Vicki H. Wysocki

Surface-induced dissociation : An effective tool to probe structure, energetics and fragmentation mechanisms of protonated peptides

The utility of surface-induced dissociation (SID) to probe the structure, energetics and fragmentation mechanisms of protonated peptides is discussed and demonstrated. High internal energy deposition provided by low-energy (eV range) ion-surface collisions yields extensive fragmentation of protonated peptides, allowing relatively uncomplicated and rapid sequence analysis of oligopeptides. SID of multiply protonated peptides is illustrated for peptides with molecular mass of up to approximately 5000 u. It is also illustrated that SID combined with electrospray ionization (ESI) provides a distinctive experimental technique to determine the energetics and mechanisms of peptide fragmentation. The relative position of ESI/SID fragmentation efficiency curves (plots of percentage fragmentation vs. laboratory collision energy) for peptides can be utilized to estimate relative energetics of peptide fragmentation and even to predict proton localization sites. The observed trends support the essential role of the mobile proton model in understanding peptide fragmentation by low-energy tandem mass spectrometry.

Internal energy distributions of isolated ions after activation by various methods

Abstract A number of activation methods have been compared by approximating internal energy distributions, P(E), of selected activated ions. The ions chosen allow simplifying assumptions to be made concerning the determination of the energy distributions since they fragment by several simple consecutive reactions. Ion abundance data, in combination with known energetics of unimolecular fragmentation, are utilized to estimate the internal energy distributions. Determination of P(E) is therefore not based on a specialized instrument or technique and may be applied to ions which do not have stable neutral counterparts. The data obtained are used to account for several important features of tandem mass spectrometry and to examine and compare different methods of varying ion internal energy. The results show, inter alia, that (i) the general shapes of the energy distributions resulting from collisional activation are relatively insensitive to ion structure; (ii) the average energy of ions activated by collision in the kiloelectron volt or electron volt range of collision energy can be comparable; (iii) in contrast to low energy collisional activation, the distribution of internal energies produced by a kiloelectron volt collision is characterized by a finite probability of depositing very high energies; (iv) the average energy of fragmenting (C2H5)4Si+. ions appears to vary linearly with the collision energy in the 5–28 eV range. As the collision energy increases, the width of the internal energy distribution appears to broaden.

Refining the model for selective cleavage at acidic residues in arginine-containing protonated peptides

Abstract Simple glycine-based peptides containing acidic [aspartic (D) and glutamic acid (E)] and basic residues [arginine (R)] were dissociated by surface-induced dissociation (SID) both in a tandem double quadrupole (Q1Q2) and a hybrid sector/time-of-flight (TOF) mass spectrometer, as well as by low-energy collision-induced dissociation in an ion trap mass spectrometer. The synthetic peptides investigated were G D GGG D G, G D GGG D GR, RG D GGG D G, RG D GGG D GR, GGG D GR, GGG E GR, and G D GGG E GR. The mass spectral results obtained support and extend our previous findings that selective cleavages at the C–(O)–N bond adjacent to the acidic residues (C-side) predominate in the spectra when the number of ionizing protons equals or is less than the number of arginine residues. They also support our conclusion that these cleavages are induced by the side-chain acidic hydrogens of D or E residues. Stochastic molecular modeling procedures have been employed in this work to probe the gas-phase conformations for these protonated peptides. These searches have revealed possible conformers of singly protonated GGG D GR, and RG D GGG D G peptide ions where the protonated arginine is solvated by nearby carboxylic and carbonyl oxygens along with simultaneous intramolecular H bonding between the D side-chain acidic hydrogen(s) and the adjacent C-side peptide bonds. Electrospray ionization/surface-induced dissociation fragmentation efficiency curves (percent fragmentation versus SID laboratory collision energy) are also presented for some of these peptides. The relative position of these curves both with the Q1Q2 and sector/TOF instruments along with less pronounced selective cleavages for the E-containing peptides support our previous conclusion that selective cleavage at E residues require longer time frames for dissociation than for D-containing peptides. The total sum of these findings underscores the idea that gas-phase secondary structure (i.e. conformation) can have an influence in peptide fragmentation.

