein, is performed by searching an Internet-accessible protein database. Convoluting the lists for all ions and ranking the organisms corresponding to matched ions results in the identification of the microorganism. The method has been successfully demonstrated on B. subtilis and E. coli, two organisms with completely sequenced genomes. The method has been also tested for identification from mass spectra of mixtures of microorganisms, from spectra of an organism at different growth stages, and from spectra originating at other laboratories. Experimental factors such as MALDI matrix preparation, spectral reproducibility, contaminants, mass range, and measurement accuracy on the database search procedure are addressed too. The proposed method has several advantages over other MS methods for microorganism identification.

Covalent sequestration of phosphoramide mustard by metallothionein--an in vitro study.

Acquired drug resistance is one of the most important problems in cancer chemotherapy. One of the proposed mechanisms for these phenomena is the sequestration of alkylating agents by metallothionein in vivo. This research shows that metallothionein can covalently sequester phosphoramide mustard, the active form of cyclophosphamide in vitro. On-line electrospray mass spectrometry reveals that it is phosphoramide, not nornitrogen mustard that alkylates metallothionein, although the metallothionein/nornitrogen mustard adduct was isolated as the major adduct. Tandem mass spectrometric experiments were performed on an isolated drug-modified tryptic peptide. The alkylation occurred predominantly at Cys48 of metallothionein. These results provide further evidence that overexpression of metallothionein can detoxify the active form of the drugs.