Cation Recombination Energy/Coulomb Repulsion Effects in ETD/ECD as Revealed by Variation of Charge per Residue at Fixed Total Charge

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) experiments in electrodynamic ion traps operated in the presence of a bath gas in the 1-10 mTorr range have been conducted on a common set of doubly protonated model peptides of the form X(AG)nX (X = lysine, arginine, or histidine, n = 1, 2, or 4). The partitioning of reaction products was measured using thermal electrons, anions of azobenzene, and anions of 1,3-dinitrobenzene as reagents. Variation of n alters the charge per residue of the peptide cation, which affects recombination energy. The ECD experiments showed that H-atom loss is greatest for the n = 1 peptides and decreases as n increases. Proton transfer in ETD, on the other hand, is expected to increase as charge per residue decreases (i.e., as n increases). These opposing tendencies were apparent in the data for the K(AG)nK peptides. H-atom loss appeared to be more prevalent in ECD than in ETD and is rationalized on the basis of either internal energy differences, differences in angular momentum transfer associated with the electron capture versus electron transfer processes, or a combination of the two. The histidine peptides showed the greatest extent of charge reduction without dissociation, the arginine peptides showed the greatest extent of side-chain cleavages, and the lysine peptides generally showed the greatest extent of partitioning into the c/z•-product ion channels. The fragmentation patterns for the complementary c- and z•-ions for ETD and ECD were found to be remarkably similar, particularly for the peptides with X = lysine

Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

AbstractBiomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC=0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB

Gas-phase ion/ion reactions in a triple quadrupole linear ion trap mass spectrometer: The roles of ion type and dissociation methods in biomolecule analysis

The advent of soft ionization methods that can generate multiply charged gaseous biomolecular ions has enabled the development of gas-phase ion/ion reactions in analytical mass spectrometry. Gas-phase ion/ion reactions have proven to be particularly effective in transforming one gaseous ion type to another. Coupled with tandem mass spectrometry, ion transformation reactions, such as proton transfer, electron transfer, metal cationization, and complex formation have shown to be useful in the identification and characterization of biomolecules. The thesis work described below surveys a variety of ion/ion reactions implemented in a triple-quadrupole linear ion trap mass spectrometer, demonstrating ion manipulation in the gas phase and evaluation of different structural characterization methods for biomolecules. Cations of disulfide bond-containing polypeptide hormones, such as oxytocin, somatostatin, and insulin, were involved in gold-cationization ion/ion reactions followed by supplemental collision-induced dissociation (CID) of the peptide-gold complexes. The incorporation of Au(I) or Au(III) cations into the biomolecule demonstrated a specific and efficient method for cleaving disulfide bonds and enabled sequencing of intact peptides and proteins. Furthermore, gaseous polypeptide cations and anions were covalently modified and cross-linked in the gas phase via ion/ion reactions using N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sulfo-NHS) based reagents. The ability to covalently modify primary amine groups in the gas phase with NHS (or sulfo-NHS) reagents opens up the possibility of attaching a wide range of chemical groups to gaseous peptides and proteins and also for selectively modifying other analytes containing free primary amine groups. This project was further extended by comparing solution vs. gas phase modification of biomolecules and evaluating the different factors influencing the modification chemistry. Lastly, the effect of cation recombination energy/coulomb repulsion on product ion channel partitioning in electron transfer dissociation (ETD) and electron capture dissociation (ECD) was examined. The goal of this project was to better understand the mechanisms governing the ETD/ECD phenomena

Gas-Phase Conjugation to Arginine Residues in Polypeptide Ions via <i>N</i>-Hydroxysuccinimide Ester-Based Reagent Ions

Gas-phase conjugation to unprotonated arginine side-chains via <i>N</i>-hydroxysuccinimide (NHS) esters is demonstrated through both charge reduction and charge inversion ion/ion reactions. The unprotonated guanidino group of arginine can serve as a strong nucleophile, resulting in the facile displacement of NHS from NHS esters with concomitant covalent modification of the arginine residue. This reactivity is analogous to that observed with unprotonated primary amines such as the N-terminus or ε-amino group of lysine. In solution, however, the arginine residues tend to be protonated at pH values low enough to prevent hydrolysis of NHS esters, which would render them relatively unreactive with NHS esters. This work demonstrates novel means for gas-phase conjugation to arginine side chains in polypeptide ions

Identification of Host Proteins Predictive of Early Stage Mycobacterium tuberculosis Infection

The objective of this study was to identify blood-based protein biomarkers of early stage Mycobacterium tuberculosis (Mtb) infection. We utilized plasma and serum specimens from TB patients and their contacts (age ≥ 12) enrolled in a household contact study in Uganda. In the discovery phase cross-sectional samples from 104 HIV-uninfected persons classified as either active TB, latent Mtb infection (LTBI), tuberculin skin test (TST) converters, or persistent TST-negative were analyzed. Two hundred eighty-nine statistically significant (false discovery rate corrected p  0.85. Panel performance was confirmed with an independent validation set of longitudinal samples from 16 subjects. These candidate protein biomarkers may allow for the identification of recently Mtb infected individuals at highest risk for developing active TB and most likely to benefit from preventive therapy