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.

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.

Na+ and K+ gas-phase affinities of the three aromatic amino acids Phe, Tyr, and Trp were measured by the kinetic method. Na+ binds these amino acids much more strongly than K+, and for both metal ions the binding strength was found to follow the order Phe < or = Tyr < Trp. Quantum chemical calculations by density functional theory (DFT) gave the same qualitative ordering, but suggested a somewhat larger Phe/Trp increment. These results are in acceptable agreement with predictions based on the binding of Na+ and K+ to the side chain model molecules benzene, phenol, and indole, and are also in reasonable agreement with the predictions from purely electrostatic calculations of the side-chain binding effects. The binding energies were compared with those to the aliphatic amino acids glycine and alanine. Binding to the aromatic amino acids was found to be stronger both experimentally and computationally, but the DFT calculations indicate substantially larger increments relative to alanine than shown by the experiments. Possible reasons for this difference are discussed. The metal ion binding energies show the same trends as the proton affinities.

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 (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.

Various aspects of the theory and modeling of ion–molecule radiative association are discussed. A general formalism for evaluating the effective rate constant for radiative and collisional association is reviewed. The implementation of variable reaction coordinate transition state theory estimates within this formalism is described. A detailed discussion is given of the limiting cases of high and low stabilization efficiency. The basic validity of the algorithm is illustrated through sample calculations for the high efficiency limit. The low efficiency limit allows for the determination of binding energies which are independent of any transition state model. The relation between the predicted and observed temperature dependence in the low efficiency limit is explored. Sample calculations employing the general formalism illustrate the usefulness of this modeling in estimating the binding energy of the complex. Modest levels of quantum chemistry (e.g., MP2/6-31G*) are found to provide satisfactory estimates of the vibrational frequencies and intensities required in the modeling. Overall, the modeling provides estimated binding energies for the protonated acetone dimer, NO+...3-pentanone, and Al+...C6H6 complexes which agree with the available literature values to within 2 kcal/mol.

The binding energies of a number of metal cations with phenol and indole in the gas phase were studied experimentally by radiative association kinetics analysis, supplemented by density functional calculations. Radiative association kinetics measurements were carried out in the Fourier transform ion cyclotron resonance mass spectrometer. Reaction in most cases resulted in adduct formation often followed by sequential addition of a second neutral molecule to the ion. The association kinetics were analyzed to yield binding energy values either by using a variational transition state theory-based approach, incorporating quantum-chemical calculations of vibrational frequencies and infrared intensities, or by using the semiquantitative standard hydrocarbon approach. The competitive collision-induced dissociation (CID) technique was used to confirm the order of relative binding energies for several complexes. Calculations of the structures and energies of a number of complexes were performed, by means of the B3LYP-density functional approach, both to complement and compare with the experimental binding energies and also to address the question of π versus heteroatom bonding. With phenol, the two binding sites are close in energy for nontransition metals, but the π-site is relatively more favorable for Fe + and probably Cr + . Experimental, literature, and calculated values were combined to give best-estimate binding energies for a variety of metal ions. For the nontransition-metal ions characterized, phenol binding is similar to benzene binding, whereas for Cr + and especially Fe + phenol binding is stronger than benzene binding. With indole, binding is always enhanced by ∼5-10 kcal mol -1 relative to benzene. Formation of dimer ML 2 + complexes followed patterns similar to those of previous benzene results, except that phenol dimer complexes with Mg + and Al + were unexpectedly observed, suggesting involvement of an oxygen binding site in these cases.

In this work, we describe two different methods for generating protonated S-nitrosocysteine in the gas phase. The first method involves a gas-phase reaction of protonated cysteine with t-butylnitrite, while the second method uses a solution-based transnitrosylation reaction of cysteine with S-nitrosoglutathione followed by transfer of the resulting S-nitrosocysteine into the gas phase by electrospray ionization mass spectrometry (ESI-MS). Independent of the way it was formed, protonated S-nitrosocysteine readily fragments via bond homolysis to form a long-lived radical cation of cysteine (Cys •ϩ ), which fragments under collision-induced dissociation (CID) conditions via losses in the following relative abundance order: •COOH ӷ CH 2 S Ͼ •CH 2 SH Ϸ H 2 S. Deuterium labeling experiments were performed to study the mechanisms leading to these pathways. DFT calculations were also used to probe aspects of the fragmentation of protonated S-nitrosocysteine and the radical cation of cysteine. NO loss is found to be the lowest energy channel for the former ion, while the initially formed distonic Cys•ϩ with a sulfur radical site undergoes proton and/or H atom transfer reactions that precede the losses of T here has been renewed interest in the gas-phase formation and reactions of radical ions of biomolecules. The motivation for these studies range from fundamental interest in species related to biological processes such as enzyme catalysis [1] and oxidative chemistry associated with damage to biomolecules such as DNA [2] and proteins [3] through to the potential for developing novel mass spectrometry based analytical applications [4]. With regards to the formation of radical ions of amino acids and peptides, several methods have been developed as alternatives to electron ionization (EI) [5]. These include UV photodissociation [6 -11] and ion-electron based techniques such as electron capture dissociation (ECD) [12][13][14][15]. Chemical-based methods that involve low-energy collisioninduced dissociation (CID) [16] of ions generated via electrospray ionization and which can thus be carried out on a wide range of mass spectrometers, merit special discussion. The first, pioneered by Siu, involves carrying out CID of ternary metal complexes to form radical cations of peptides via the redox reaction shown in eq 1 [17][18][19]. To date, doubly charged copper(II) ternary metal complexes (eq 1, where Metal ϭ Cu; L ϭ a range of neutral ligands, x ϭ 2, y ϭ 0) and singly charged metal(III) complexes (eq 1, where Metal ϭ Cr, Mn, Fe, and Co; L ϭ a salen ligand, x ϭ 3, y ϭ 2) have been used to study the formation and reactions of peptide radical ions, including recent contributions from Julia Laskin [20 -23].The second method involves carrying out CID on a cationized peptide containing a functional group with a weak bond, which is susceptible to bond homolysis. A number of different functional groups have been examined to date including: (1) N-terminal azo derivatives