Jennifer S. Brodbelt

Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation

The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimers disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissociation methods.

Analytical applications of ion-molecule reactions

This review covers applications of ion-molecule reactions for solving increasingly complex analytical problems. Because gas-phase reactions are frequently fast and efficient, the use of ion-molecule reactions provides a diverse frontier for extending the boundaries of mass spectrometry. Product distributions from ion-molecule reactions may provide key diagnostic information for structure identification, and particular product ions may afford more structurally informative fragmentation patterns than those patterns of ions that are generated by conventional methods. Applications range from those areas involving the development of novel chemical ionization reagents that show structural specificity upon reactions with analytes, to those areas in which ion-molecule reactions are combined with collisionally activated dissociation in unusual sequences, to those areas involving ion-molecule reactions of species formed by laser desorption or electrospray ionization. In electrospray ionization applications, the use of ion-molecule reactions allows the concentration of ion current into fewer multicharged ions, permits the counting of acidic or basic sites, and provides indirect information about protein structures and thermochemical data about individual sites in large molecules.

Probing molecular recognition by mass spectrometry

Abstract The use of mass spectrometry for the study of host–guest complexation and molecular recognition involving either synthetic hosts or biological hosts has been a growing area of research over the past decade. Mass spectrometry has allowed the first studies of host–guest chemistry in a solvent-free environment in which both size-selectivity and electronic effects influence the formation, reactions and stabilities of gas-phase host–guest complexes. Aspects of solution equilibria, such as the determination of binding selectivities of hosts and binding constants, may be examined by using electrospray ionization to transfer noncovalent complexes from solution to the gas phase for analysis. This article will review some of the highlights involving the application of mass spectrometry for solving problems in the area of molecular recognition.

Photodissociation mass spectrometry: new tools for characterization of biological molecules

Photodissociation mass spectrometry combines the ability to activate and fragment ions using photons with the sensitive detection of the resulting product ions by mass spectrometry. This combination affords a versatile tool for characterization of biological molecules. The scope and breadth of photodissociation mass spectrometry have increased substantially over the past decade as new research groups have entered the field and developed a number of innovative applications that illustrate the ability of photodissociation to produce rich fragmentation patterns, to cleave bonds selectively, and to target specific molecules based on incorporation of chromophores. This review focuses on many of the key developments in photodissociation mass spectrometry over the past decade with a particular emphasis on its applications to biological molecules.

Infrared multiphoton dissociation in quadrupole ion traps

The development of new ion activation techniques continues to be a dynamic area of scientific discovery, in part to complement the tremendous innovations in ionization methods that have allowed the mass spectrometric analysis of an enormous array of molecules. Ion activation/dissociation provides key information about ion structures, binding energies, and differentiation of isomers, as well as affording a primary means of identifying compounds in mixtures. Numerous new activation methods have emerged over the past two decades in an effort to develop alternatives to collisional activated dissociation, the gold standard for providing structurally diagnostic fragmentation patterns. Collisional activated dissociation does not always offer sufficiently high or controllable energy deposition, thus rendering it less useful for certain classes of molecules, such as large proteins or macromolecular complexes. Photodissociation is one of the most promising alternatives and is readily implemented in ion trapping and time-of-flight mass spectrometers. Photodissociation generally entails using a laser to irradiate ions with UV, visible, or IR photons, thus resulting in internal energy deposition based on the number and wavelengths of the photons. The activation process can be extremely rapid and efficient, as well as having the potential for high total energy deposition. This review describes infrared multiphoton dissociation in quadrupole ion trap mass spectrometry. A comparison of photodissociation and collisional activated dissociation is covered, in addition to some of the methods to increase photodissociation efficiency. Numerous applications of IRMPD are discussed as well, including ones related to the analysis of drugs, peptides, nucleic acids, and oligosaccharides.

Determination of barbiturates by solid-phase microextraction (SPME) and ion trap gas chromatography-mass spectrometry

Solid-phase microextraction (SPME) in conjunction with quadrupole ion trap GC-MS was applied to the determination of a series of barbiturates. A 65 microns Carbowax-divinylbenzene (DVB) SPME fiber was used to successfully extract a series of eight barbiturates from aqueous solution. Absorption kinetics and distribution coefficients for the 65 microns Carbowax-DVB SPME fiber were determined for the compounds. In addition the method was evaluated with respect to linearity, limit of detection, precision, desorption time, and the effect of salt. Limits of detection reached 1 ng/ml for the barbiturates. Linearity was established for the barbiturates over a concentration range of 10-1000 ng/ml, with coefficients of correlation 0.99. Overall, the precision of the method fell between 2.2%-6.5%, depending on the barbiturate. SPME was applied to the identification and quantitation of the barbiturates in a urine matrix. The method was validated by analyzing a reference standard pentobarbital-spiked urine sample. Both standard addition and internal standard with [2H5]-pentobarbital techniques were evaluated, with recoveries found to be 93% and 104%, respectively SPME was then used to rapidly screen a urine specimen tested positive for barbiturates, and butalbital was detected and quantified.

