Browsing by Subject "mass spectrometry"
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Item Development and implementation of a FT-ICR mass spectrometer for the investigation of ion conformations of peptide sequence isomers containing basic amino acid residues by gas-phase hydrogen/deuterium exchange(Texas A&M University, 2004-09-30) Marini, Joseph ThomasThe gas-phase hydrogen/deuterium (H/D) exchange of protonated di- and tripeptides containing a basic amino acid residue has been studied with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Bimolecular reactions are monitored as a function of time providing exchange efficiencies and temporal distributions for the peptide ions. Results from these experiments indicated that position of the basic residue within the peptide (i.e. N-terminal, internal, or C-terminal) influences gas-phase H/D exchange, suggesting unique peptide ion conformations. The FT-ICR mass spectrometer employed for these gas-phase H/D exchange studies was modified from its original design. Instrument modifications include development of an internal matrix assisted laser desorption ionization (MALDI) source for peptide protonation. In addition, a two-section cell was utilized, allowing control of ion motion and factors affecting gas-phase ion molecule reactions. Systems investigated in these gas-phase H/D exchange studies are peptides containing the same amino acid residues but different sequences. These sequence isomers display dissimilar reaction efficiencies and temporal distributions for deuterium incorporation depending on the primary structure of the peptide ion. Specifically, [M+H]+ peptide ions containing a N-terminal basic residue demonstrate unique H/D exchange behavior when compared to their internal and C-terminal counterparts. These differences are attributed to dissimilar intramolecular bridging interactions involved with inductive stabilization of the charge site. Gas-phase H/D exchange of peptide sequence isomers was also probed with various deuterium reagents. Findings suggest that different reagents also influence H/D exchange reaction rate efficiencies and temporal distributions. These dissimilarities are ascribed to relative gas-phase basicity and proposed mechanistic exchange differences for the deuterium reagents.Item Development of Ion Mobility-mass Spectrometry Instrumentation to Probe the Conformations and Capture the Solution to Gas Phase Transition of Electrosprayed Biomolecules(2013-11-22) Silveira, Joshua ARecent progress has been made developing ion mobility-mass spectrometry (IM-MS) instruments for biophysical studies; however, experimental techniques that can probe the structure and/or dynamics of biomolecules at intermediate extents of hydration are limited and little is known about the final stages of desolvation during electrospray ionization (ESI). Here, ion optical devices, analytical methodology, and instrument platforms are developed to study the conformations of structurally labile biomolecules (i.e., peptides and proteins) produced upon ESI and provide new insight into their solution to gas phase evolution. First, fundamental principles of periodic focusing ion mobility spectrometry are comprehensively discussed. Radial ion confinement is attributed to a collisionally dampened effective potential that ultimately results in high ion transmission. Detailed equations of motion are derived that culminate into useful methodology for accurate determination of peptide and protein collision cross section values via inclusion of a mobility dampening coefficient. Second, evaporation of water from extensively hydrated protons and peptides formed by ESI is examined for the first time using a new cryogenic (80 K) IM-MS instrument platform. Key parameters that influence the cluster distributions are critically examined. In agreement with previous studies, the findings indicate that water evaporation is largely dependent upon the particular charge-carrying species within the cluster. IM-MS results for protonated water clusters suggest that the special stability of the well-known H^(+)(H_(2)O)_(n) (n = 21) ?magic number? cluster is attributed to the presence of a compact clathrate cage isomer produced upon ESI. Peptide studies are also presented in which specific and nonspecific solvation is observed for gramicidin S [GS + 2H]^(2+) (H_(2)O)_(n) (n = 0 to 26) and bradykinin [BK + 2H]^(2+) (H_(2)O)_(n) (n = 0 to 73), respectively. However in the case of substance P, [SP + 3H]^(3+), the results demonstrate that a compact dehydrated conformer population (resulting from the evaporative ESI process) can be kinetically trapped on the time scale of several milliseconds, even when an extended coil conformation is energetically favorable in the gas phase.Item Factors Affecting the Fragmentation of Peptide Ions: Metal Cationization and Fragmentation Timescale(2012-10-19) Kmiec, KevinThe factors affecting peptide fragmentation have been extensively studied in the literature in order to better predict the fragment ion spectra of peptides and proteins. While there are countless influences to consider, metal cation binding in the gas-phase is particularly interesting. Herein, a comparison of fragmentation patterns of a model peptide series with various charge carriers (H+, Li+, Na+, K+, and Cu+) will assist in determining the location of the preferred binding site of the metal cation and in assessing differences in the fragmentation pattern as a result of this binding site. An interesting observation from these studies reveals abundant x-type fragment ions occurring from the fragmentation of alkali-metal cationized peptides. As these fragment ions have been observed in previous studies by others but not addressed, the factors affecting the formation of these x-type fragment ions are explored. Additionally, a home-built 193-nm photodissociation tandem time-of-flight mass spectrometer is utilized to study how peptide fragmentation kinetics affect the fragmentation pattern observed. Initially, the fragmentation timescales of various peptides are investigated. Results indicate that longer fragmentation timescales (~10 microseconds) result in an increased number of identified peaks with internal and ammonia loss fragment ions being the most common in comparison to 'prompt' fragmentation timescales (~1 microsecond). Furthermore, b-type fragment ion formation is also favored at longer timescales for the arginine containing peptides investigated. The fragmentation pattern of several proline containing peptides is examined by collision-induced dissociation and 193-nm photodissociation. Unique fragment ions are observed with each occurring at a proline residue. Few differences are detected between CID and 193-nm photodissociation spectra, indicating that the proline residues direct fragmentation rather than the dissociation method. In an effort to improve the performance of the photodissociation tandem TOF instrument, the addition of a second source and a dual-stage reflectron are incorporated. The modifications result in improved mass range, signal-to-noise, and increased fragment ion collection efficiencies. High quality mass spectra are acquired across a range of mass-to-charge ratios from ~600 to 1900. Furthermore, the modifications continue to allow investigation of various fragmentation timescales with the addition of an additional timeframe of ~3 microseconds.Item Gas-phase and Solution-phase Peptide Conformations Studied by Ion Mobility-mass Spectrometry and Molecular Dynamics Simulations(2012-10-19) Chen, LiuxiIon mobility spectrometry (IMS) separates ions on the basis of ion-neutral collision cross-sections (CCS, [omega]), which are determined by the geometry or conformation of the ions. The size-based IM separation can be extended to distinguish conformers that have different shapes in cases where shape differences influence the accessible surface area of the molecule. In recent years, IM has rapidly evolved as a structural characterization technique, which has applied on various structural biology problems. In this work, IMS is combined with molecular dynamics simulation (MDS), specially the integrated tempering sampling molecular dynamics simulation (ITS-MDS) to explore the gas-phase conformation space of two molecular systems (i) protonated tryptophan zipper 1 (trpzip1) ions and its six derivatives (ii) alkali metal ion (Na, K and Cs) adducts of gramicidin A (GA). The structural distributions obtained from ITS-MDS are compared well with results obtained from matrix-assisted laser desorption ionization-ion mobility-mass spectrometry (MALDI-IM-MS) for trpzip 1 series and electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) for alkali metal ion adducts of GA. Furthermore, the solvent dependence on conformational preferences of the GA dimer is investigated using a combination of mass spectrometry techniques, viz. ESI-IM-MS and hydrogen/deuterium exchange (HDX)-MS, and MDS. The IM experiments reveal three distinct gramicidin A species, detected as the sodium ion adduct ions, [2GA + 2Na]??, and the equilibrium abundances of the dimer ions varies with solvent polarity. The solution phase conformations are assigned as the parallel and anti-parallel [beta]-helix dimer, and the anti-parallel dimer is the preferred conformation in non-polar organic solvent. The calculated CCS profiles by ITS-MDS agree very well with the experimentally measured CCS profiles, which underscore the utility of the method for determining candidate structures as well as the relative abundances of the candidate structures. The benefit of combining ion mobility measurements with solution-phase H/D exchange is allowing identifications and detail analysis of the solution-phase subgroup conformations, which cannot be uncovered by one method alone.Item Improving the Flavor of Ground Beef by Selecting Trimmings from Specific Locations(2012-08-17) Harbison, Amanda 1989-We hypothesized that carcass subcutaneous fat location would affect sensory and quality traits. Five carcass fat sources were tested: brisket, chuck, plate, flank, and round. Ground beef was formulated using each fat source and extra-lean beef trim (>95% lean) to contain 80% lean trim and 20% fat trim. Patties (100 g) were evaluated for color, lipid oxidation, fatty acid composition and consumer evaluation. Flavor was analyzed using a Gas chromatography with mass spectrometry (GC/MS) on the headspace above a cooked (74 degrees C) patty in a heated (60 degrees C) 473 mL glass jar with a solid phase micro-extraction (SPME) fiber. Color, thiobarbituric acid reactive substance assay (TBARS), consumer sensory, and cook/freezer loss data showed no differences (P > 0.05) among carcass locations. Percentage stearic acid was lower (P = 0.044) in the brisket than in the chuck and flank. The brisket was higher in percentage cis-vaccenic acid (P = 0.016) and in the saturated fatty acid to monounsaturated fatty acid ratio (P = 0.018), and lower (P = 0.004) in the percentage of total saturated fatty acids than all other sources of subcutaneous fat. Butanedione was highest (P = 0.013) in the flank and plate fat. Brisket tended to be higher (P = 0.054) than flank, plate, and round in 1-octen-3-ol. Brisket was higher (P = 0.008) than chuck, flank, and round, but not different (P > 0.05) than plate in octanedione. Brisket was higher (P = 0.003) than all other sources for beefy aroma. Flank was higher (P = 0.047) than chuck and round for chemical aroma. Brisket was higher (P = 0.004) than all other sources except flank for floral aromas. Plate was higher (P = 0.029) than all other sources for heated oil aromas. For secondary aroma descriptor, round was higher (P < 0.001) than flank, plate, and chuck for dairy. While differences in some key fatty acids and aromatics existed among carcass locations, when the fat was diluted with a common lean source, fat source did not have a negative effect on sensory or quality traits. Therefore, formulating ground beef using subcutaneous fat from specific locations on a carcass may improve the beef aromatics without negatively affecting sensory or quality traits.Item Investigation of Metalloproteins Utilizing High Resolution Mass Spectrometry(2011-08-08) Wu, ZhaoxiangCopper ions (Cu?, Cu??) play important roles in many biological processes (i.e., oxidation, dioxygen transport, and electron transfer); many of the functions in these processes result from copper ions interacting with proteins and peptides. Previous studies using matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) have shown that Cu? ions preferentially bind to electron rich groups in gas phase (i.e., N-terminal amino group, the side-chains of lysine, histidine and arginine). For peptides with more than one Cu? ligand, the interaction between Cu? ions and ligands is described in terms of competitive binding; however, Cu? coordination chemistry for multiple Cu?-containing proteins and peptides in gas phase is still not fully understood. In addition, no studies on the fragmentation chemistry for multiple Cu?-binding peptides, such as [M + 2Cu - H]? ions, have been reported. The synthesized dinuclear copper complex (alpha-cyano-4-hydroxycinnamic acid (CHCA) copper salt (CHCA)?Cu?) enhances the ion abundances for [M + xCu - (x-1)H]? (x = 1-6) ions in gas-phase when used as a MALDI matrix. Using this matrix we have investigated site-specific copper binding of several peptides using fragmentation chemistry of [M + Cu]? and [M + 2Cu - H]? ions. The fragmentation studies reveal that the binding of a single Cu? ion and two Cu? ions are different, and these differences are explained in terms of intramolecular interactions of the peptide-Cu ionic complex. The competitive Cu? binding to C-terminus (i.e., amide, carboxyl, methyl ester) versus lysine, as well as cysteine (SH/SO?H) versus arginine (guanidino), was also examined by MALDI MS and theoretical calculations (Density Functional Theory (DFT)). For example, results from theoretical and experimental (fragmentation reactions) studies on [M + Cu]? and [M + 2Cu - H]? ions suggest that cysteine side chains (SH/SO?H) are important Cu? ligands. Note that, the proton of the SH/SO?H group is mobile and can be transferred to the arginine guanidino group. For [M + 2Cu - H]? ions, deprotonation of the -SH/SO?H group is energetically more favorable than that of the carboxyl group, and the resulting thiolate/sulfonate group plays an important role in the coordination structure of [M + 2Cu - H]? ions.Item Investigation of the effect of intra-molecular interactions on the gas-phase conformation of peptides as probed by ion mobility-mass spectrometry, gas-phase hydrogen/deuterium exchange, and molecular mechanics(Texas A&M University, 2006-04-12) Sawyer, Holly AnnIon mobility-mass spectrometry (IM-MS), gas-phase hydrogen/deuterium (H/D) exchange ion molecule reactions and molecular modeling provide complimentary information and are used here for the characterization of peptide ion structure, including fine structure detail (i.e., cation-π interactions, β-turns, and charge solvation interactions). IM-MS experiments performed on tyrosine containing tripeptides show that the collision cross-sections of sodiated, potassiated and doubly sodiated species of gly-gly-tyr are smaller than that of the protonated species, while the cesiated and doubly cesiated species are larger. Conversely, all of the alkali-adducted species of try-gly-gly have collision cross-sections that are larger than that of the protonated species. The protonated and alkali metal ion adducted (Na+, K+ and Cs+) species of bradykinin and bradykinin fragments 1-5, 1-6, 1-7, 1-8, 2-7, 5-9 and 2-9 were also studied using IM-MS and the alkali metal ion adducts of these species were found to have cross-sections very close to those of the protonated species. Additionally, multiple peak features observed in the ATDs of protonated bradykinin fragments 1-5, 1-6 and 1-7 are conserved upon alkali metal ion adduction. It was observed from gas-phase H/D ion molecule reactions that alkali adducted species exchange slower and to a lesser extent than protonated species in the tyrosine- and arginine-containing peptides. Experimental and computational results are discussed in terms of peptide ion structure, specifically the intra-molecular interactions present how those interactions change upon alkali salt adduction, as well as with the sequence of the peptide. Additionally, IM-MS data suggests the presence of a compact conformation of bradykinin fragment 1-5 (RPPGF) when starting from organic solvent conditions. As water is added stepwise to methanolic solutions, a more extended conformation is populated. When the starting solution is composed of ≈90% water, two distinct mobility profiles are observed as well as a shoulder, indicating the presence of three gas-phase conformations for RPPGF. Gas-phase H/D exchange of [M+H]+ ions prepared from aqueous solvents show a bi-exponential decay, whereas samples prepared from organic solvents show a single exponential decay. The effect of solvent on gas-phase peptide ion structure, i.e., solution-phase memory effects, is discussed and gas-phase structures are compared to know solution-phase structures.Item Proteomics of Oxidative Stress Using Inducible CYP2E1 Expressing HepG2 Cells and 3T3-L1 Adipocytes as Model Systems(2012-07-16) Newton, Billy WalkerThe overall goal of this research was to investigate oxidative stress related changes to the proteomes of 3T3-L1 adipocytes and an inducible CYP2E1 expressing HepG2 cells. Enhanced oxidative stress in hypertrophic adipocytes is associated with metabolic dysregulation and insulin resistance. Because mitochondria generate reactive oxygen species (ROS), we monitored changes to the adipocyte mitochondrial proteome during differentiation and enlargement. We labeled mitochondrial extracts from 3T3-L1 cells that were 0, 4, 7, 10, 14, and 18 days post differentiation with iTRAQ, followed by MS based identification. We found citric acid cycle proteins such as pyruvate carboxylase, citrate synthase, as well as beta-oxidation enzymes; cartinine acyl transferase and long-chain enoyl-CoA hydratase up-regulated from 7 through 18 days post differentiation onset. These data indicate TCA up-regulation for enhanced metabolic and citrate output necessary for lipid synthesis in adipocytes. Paradoxically, the data also show the simultaneous increase in the fatty acid oxidation, indicating a metabolic overdrive state. Biochemical assays showing peaks in ATP and ROS generation in 3 day old adipocytes provide further evidence of this overdrive state. A second peak in ROS generation occurred in 10 day old adipocytes; concurrent ATP generation reduced to near pre-adipocyte levels and this may indicate a metabolic shift that may be responsible for increased oxidative stress in hypertrophic adipocytes. We developed a doxycycline inducible CYP2E1 expressing HepG2 cell line using the pTet-On/pRevTRE expression system to allow greater control and sensitivity in the generation CYP2E1 mediated oxidative stress. Our cell line (RD12) demonstrated stability and tight expression control. After induction, RD12 cells showed 30 percent higher CYP2E1 activity when compared to the constitutive E47 cell line. RD12 cells showed 30 percent greater toxicity than E47 cells and 25 percent less free glutathione when exposed to 20 mM acetaminophen, indicating RD12 cells are more sensitive to the effects reactive intermediates and oxidative stress generated by CYP2E1. We conducted a survey of the toxicity of dietary fatty acids (oleic, linoleic, and palmitic) on HepG2 cells to determine fatty acid doses that induced metabolic changes, but did not cause excessive cell death. The dose of 0.20 mM linoleic and palmitic acid for 48 hours produced low toxicity, but oleic acid actually produced lower toxicity than untreated cells. After exposure cells were treated with a pro-oxidant to determine which fatty acid increased the susceptibility to protein carbonylation. The carbonylated protein isolation procedure indicated the palmitic acid may induce more carbonylation than oleic acid, but greater efficiency in the isolation procedure is required for a confidant determination.Item Structure-property relationships in gas-phase protonated and metalated peptide ions(2009-05-15) Slaton, James GarrettPeptide synthesis and metal doping, combined with mass spectrometric and ion mobility spectrometric techniques, have provided a picture of the fragmentation behavior of a large field of homologous peptide ions, represented as XVGVAZG, where the X amino acid is either arginine, histidine, lysine, aspartic acid or tryptophan and the Z amino acid is proline, glycine, serine, or histidine. These homologous peptide ions have been carefully selected to probe the effects of charge site location and secondary interactions upon the fragmentation chemistry of peptides. Peptides were synthesized on solid support, doped with appropriate metal salts to attach Li+, Na+, K+, Cu+ and Ag+ , and then examined using ion mobility spectrometry, and tandem mass spectrometry, both high energy collision induced dissociation (CID) and photodissociation using 193- nm laser light. Molecular dynamics calculations enabled me to derive candidate structures for these ions that agree with the ion mobility data for the ions. The fragmentation chemistry and structure selection of the first group of peptides, those that contain a proline residue, indicate that the presence of high proton and high metal ion affinity residues at the N-terminal position of the peptide direct the fragmentation of the highly charge-solvated ions according to a charge site directed mechanism. Further examples of charge-solvated structures and charge-directed fragmentation are shown for peptides where the sixth amino acid residue has been replaced with glycine or serine, eliminating the influence of the proline residue in the sixth position. Photodissociation of the peptides indicates that the position of valine residues along the peptide backbone influences the types of abundant fragment ions observed and ai and dai ions are observed exclusively at the site of valine residues. This observation continued, even when the position of the valine residues were altered by synthesis, leading me to the conclusion that the fragmentation of these peptides. The study was expanded to include significantly more complex peptides, those containing second high proton and high metal ion affinity residues, and though the data are complex, the influence of charge solvation in those systems is strong as well, according to my analysis of the candidate structures obtained and the types of fragment ions observed.