Brian T. Luke

The use of urine proteomic and metabonomic patterns for the diagnosis of interstitial cystitis and bacterial cystitis

The advent of systems biology approaches that have stemmed from the sequencing of the human genome has led to the search for new methods to diagnose diseases. While much effort has been focused on the identification of disease-specific biomarkers, recent efforts are underway toward the use of proteomic and metabonomic patterns to indicate disease. We have developed and contrasted the use of both proteomic and metabonomic patterns in urine for the detection of interstitial cystitis (IC). The methodology relies on advanced bioinformatics to scrutinize information contained within mass spectrometry (MS) and high-resolution proton nuclear magnetic resonance (1H-NMR) spectral patterns to distinguish IC-affected from non-affected individuals as well as those suffering from bacterial cystitis (BC). We have applied a novel pattern recognition tool that employs an unsupervised system (self-organizing-type cluster mapping) as a fitness test for a supervised system (a genetic algorithm). With this approach, a training set comprised of mass spectra and 1H-NMR spectra from urine derived from either unaffected individuals or patients with IC is employed so that the most fit combination of relative, normalized intensity features defined at precise m/z or chemical shift values plotted in n-space can reliably distinguish the cohorts used in training. Using this bioinformatic approach, we were able to discriminate spectral patterns associated with IC-affected, BC-affected, and unaffected patients with a success rate of approximately 84%.

Theoretical investigation of the role of clay edges in prebiotic peptide bond formation. II - Structures and thermodynamics of the activated complex species

Amino acid activation by anhydride formation in model tetrahedral silicate and aluminate sites in clays and neutral phosphates have been studied by semi-empirical molecular orbital calculations. the results have been compared to previousab initio studies on the reactant species and were found to be in good agreement. The geometries of all species were totally optimized and heats of formation obtained. Relative heats of formation of the anhydrides indicate the extent of anhydride formation to be Al > Si > P which is the same order as the stability of hydrolysis. The relative efficacy of the anhydrides in promoting peptide bond formation has been evaluated using both thermodynamic and chemical reactivity criteria. Heats of reaction for model reactions were calculated from calculated enthalpies of formation of the products and reactants. The electrophilicity of the carbonyl carbon and the nucleophilicity of the oxygen were specifically used as indicators of chemical reactivity towards dipeptide formation by the activated amino acids. Our results indicate that if the reaction mechanism is dominated by the nucleophilic character of the oxygen, tetrahedral Al sites should be more active than Si, and if the electrophilic character dominates, the order would be reversed.

Theoretical study of the carbenoid CH2FLi: Structures and energies of the stable configurations and transition states

Abstract The potential energy surface of the singlet carbenoid CH 2 FLi has been examined using ab initio methods including electron correlation and zero-point energy corrections. The three non-tetrahedral structures found earlier are confirmed to be minima, but only the lowest energy structure 1 should be experimentally observable. Two other local minima, 2 and 3 , should rearrange to 1 with minimal activation. On the other hand, 1 should be quite rigid configurationally. This structure is predicted to be stable with respect to separated singlet methylene and lithium fluoride monomer by 49.3 kcal/mol, though the reaction of two molecules of 1 to form ethylene and two lithium fluoride monomers is exothermic by 98 kcal/mol.

A theoretical comparison of the lowest-lying singlet and triplet states of HCB and HBC

Abstract The low-lying singlet and triplet states of H 2 CBe and HCBeH are examined using ab inito molecular orbital theory. In agreement with earlier results, the lowest-lying structure of H 2 CBe has C 2v symmetry and is a triplet with one π electron ( 3 B 1 ). The results presented here suggest that the lowest-energy singlet structure is the ( 1 B 1 ) open-shell singlet, also with C 2v symmetry, at least 2.5 kcal/mol higher in energy. The singlet C 2v structure with two π electrons ( 1 A 1 ) is 15.9 kcal/mol higher than 3 B 1 . All of these structures are bound with respect to the ground state of methylene and the beryllium atom. In HCBeH, linear equilibrium geometries are found for the triplet ( 3 Σ) and singlet ( 1 Δ) states. The triplet is more stable than the singlet ( 1 Δ) by 35.4 kcal/mol, and is only 2.9 kcal/mol higher in energy than triplet H 2 CBe. Since the transition structure connecting these two triplet molecules is found to be 50.2 kcal/mol higher in energy than H 2 CBe, both triplet equilibrium species might exist independently. The harmonic vibrational frequencies of all structures are also reported.