Determination of Binding Selectivities in Host-guest Complexation By Electrospray/Quadrupole Ion Trap Mass Spectrometry

The quantifiable relationship between the equilibrium solution composition and electrospray (ESI) mass spectral peak intensities of simple host-guest complexes was investigated. Specifically, host-guest complexes of simple crown ethers or glymes with alkali metals and ammonium ions were studied. Comparisons were made between the theoretical concentrations of host-guest complexes derived in solution from known stability constants and the peak intensities for the complexes observed by ESI mass spectrometry (ESI-MS). Two types of complexation experiments were undertaken. First, complexation of a single guest ion, such as an alkali metal, and two crown ethers was studied to evaluate the determination of binding selectivities. Second, complexation of two different guest ions by a single polyether host was also examined. In general, solvation was found to play an integral part in the ability to quantify binding selectivities by ESI-MS. The more similar the solvation energies of the two complexes in the mixture, the more quantifiable their binding selectivities by ESI-MS. In some cases, excellent correlation was obtained between the theoretically predicted selectivity ratios and the ESI mass spectral ratios, in particular when the ESI ratios were adjusted based on evaluation of ESI response factors for the various host-guest complexes.

An Empirical Approach to Estimation of Critical Energies by Using a Quadrupole Ion Trap

A simple energy-resolved mass spectrometric technique is described for the estimation of critical energies for dissociation of ions via threshold collisional activation measurements in a quadrupole ion trap. The method is calibrated by using compounds with well-defined dissociation energies, and separate calibration curves must be constructed for radical ions that are bound by covalent bonds versus hydrogen-bonded complexes. For these sets of experiments, the threshold point is defined as the activation voltage required for the fragment ion intensity to be 10% of the total ion intensity. A plot of threshold activation voltage of the calibrant versus literature critical energies shows a near-linear function, and accuracies are estimated as better than ± 6 kcal/mol. The qz value during activation seems to have little effect on the threshold voltages as long as very low qz values that cause ion ejection are avoided. Activation periods that are substantially longer than 10-ms result in nonlinear behavior in the calibration curves for ions that have critical energies above 30 kcal/mol. This energy-resolved method was also useful for the estimation of critical energies of complexes bound by electrostatic forces, such as hydrogen-bonding interactions. A quantitative evaluation of proton-bound polyether-amine complexes showed that the number of available hydrogen-binding sites, the gas-phase basicities of the polyether and amine components, and the ability of the complex to attain the most favorable near-linear hydrogen bonds correlate with the threshold values.

Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in Gram-positive and Gram-negative bacteria

Historically, the O1 El Tor and classical biotypes of Vibrio cholerae have been differentiated by their resistance to the antimicrobial peptide polymyxin B. However, the molecular mechanisms associated with this phenotypic distinction have remained a mystery for 50 y. Both Gram-negative and Gram-positive bacteria modify their cell wall components with amine-containing substituents to reduce the net negative charge of the bacterial surface, thereby promoting cationic antimicrobial peptide resistance. In the present study, we demonstrate that V. cholerae modify the lipid A anchor of LPS with glycine and diglycine residues. This previously uncharacterized lipid A modification confers polymyxin resistance in V. cholerae El Tor, requiring three V. cholerae proteins: Vc1577 (AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the protein machinery required for glycine addition is reminiscent of the Gram-positive system responsible for d-alanylation of teichoic acids. Such machinery was not thought to be used by Gram-negative organisms. V. cholerae O1 El Tor mutants lacking genes involved in transferring glycine to LPS showed a 100-fold increase in sensitivity to polymyxin B. This work reveals a unique lipid A modification and demonstrates a charge-based remodeling strategy shared between Gram-positive and Gram-negative organisms.

Transmission Mode Desorption Electrospray Ionization

A new mode of operation for desorption electrospray ionization (DESI) analysis of liquids or solid residues from evaporated solvents is presented. Unlike traditional DESI, the electrospray is not deflected off of a surface but instead is transmitted through a sampling mesh at a 0° angle between the electrospray tip, sample mesh, and capillary inlet of a mass spectrometer. In this configuration, deposited samples can be analyzed rapidly without rigorous optimization of spray distances or angles and without the preparation time associated with solvent evaporation. The new transmission mode desorption electrospray ionization (TM-DESI) technique is not applicable to bulk materials, but instead is a method designed to simplify the sample preparation process for liquid samples and sample extracts. The technique can reduce analysis time to seconds while consuming only microliters of sample. The results presented summarize the optimization of the technique, highlight key figures of merit for several model compounds, and illustrate potential applications to high throughput screening of liquid mixtures in both extraction solvents and biological matrices.