David H. Russell

Characterization of the Acinetobacter baumannii growth phase‐dependent and serum responsive transcriptomes

Acinetobacter baumannii has emerged as a bacterial pathogen of considerable healthcare concern. Yet, little is known about the organisms basic biological processes and the regulatory networks that modulate expression of its virulence factors and antibiotic resistance. Using Affymetrix GeneChips , we comprehensively defined and compared the transcriptomes of two A. baumannii strains, ATCC 17978 and 98-37-09, during exponential and stationary phase growth in Luria-Bertani (LB) medium. Results revealed that in addition to expected growth phase-associated metabolic changes, several putative virulence factors were dramatically regulated in a growth phase-dependent manner. Because a common feature between the two most severe types of A. baumannii infection, pneumonia and septicemia, includes the organisms dissemination to visceral organs via the circulatory system, microarray studies were expanded to define the expression properties of A. baumannii during growth in human serum. Growth in serum significantly upregulated iron acquisition systems, genes associated with epithelial cell adherence and DNA uptake, as well as numerous putative drug efflux pumps. Antibiotic susceptibility testing verified that the organism exhibits increased antibiotic tolerance when cultured in human serum, as compared to LB medium. Collectively, these studies provide researchers with a comprehensive database of A. baumanniis expression properties in LB medium and serum and identify biological processes that may contribute to the organisms virulence and antibiotic resistance.

Peptide sequencing by MALDI 193-nm photodissociation TOF MS.

Ultraviolet photodissociation time-of-flight (TOF) mass spectrometry (MS) is described as a method for determination of peptide ion primary structure. Monoisotopic selection and bond-specific activation, combined with the rapidity of TOF MS analysis, render this technique invaluable to the rapidly expanding field of proteomics. Photofragment ion spectra of model peptides acquired using both post-source decay (PSD) focusing and TOF-TOF experimental methods are exhibited. Advantages of 193-nm photodissociation for de novo sequencing of peptide ions are discussed.

Development of a Fourier-Transform Ion Cyclotron Resonance (FTICR) Mass Spectrometry Method for Studies of Metal Ion Excited States

Studies of ion-molecule reaction chemistry of gas-phase transition metal ions have rapidly expanded and the field of gas phase organometallic chemistry has emerged [1]. Eller and Schwarz have compiled an amazingly comprehensive review of this entire field that covers the period 1973 to 1992 [2]. The pioneering gas-phase ion chemistry studies emphasized the type of reactions, product ion distribution, and speculation on reaction mechanisms; however, the most important contribution of much of this work is to the understanding of reaction energetics and bond energies to metal centers [3]. In the last few years, several groups have placed considerable attention on the specific electronic state(s) of the reacting metal ion (M+) and how the reactivity of M+ might change if different excited states are formed by the ionizing process.

Desorption Ionization and Fourier Transform Mass Spectrometry for the Analysis of Large Biomolecules

The recent developments in biomolecule mass spectrometry can be traced to progress made in new ionization methods and instruments having extended mass range. Early in the development of Fourier transform mass spectrometry (FTMS) Comisarow and Marshall [1] Gross and Wilkins [2] and McIver [3] noted the applicability of this method for the analysis of large molecules. The principle limitation to developing these capabilities was in adapting the available desorption ionization methods to FTMS. High-mass FTMS requires the use of super-conducting magnets, the design of which imposes mechanical limitations in designing such systems, and ultra-high vacuum (less than 10−8 torr) which is not compatible with many desorption ionization sources and methods, e.g., gaseous discharge primary ion sources and liquid matrices. The first successful demonstration of desorption ionization with FTMS was the laser desorption ionization by Gross [4]. Subsequent to this work our laboratory reported on the use of Cs+ ion SIMS with FTMS detection of high-mass ions, e.g., greater than m/z 2000 [5]. These preliminary results demonstrated the feasibility for performing desorption ionization with FTMS,and more recent work from these two laboratories clearly establishes the analytical utility of the method. Also, Wilkins [6] has reported impressive results for laser desorptionFTMS of a variety of biomolecules, and McIver and Hunt [7,8] have obtained excellent data on biomolecules with tandem quadrupole-FTMS system and fast-atom bombardment (FAB) ionization.

Mass Spectrometry of Biomolecules

This paper reviews the present status of mass spectrometry of large molecules. Emphasis is given to the use of ionization methods for nonvolatile, thermally labile molecules and to the utilization of tandem mass spectrometry for structural characterization. The combination of desorption ionization and tandem mass spectrometry is relatively new and must undergo additional development before it is a routine analytical method. This review discusses areas where further developments are required and the potential effects that such developments could have on the problems of molecular characterization by mass spectrometry-based methods.

Protein mixture analysis by MALDI/mobility/time-of-flight mass spectrometry

Progress in the development of ion mobility (IM) orthogonal time-of-flight (oTOF) mass spectrometry for rapid analysis of biological samples is presented. The IM-oTOF apparatus described consists of a short drift tube (1 to 15 cm) designed for ion mobility measurement in the low-field limit and a low resolution linear (20 cm) TOF mass spectrometer. Proof of concept is demonstrated by analysis of peptide mixtures generated by proteolytic digestion of proteins.

Fourier Transform Mass Spectrometry for High Mass Applications

Fourier transform mass spectrometry (FTMS) has enormous potential as an analytical mass spectrometry, especially for the analysis of large molecules [1,2,3]. The potential advantages of this instrument over magnetic sector and time-of-flight instruments are the available mass range and ultra-high mass resolution. In addition, as with the time-of-flight system, the FTMS experiment utilizes a pulsed, low duty cycle ionization source which should result in greater sensitivity than can be obtained with CW ionization sources.

Gas-Phase Polar Cycloaddition Reactions

The ion-molecule reaction chemistry of simple alkenes has been extensively studied in many laboratories. At low ion translational energies, as found in the cell of an ion cyclotron resonance spectrometer, the reactions undoubtedly proceed via an intermediate complex. Although much is known about the product distributions and rates of reaction of these processes, little is known about the structure(s) of the intermediate. This is a difficult question because, in many cases, the intermediate cannot be observed. Its properties can be only inferred by indirect methods such as studies involving isotopic labels or comparison of fragmentation channels with the mass spectral decomposition of suspected intermediate. Unfortunately, deuterium labelling in simple hydrocarbon systems is not very informative because of extensive isomerizations of hydrogen atoms in the intermediate complex prior to subsequent fragmentation.