J. Mitchell Wells

Collision‐Induced Dissociation (CID) of Peptides and Proteins

The most commonly used activation method in the tandem mass spectrometry (MS) of peptides and proteins is energetic collisions with a neutral target gas. The overall process of collisional activation followed by fragmentation of the ion is commonly referred to as collision-induced dissociation (CID). The structural information that results from CID of a peptide or protein ion is highly dependent on the conditions used to effect CID. These include, for example, the relative translational energy of the ion and target, the nature of the target, the number of collisions that is likely to take place, and the observation window of the apparatus. This chapter summarizes the key experimental parameters in the CID of peptide and protein ions, as well as the conditions that tend to prevail in the most commonly employed tandem mass spectrometers.

A quadrupole ion trap with cylindrical geometry operated in the mass-selective instability mode.

A cylindrical geometry ion trap is used to record mass spectra in the mass-selective instability mode. The geometry of the cylindrical ion trap has been optimized to maximize the quadrupole field component relative to the higher-order field content through field calculations using the Poisson/Superfish code and through experimental variation of the electrode structure. The results correspond well with predictions of the calculations. The trap has been used to record mass spectra with better than unit mass resolution, high sensitivity, and a mass/charge range of ∼600 Th. Multistage (MS(3)) experiments have been performed, and the Mathieu stability region has been experimentally mapped. The performance of this device compares satisfactorily with that of the hyperbolic ion trap.

Implementation of DART and DESI ionization on a fieldable mass spectrometer.

A recently developed prototype mobile laboratory mass spectrometer, incorporating an atmospheric pressure ionization (API) interface, is described. This system takes advantage of the small size, lower voltage requirements, and tandem MS abilities of the cylindrical ion trap mass analyzer. The prototype API MS uses small, low-power pumps to fit into a 0.1-m3 self-contained package weighing <45 kg. This instrument has been adapted to allow rapid interfacing to electrospray ionization, desorption electrospray ionization, and direct analysis in real-time sources. Initial data indicate that these techniques provide rapid detection and identification of compounds for quality control, homeland security, and forensic applications. In addition, this instrument is self-contained and compact, making it ideally extensible to mobile laboratory and field analyses. Initial MS and MS/MS data for analyses of drugs, food, and explosives are presented herein.

Charge dependence of protonated insulin decompositions

Abstract Ion trap collisional activation is used to study the effects of charge state on protonated insulin decompositions for three forms of insulin: bovine, porcine, and human. Tandem mass spectrometry data are presented for ions with one to five protons dissociated under identical resonance excitation conditions. The (M+5H)5+ and (M+4H)4+ ions fragment exclusively by peptide bond cleavage of bonds outside the cycles formed by the disulfide linkages present in the insulin molecule, whereas the (M+3H)3+ and (M+2H)2+ ions appear to show a mixture of peptide bond cleavage and fragments arising from mechanisms associated with disulfide bond cleavage. The (M+H)+ ion fragments almost exclusively by way of disulfide bond cleavage, with the only major exception being cleavage on the C-terminal side of glutamic acid residues external to the cycles formed by the disulfide linkages.

Ion/ion chemistry as a top-down approach for protein analysis.

Developing methodology for analyzing complex protein mixtures in a rapid fashion is one of the most challenging problems facing analytical biochemists today. Recent advances in mass spectrometry for the analysis of intact proteins (i.e. the top-down approach) show great promise for rapid protein identification. The ion/ion chemistry approach for the detection and identification of target proteins in complex matrices, determination of fragmentation channels as a function of precursor ion charge state, and post-translational modification characterization are discussed with particular emphasis on tandem mass spectrometry of intact proteins.

“Dueling” ESI: Instrumentation to study ion/ion reactions of electrospray-generated cations and anions

Novel instrumentation has been developed which allows for the sequential injection and subsequent reaction of oppositely-charged ions generated via electrospray ionization (ESI) in a quadrupole ion trap mass spectrometer. The instrument uses a DC turning quadrupole to sequentially direct the two ion polarities into the ion trap from ESI sources which are situated 90° from the axial (z) dimension of the trap, and 180° from one another. This arrangement significantly expands the range of ionic reactants amenable to study over previously-used instrumentation. For example, ion/ion reactions of multiply-charged positive ions with multiply-charged negative ions can be studied. Also, reactions of multiply-charged ions with singly-charged ions of opposite polarity that could not be generated by previously used ionization methods, or that could not be efficiently injected through the ion trap ring electrode, can be studied with the new instrument. This capability allows, for example, the charge state manipulation of negatively-charged precursor and product ions derived from proteins and oligonucleotides via proton transfer reactions with singly-charged cations generated by ESI.