Use of PCR Coupled with Electrospray Ionization Mass Spectrometry for Rapid Identification of Bacterial and Yeast Bloodstream Pathogens from Blood Culture Bottles

ABSTRACT Sepsis is among the top 10 causes of mortality in the United States. Rapid administration of antibiotics is one of the most important contributors to patient survival, yet only a limited number of methods exist for rapid identification of microbes cultivated from bloodstream infections, which can lead to sepsis. While traditional single-target molecular methods have been shown to greatly improve survival for septic patients by enabling rapid deescalation of broad-spectrum antibiotics, multiplex methods offer even greater possibilities. A novel multiplex method, PCR coupled to electrospray ionization mass spectrometry (PCR/ESI-MS), was used to identify the genus and species of microorganisms found to cause human bloodstream infections. DNA was directly extracted from 234 BacT-Alert blood culture bottles, and results were compared to those obtained by clinical reference standard methods. The study results demonstrated 98.7% and 96.6% concordance at the genus and species levels, respectively. Mixtures of microbes were identified in 29 blood culture bottles, including mixed species of the same genus, as well as mixtures containing Gram-positive and Gram-negative organisms, exemplifying the PCR/ESI-MS capability to identify multiple organisms simultaneously without the need for cultivation. This study demonstrates high analytical accuracy in comparison to routine subculture of blood culture bottles and phenotypic identification of microbes. Without foreknowledge of the microorganisms potentially present, the PCR/ESI-MS methods can deliver accurate results in as little as 5 to 6 h after a positive alarm from the automated blood culture system; however, current batch mode testing limits the methods clinical utility at this time.

IRMPD spectroscopy shows that AGG forms an oxazolone b2+ ion.

Infrared multiple photon dissociation (IRMPD) spectroscopy combined with theoretical vibrational spectra provides a powerful tool for probing structure. This technique has been used to probe the structure of protonated cyclic AG and the b(2)(+) ion from AGG. The experimental spectrum for protonated cyclo AG compares very well with the theoretical spectra for a diketopiperazine. The spectrum corresponds best to a combination of two structures protonation at the alanine and glycine amide oxygens. The experimental spectrum for the b(2)(+) ion from protonated AGG matches best to the theoretical spectrum for an oxazolone structure protonated on the ring nitrogen. In particular, the carbonyl stretching band at 1970 cm(-1) is blue-shifted by approximately 200 cm(-1) compared to the experimental spectrum for protonated cAG, indicating that these two structures are distinct. This is the first time that an IRPD spectrum of a b(2)(+) ion has been obtained and, for this ion, the oxazolone structure proposed based on prior calculations and experiments is confirmed by the spectroscopic method.

Charge-remote fragmentation of gas-phase ions: mechanistic and energetic considerations in the dissociation of long-chain functionalized alkanes and alkenes

Abstract The collision-activated dissociation pathways of a number of long-chain functionalized alkane ions, CH3 (CH2)nX+, and alkene ions have been examined by tandem mass spectrometry. Major dissociation pathways observed following collisional activation include the loss of CnH2n+2 units, the loss of CnH2n+1 units, the formation of CnH+2n+1, and the formation of X+ or XH+. Isotopic labelling data for [(CD3)3 N(CD2)13CH3]+ show that no hydrogen/deuterium scrambling occurs between the charge site and the terminal methyl group for the losses of CnH2n+2 or CnH2n+1 from [(CH3)3N(CH)2)13CH3]+. The collision energy required to detect charge-remote fragmentation in low-energy (electronvolt) collisional activation experiments was found to vary with compound type, ion formation method, mass of collision target, and target gas pressure. It is proposed that the dependence of the product distribution on the type of compound is caused by competition between charge-remote fragmentation and charge-directed processes such as the formation of CnH+2n+1 or the formation of X+ or XH+. A radical mechanism is proposed to account for several charge-remote dissociation pathways that cannot be accounted for by a previously proposed cyclic elimination mechanism. These processes include the loss of methane, the formation of odd- and even-electron product ions separated by 1 u, andthe enhancement of certain dissociation products (e.g. allylic cleavage product ions) when unsaturation or substituent sites are introduced into the molecule.

