Characterization of microbial mixtures by mass spectrometry.

MS applications in microbiology have increased significantly in the past 10 years, due in part to the proliferation of regulator-approved commercial MALDI MS platforms for rapid identification of clinical infections. In parallel, with the expansion of MS technologies in the "omics" fields, novel MS-based research efforts to characterize organismal as well as environmental microbiomes have emerged. Successful characterization of microorganisms found in complex mixtures of other organisms remains a major challenge for researchers and clinicians alike.

Ion Mobility Spectrometry - High Resolution LTQ-Orbitrap Mass Spectrometry for Analysis of Homemade Explosives.

The detailed chemical characterization of homemade explosives (HMEs) and other chemicals that can mimic or mask the presence of explosives is important for understanding and improving the performance of commercial instrumentation used for explosive detection. To that end, an atmospheric-pressure drift tube ion mobility spectrometry (IMS) instrument has been successfully coupled to a commercial tandem mass spectrometry (MS) system. The tandem MS system is comprised of a linear ion trap and a high resolution Orbitrap analyzer.

Establishing drug resistance in microorganisms by mass spectrometry.

A rapid method to determine drug resistance in bacteria based on mass spectrometry is presented. In it, a mass spectrum of an intact microorganism grown in drug-containing stable isotope-labeled media is compared with a mass spectrum of the intact microorganism grown in non-labeled media without the drug present. Drug resistance is determined by predicting characteristic mass shifts of one or more microorganism biomarkers using bioinformatics algorithms.

Enhanced in-source fragmentation in MALDI-TOF-MS of oligonucleotides using 1,5-diaminonapthalene.

The capability to rapidly and confidently determine or confirm the sequences of short oligonucleotides, including native and chemically-modified DNA and RNA, is important for a number of fields. While matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been used previously to sequence short oligonucleotides, the typically low fragmentation efficiency of in-source or post-source decay processes necessitates the accumulation of a large number of spectra, thus limiting the throughput of these methods. Here we introduce a novel matrix, 1,5-diaminonapthalene (DAN), for facile in-source decay (ISD) of DNA and RNA molecular anions, which allows for rapid sequence confirmation.

Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant.

Background: Despite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents.

Top-down identification of protein biomarkers in bacteria with unsequenced genomes.

MALDI mass spectrometry-based systems for rapid characterization of microorganisms in biodefense or medical diagnostics usually detect intact proteins in the 5000-20,000 Da range. To evaluate the reliability of species discrimination, and also for forensic applications, it is important that these biomarker proteins be identified. In the present study we apply high resolution tandem mass analysis on an Orbitrap and top-down bioinformatics to identify major biomarker proteins observed in MALDI spectra of intact bacteria for which little genomic or protein sequence information is available.

Mass spectrometry in biodefense.

Potential agents for biological attacks include both microorganisms and toxins. In mass spectrometry (MS), rapid identification of potential bioagents is achieved by detecting the masses of unique biomarkers, correlated to each agent. Currently, proteins are the most reliable biomarkers for detection and characterization of both microorganisms and toxins, and MS-based proteomics is particularly well suited for biodefense applications.

Mass spectrometry for rapid characterization of microorganisms.

Advances in instrumentation, proteomics, and bioinformatics have contributed to the successful applications of mass spectrometry (MS) for detection, identification, and classification of microorganisms. These MS applications are based on the detection of organism-specific biomarker molecules, which allow differentiation between organisms to be made. Intact proteins, their proteolytic peptides, and nonribosomal peptides have been successfully utilized as biomarkers.

Top-down proteomics for rapid identification of intact microorganisms.

We apply MALDI-TOF/TOF mass spectrometry for the rapid and high-confidence identification of intact Bacillus spore species. In this method, fragment ion spectra of whole (undigested) protein biomarkers are obtained without the need for biomarker prefractionation, digestion, separation, and cleanup. Laser-induced dissociation (unimolecular decay) of higher mass (>5 kDa) precursor ions in the first TOF analyzer is followed by reacceleration and subsequent high-resolution mass analysis of the resulting sequence-specific fragments in a reflectron TOF analyzer.

Detection of Plasmodium falciparum in pregnancy by laser desorption mass spectrometry.

Detection of Plasmodium falciparum malaria during pregnancy is complicated by sequestration of parasites in the placenta, which reduces peripheral blood microscopic detection. Laser desorption mass spectrometry (LDMS) has previously demonstrated sensitive detection of hemozoin from P. falciparum blood cultures and the ability to track parasitemia in a Plasmodium yoelii malaria mouse model.

Arrayed time-of-flight mass spectrometry for time-critical detection of hazardous agents.

The design and operation of an arrayed time-of-flight (TOF) mass spectrometer for simultaneous data acquisition from multiple samples is described. Versions of the instrument employ sets of two or four linear or reflectron mass analyzers. They are housed in the same vacuum chamber and utilize the same laser for ion desorption.

Rapid detection of malaria infection in vivo by laser desorption mass spectrometry.

Rapid diagnosis leading to effective treatment is essential to control escalating infectious diseases such as malaria. Malaria pigment (hemozoin) detection by laser desorption mass spectometry (LDMS) was recently shown to be a sensitive (<10 parasites/muL) technique for detecting Plasmodium falciparum parasites cultured in human blood. To examine the use of LDMS in a rapid new malaria screening assay, we followed the time course of P.

Mass spectrometry for malaria diagnosis.

A physical method currently being developed for malaria parasite detection and diagnosis in blood is reviewed in this article. The method - direct laser desorption mass spectrometry - is based on the detection of heme (iron protoporphyrin) as a unique qualitative and quantitative molecular biomarker for malaria. In infected erythrocytes, the parasite sequesters heme in a molecular crystal (hemozoin) - a volume of highly concentrated and purified biomarker molecules.

Enhanced specificity of bacterial spore identification by oxidation and mass spectrometry.

Addition of an oxidizing agent (e.g., hydrogen peroxide) to intact spores selectively and completely oxidizes Met-containing biomarker proteins by formation of Met sulfoxides.

Microorganism identification by matrix-assisted laser/desorption ionization mass spectrometry and model-derived ribosomal protein biomarkers.

An improved data analysis method is described for rapid identification of intact microorganisms from MALDI-TOF-MS data. The method makes no use of mass spectral fingerprints. Instead, a microorganism database is automatically generated that contains biomarker masses derived from ribosomal protein sequences and a model of N-terminal Met loss.

Covariance mapping in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A novel method for acquisition and numerical analysis of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectral data is described. The digitized ion current transient from each consecutive laser shot is first acquired and stored independently. Subsequently, statistical correlation parameters between all stored transients are computed.

Detection of malaria parasites in blood by laser desorption mass spectrometry.

A novel method for the in vitro detection of the protozoan Plasmodium, the causative agent of malaria, has been developed. It comprises a protocol for cleanup of whole blood samples, followed by direct ultraviolet laser desorption (LD) time-of-flight mass spectrometry. Intense ion signals are observed from intact ferriprotoporphyrin IX (heme), sequestered by malaria parasites during their growth in human red blood cells.

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.

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.

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.

Bioinformatics and mass spectrometry for microorganism identification: proteome-wide post-translational modifications and database search algorithms for characterization of intact H. pylori.

MALDI-TOF mass spectrometry has been coupled with Internet-based proteome database search algorithms in an approach for direct microorganism identification. This approach is applied here to characterize intact H. pylori (strain 26695) Gram-negative bacteria, the most ubiquitous human pathogen.

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.

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.