Gaseous apomyoglobin ion dissociation in a quadrupole ion trap: [M + 2H]2+-[M + 21H]21+1

Abstract The dissociation of the multiply protonated ions of apomyoglobin ranging in charge from [M + 2H] 2+ to [M + 21H] 21+ have been studied using collisional activation and ion/ion reactions in a quadrupole ion trap. A variety of collisional activation conditions were explored for each charge state to determine optimal conditions for yielding the highest quality product ion spectra. Product ion spectra for charge states greater than [M + 6H] 6+ were acquired using both on-resonance and off-resonance collisional activation, with on-resonance activation conditions providing the highest quality spectra. Product ion spectra for the lowest charge states could only be acquired using on-resonance collisional activation. The lowest charge states show a high propensity for losses of small molecules, as well as a number of favored amide bond cleavages, such as fragmentation C-terminal to aspartic acid residues. A novel, dominant cleavage between adjacent lysine-histidine residues was also observed, particularly for charge states higher than [M + 6H] 6+ . The largest number of structurally informative fragments, corresponding to b-type or y-type product ions, were produced from the intermediate charge states of [M + 10H] 10+ to [M + 14H] 14+ . The product ion spectra for the charge states of [M + 15H] 15+ and higher were dominated by the y 151 ion, which appeared to be related to protonation of the N-terminus and, possibly, a secondary structure effect. The overall charge state dependent fragmentation behavior of apomyoglobin ions parallels that of other protein ions studied to date using a quadrupole ion trap in that the most extensive structural information is yielded by parent ions of intermediate charge states. This behavior is consistent with these intermediate charge states either being comprised of a diversity of parent ion structures, having a relatively high degree of proton mobility, or a combination of both.

Dissociation reactions of gaseous ferro-, ferri-, and apo-cytochrome c ions

Electrochemical reduction of the iron bound in the heme group of cytochrome c is shown to occur in the nano-electrospray capillary if the protein is sprayed from neutral water using a steel wire as the electrical contact. Quadrupole ion trap collisional activation is used to study the dissociation reactions of cytochrome c as a function of the oxidation state of the iron. Oxidized (Fe(III)) cytochrome c dissociates via sequence-specific amide bond cleavage, while the reduced (Fe(II)) form of the protein dissociates almost exclusively by loss of protonated heme. Apo-cytochrome c, from which the heme has been removed either via gas-phase dissociation of the reduced holo-protein or via solution chemistry, dissociates via amide bond cleavage in similar fashion to the oxidized holo-protein.

Differential non-destructive image current detection in a fourier transform quadrupole ion trap

Dual-detector differential non-destructive Fourier transform detection in a quadrupole ion trap is shown to improve signal intensity and reduce noise compared with spectra recorded using a single detector. A larger area detector in each end-cap electrode is machined to fit its hyperbolic shape and so minimize field imperfections on the z-axis. Argon, acetophenone and bromobenzene spectra were recorded to allow a comparison between single- and dual-detector (differential) modes of detection and to demonstrate the improvement achieved with differential detection. Copyright 1999 John Wiley & Sons, Ltd.

Novel quadrupole ion trap methods for characterizing the chemistry of gaseous macro-ions

Abstract A variety of ion manipulation techniques that have been developed to study the chemistry of gas-phase macro-ions are described and illustrated. These techniques take advantage of several unique characteristics of the quadrupole ion trap, including operation at relatively high (1 mTorr) bath gas pressure and the ability to simultaneously store positive and negative ions in overlapping regions of space. Efforts to characterize unimolecular dissociation in the ion trap via resonance excitation and thermal dissociation at elevated bath gas temperatures are described; such dissociation is of interest as a means for obtaining primary structural information and Arrhenius activation parameters for macro-ions. The use of ion/molecule chemistry to obtain composition and higher level structural information is illustrated with a description of novel HI attachment chemistry which yields information on the number and accessibility of neutral basic sites in macro-ions. Ion/ion chemistry for the manipulation of parent and product ion charge states is shown to expand the range of charge states accessible for tandem mass spectrometry (MS/MS) analysis, and to greatly simplify the interpretation of MS/MS spectra obtained from multiply charged parent ions.