Comparative analysis of PCR-electrospray ionization/mass spectrometry (MS) and MALDI-TOF/MS for the identification of bacteria and yeast from positive blood culture bottles

BACKGROUND Emerging technologies for rapid identification of microbes demonstrate a shift from traditional biochemical and molecular testing algorithms toward methods using mass spectrometry (MS) for the semiquantitative analysis of microbial proteins and genetic elements. This study was performed to assess the diagnostic accuracy of 2 such technologies, PCR–electrospray ionization (ESI)/MS and MALDI-TOF/MS, with respect to phenotypic and biochemical profiling as a reference standard method. A positive challenge set of blood culture bottles was used to compare PCR-ESI/MS and MALDI-TOF/MS performance on a matched set of samples. METHODS We performed characterization of bloodstream infections from blood cultures using the Ibis T5000 PCR-ESI/MS and the Bruker MALDI Biotyper 2.0 (MALDI-TOF/MS) platforms for microbial identification. Diagnostic accuracy was determined by independent comparison of each method to phenotypic and biochemical characterization with Vitek2 analysis as the reference standard identification. RESULTS The diagnostic accuracy, represented as positive agreement, at the genus level was 0.965 (0.930–0.984) for PCR-ESI/MS and 0.969 (0.935–0.987) for MALDI-TOF/MS, and at the species level was 0.952 (0.912–0.974) with PCR-ESI/MS and 0.943 (0.902–0.968) for MALDI-TOF/MS. No statistically significant difference was found between PCR-ESI/MS and MALDI-TOF/MS in the ability to rapidly identify microorganisms isolated from blood culture. CONCLUSIONS Our results demonstrate that PCR-ESI/MS and MALDI-TOF/MS are equivalent in their ability to characterize bloodstream infections with respect to the reference standard, and highlight key differences in the methods that allow for each method to have a unique niche as a tool for rapid identification of microbes in blood cultures.

Surface-Induced Dissociation of Small Molecules, Peptides, and Non-covalent Protein Complexes

This article provides a perspective on collisions of ions with surfaces, including surface-induced dissociation (SID) and reactive ion scattering spectrometry (RISS). The content is organized into sections on surface-induced dissociation of small ions, surface characterization of organic thin films by collision of well-characterized ions into surfaces, the use of SID to probe peptide fragmentation, and the dissociation of large non-covalent complexes by SID. Examples are given from the literature with a focus on experiments from the authors’ laboratory. The article is not a comprehensive review but is designed to provide the reader with an overview of the types of results possible by collisions of ions into surfaces.

Evidence of diketopiperazine and oxazolone structures for HA b2+ ion.

Peptide fragmentation can lead to an oxazolone or diketopiperazine b(2)(+) ion structure. IRMPD spectroscopy combined with computational modeling and gas-phase H/D exchange was used to study the structure of the b(2)(+) ion from protonated HAAAA. The experimental spectrum of the b(2)(+) ion matches both the experimental spectrum for the protonated cyclic dipeptide HA (a commercial diketopiperazine) and the theoretical spectrum for a diketopiperazine protonated at the imidazole pi nitrogen. A characteristic band at 1875 cm(-1) and increased abundance of the peaks at 1619 and 1683 cm(-1) indicate a second population corresponding to an oxazolone species. H/D exchange also shows a mixture of two populations consistent with a mixture of b(2)(+) ion diketopiperazine and oxazolone structures.

Surface-induced dissociation in tandem quadrupole mass spectrometers: A comparison of three designs

Three different devices that-can be used for surface-induced dissociation (SID) m tandem quadrupole instruments are compared here. The designs were compared by examining the fragmentation of several compounds including benzene, W(CO)6, and (CH3)4N+. These studies show that SID can be readily implemented on a variety of tandem quadrupoIe instruments and that the spectra obtained with the in-line and 90° instruments are similar. Evidence is presented that confirms that high average internal energies and narrow distributions of internal energy are available by this technique. Efficiencies for fragmentation of odd-electron ions are on the order of those previously reported by others. The overall SID efficiency for even-electron ions is higher than that for odd-electron ions of similar structure.