Theoretical investigations of the low-lying electronic states of the HeC2+ dication

Abstract Extended MC SCF computations of the CAS SCF type have been performed on four energetically low-lying electronic states of HeC 2+ dications. The X 1 Σ + ground state is predicted to be thermodynamically stable by 0.72 eV, while the a 3 Π and A 1 Π excited states represent metastable species with barrier heights of 2.19 and 0.20 eV, respectively. The b 3 Σ + state exhibits merely a very shallow potential dip with a well depth of only 0.06 eV. The HeC 2+ dication is therefore predicted to be experimentally observable in the gas phase. Bonding in these unusual dications is discussed and compared to the isoelectronic CH + cation.

Trypsin-Mediated 18 O/ 16 O Labeling for Biomarker Discovery

Differential (18)O/(16)O stable isotopic labeling that relies on post-digestion (18)O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two (18)O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between (18)O- and (16)O-labeled peptides. This allows for wide-range relative quantitation of proteins in complex mixtures using shotgun proteomics. Because of minimal sample consumption and unrestricted peptide tagging, the post-digestion (18)O exchange is suitable for labeling of low-abundance membrane proteins enriched from cancer cell lines or clinical specimens, including tissues and body fluids. This chapter describes a protocol that applies post-digestion (18)O labeling to elucidate putative endogenous tumor hypoxia markers in the plasma membrane fraction enriched from a hypoxia-adapted malignant melanoma cell line. Plasma membrane proteins from hypoxic and normoxic cells were differentially tagged using (18)O/(16)O stable isotopic labeling. The initial tryptic digestion and solubilization of membrane proteins were carried out in a buffer containing 60 % methanol followed by post-digestion (18)O exchange/labeling in buffered 20 % methanol. The differentially labeled peptides were mixed in a 1:1 ratio and fractionated using off-line strong cation exchange (SCX) liquid chromatography followed by on-line reversed-phase nano-flow RPLC-MS identification and quantitation of peptides/proteins in respective SCX fractions. The present protocol illustrates the utility of (18)O/(16)O stable isotope labeling in the context of quantitative shotgun proteomics that provides a basis for the discovery of hypoxia-induced membrane protein markers in malignant melanoma cell lines.

A Comparison of Biomarker and Fingerprint-Based Classifiers of Disease

Early detection of a disease is very important since it greatly improves the individual’s chance of responding well to treatment. For example, the 5-year survival rate from prostate cancer is nearly 100% if it is detected early [http://www.toacorn.com/news/2005/1027/ Health_and_Wellness/077.html]. Similarly, the 5-year survival rate for ovarian cancer is 95% if caught early, but since 75% of the cases are first observed in the later stages of the disease, the overall 5-year survival rate is less than 50% [http://www.information-aboutovarian-cancer.com/]. It would be nice if there was a single test to determine if an individual had cancer somewhere in their body, but unfortunately such a test does not exist. While all cancers have many factors in common, tissue differences and the body’s response to different cancers make the test for ovarian cancer (CA125) very different from the test for prostate cancer (PSA). The lack of sufficient sensitivity and specificity has recently resulted in the recommendation that PSA no longer be used as a potential marker of prostate cancer [http://www.uspreventiveservicestaskforce.org/uspstf/uspsprca.htm].

Theoretical investigations of the role of clay edges in prebiotic peptide bond formation 2. Structure and electron distribution of activated amino acid esters

Activation of amino acids appears to have played a crucial role in prebiotic peptide bond formation.(White and Erickson 1980, 1981) As a model of this process in living systems, phosphates have been used as amino acid activators. The possible role of clay minerals under fluctuating hot/cold:wet/dry conditions has also been investigated. (Lahav and Chang, 1976, Lahav and White 1980, White, Kennedy and Macklin 1984) We present here results of semi-empircal molecular orbital studies investigating the possible activation of amino acids by these agents, as an initial step in peptide bond formation. Using a model of this activation process, described by the reaction: