Peak capacity of ion mobility mass spectrometry: separation of peptides in helium buffer gas

Advances in the field of proteomics depend upon the development of high-throughput separation methods. Ion mobility-mass spectrometry is a fast separation method (separations on the millisecond time-scale), which has potential for peptide complex mixture analysis. Possible disadvantages of this technique center around the lack of orthogonality between separation based on ion mobility and separation based on mass. In order to examine the utility of ion mobility-mass spectrometry, the peak capacity (phi) of the technique was estimated by subjecting a large dataset of peptides to linear regression analysis to determine an average trend for tryptic peptides. This trend-line, along with the deviation from a linear relationship observed for this dataset, was used to define the separation space for ion mobility-mass spectrometry. Using the maximum deviation found in the dataset (+/-11%) the peak capacity of ion mobility-mass spectrometry is approximately 2600 peptides. These results are discussed in light of other factors that may increase the peak capacity of ion mobility-mass spectrometry (i.e. multiple trends in the data resulting from multiple classes of compounds present in a sample) and current liquid chromatography approaches to complex peptide mixture analysis.     

Conformational and molecular modeling studies of sulfated cholecystokinin-15

Conformational features of the C-terminal carboxyamidated pentadecapeptide of CCK (S(19)HRISDRD[SO(4)]-YMGWMDF(33)-NH(2)) were determined by NMR spectroscopy in a zwitterionic membrane-mimetic solvent system, composed of DPC micelles. The C-terminal octapeptide consisted of a well-defined pseudohelix that was nearly identical to the structure previously reported for nonsulfated CCK-8 in the same solvent system. N-terminal amino acids of CCK-15 were highly disordered, with no clear conformational preference. Extensive NOE-restrained molecular dynamics simulations of the CCK-15/CCK(1)-R complex suggested that almost all the experimentally determined intermolecular contact points provided by NMR, site-directed mutagenesis, and photoaffinity labeling could be simultaneously satisfied, when the N-terminus of the ligand is placed in close spatial proximity to the N-terminus of the receptor.     

Comparison of statistical methods for classification of ovarian cancer using mass spectrometry data

MOTIVATION: Novel methods, both molecular and statistical, are urgently needed to take advantage of recent advances in biotechnology and the human genome project for disease diagnosis and prognosis. Mass spectrometry (MS) holds great promise for biomarker identification and genome-wide protein profiling. It has been demonstrated in the literature that biomarkers can be identified to distinguish normal individuals from cancer patients using MS data. Such progress is especially exciting for the detection of early-stage ovarian cancer patients. Although various statistical methods have been utilized to identify biomarkers from MS data, there has been no systematic comparison among these approaches in their relative ability to analyze MS data. RESULTS: We compare the performance of several classes of statistical methods for the classification of cancer based on MS spectra. These methods include: linear discriminant analysis, quadratic discriminant analysis, k-nearest neighbor classifier, bagging and boosting classification trees, support vector machine, and random forest (RF). The methods are applied to ovarian cancer and control serum samples from the National Ovarian Cancer Early Detection Program clinic at Northwestern University Hospital. We found that RF outperforms other methods in the analysis of MS data.     

Carbohydrate supplementation and resistance training

There is a growing body of evidence suggesting that the performance of resistance-training exercises can elicit a significant glycogenolytic effect that potentially could result in performance decrements. These decrements may result in less than optimal physiological adaptations to training. Currently some scientific evidence suggests that carbohydrate supplementation prior to and during high-volume resistance training results in the maintenance of muscle glycogen concentration, which potentially could result in the maintenance or increase of performance during a training bout. Some researchers suggest that ingesting carbohydrate supplements prior to and during resistance training may improve resistance-training performance. Additionally, the ingestion of carbohydrates following resistance exercise enhances the resynthesis of muscle glycogen, which may result in a faster time of recovery from resistance training, thus possibly allowing for a greater training volume. On the basis of the current scientific literature, it may be advisable for athletes who are performing high-volume resistance training to ingest carbohydrate supplements before, during, and immediately after resistance training.     

Reliability of power output during short-duration maximal-intensity intermittent cycling

The aims of the present study were: (a) to determine the number of familiarization trials required to establish a high degree of reliability in measures of power output during maximal intermittent cycling; and (b) to examine the reliability of those same measures after familiarization had been established. On separate days over a 3-week period, 2 groups of 7 recreationally active men completed 8 trials of 1 of 2 maximal (20 x 5-second) intermittent cycling tests with contrasting recovery periods (10-seconds or 30-seconds). Significant (p < 0.05) between-trial differences were detected in post-hoc tests involving trials 1 and 2 only. Within-subject test-retest reliability was therefore assessed across trials 3-8. Apart from values of maximum power output in Protocol 1 (10-second recovery periods), all remaining measures of power output showed high degrees of within-subject test-retest reliability (coefficient of variation: 2.4-3.7%). The results of the present study indicate that in subjects unfamiliar with maximal intermittent cycling, high degrees of reliability in many performance measures can be achieved following the completion of 2 familiarization trials.     

Vancomycin Resistance Among Strains of Staphylococcus epidermidis: Effects on Adherence to Silicone

Nosocomial device-related infections with Gram-positive cocci and their resistance to vancomycin are of increasing occurrence. We examined clinical isolates of relatively avirulent coagulase-negative staphylococci for their resistance to vancomycin and for their capabilities to adhere in vitro to medical grade silicone. Vancomycin resistance was found in 9 of 20 isolates, but there was no correlation between adherence capacity to silicone in the absence of vancomycin and vancomycin resistance for a given strain. Vancomycin in the medium, adsorbed to the surface of medical grade silicone or adsorbed on nongrowing cells, reduced adherence of representative Staphylococcus epidermidis to medical grade silicone. Received: 27 November 2000 / Accepted: 10 January 2001     

Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase

Formaldehyde, a major industrial chemical, is classified as a carcinogen because of its high reactivity with DNA. It is inactivated by oxidative metabolism to formate in humans by glutathione-dependent formaldehyde dehydrogenase. This NAD(+)-dependent enzyme belongs to the family of zinc-dependent alcohol dehydrogenases with 40 kDa subunits and is also called ADH3 or chi-ADH. The first step in the reaction involves the nonenzymatic formation of the S-(hydroxymethyl)glutathione adduct from formaldehyde and glutathione. When formaldehyde concentrations exceed that of glutathione, nonoxidizable adducts can be formed in vitro. The S-(hydroxymethyl)glutathione adduct will be predominant in vivo, since circulating glutathione concentrations are reported to be 50 times that of formaldehyde in humans. Initial velocity, product inhibition, dead-end inhibition, and equilibrium binding studies indicate that the catalytic mechanism for oxidation of S-(hydroxymethyl)glutathione and 12-hydroxydodecanoic acid (12-HDDA) with NAD(+) is random bi-bi. Formation of an E.NADH.12-HDDA abortive complex was evident from equilibrium binding studies, but no substrate inhibition was seen with 12-HDDA. 12-Oxododecanoic acid (12-ODDA) exhibited substrate inhibition, which is consistent with a preferred pathway for substrate addition in the reductive reaction and formation of an abortive E.NAD(+).12-ODDA complex. The random mechanism is consistent with the published three-dimensional structure of the formaldehyde dehydrogenase.NAD(+) complex, which exhibits a unique semi-open coenzyme-catalytic domain conformation where substrates can bind or dissociate in any order.     

Fumonisin B1-induced cell death in arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways

We have established an Arabidopsis protoplast model system to study plant cell death signaling. The fungal toxin fumonisin B1 (FB1) induces apoptosis-like programmed cell death (PCD) in wild-type protoplasts. FB1, however, only marginally affects the viability of protoplasts isolated from transgenic NahG plants, in which salicylic acid (SA) is metabolically degraded; from pad4-1 mutant plants, in which an SA amplification mechanism is thought to be impaired; or from jar1-1 or etr1-1 mutant plants, which are insensitive to jasmonate (JA) or ethylene (ET), respectively. FB1 susceptibility of wild-type protoplasts decreases in the dark, as does the cellular content of phenylalanine ammonia-lyase, a light-inducible enzyme involved in SA biosynthesis. Interestingly, however, FB1-induced PCD does not require the SA signal transmitter NPR1, given that npr1-1 protoplasts display wild-type FB1 susceptibility. Arabidopsis cpr1-1, cpr6-1, and acd2-2 protoplasts, in which the SA signaling pathway is constitutively activated, exhibit increased susceptibility to FB1. The cpr6-1 and acd2-2 mutants also constitutively express the JA and ET signaling pathways, but only the acd2-2 protoplasts undergo PCD in the absence of FB1. These results demonstrate that FB1 killing of Arabidopsis is light dependent and requires SA-, JA-, and ET-mediated signaling pathways as well as one or more unidentified factors activated by FB1 and the acd2-2 mutation.     

NMR solution structure and receptor peptide binding of the CC chemokine eotaxin-2

The human CC chemokine eotaxin-2 is a specific agonist for the chemokine receptor CCR3 and may play a role in the recruitment of eosinophils in allergic diseases and parasitic infections. We report the solution structure of eotaxin-2 determined using heteronuclear and triple resonance NMR methods. A family of 20 structures was calculated by hybrid distance geometry-simulated annealing from 854 NOE distance restraints, 48 dihedral angle restraints, and 12 hydrogen bond restraints. The structure of eotaxin-2 (73 amino acid residues) consists of a helical turn (residues 17-20) followed by a 3-stranded antiparallel beta-sheet (residues 22-26, 37-41, and 44-49) and an alpha-helix (residues 54-66). The N-loop (residues 9-16) is packed against both the sheet and the helix with the two conserved disulfide bonds tethering the N-terminal/N-loop region to the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 7-66) are 0.52 and 1.13 A, respectively. A linear peptide corresponding to the N-terminal region of CCR3 binds to eotaxin-2, inducing concentration-dependent chemical shift changes or line broadening of many residues. The distribution of these residues suggests that the peptide binds into an extended groove located at the interface between the N-loop and the beta2-beta3 hairpin. The receptor peptide may also interact with the N-terminus of the chemokine and part of the alpha-helix. Comparison of the eotaxin-2 structure with those of related chemokines indicates several structural features that may contribute to receptor specificity.