John H. Schwacke

A Statistical Model for iTRAQ Data Analysis

We describe biological and experimental factors that induce variability in reporter ion peak areas obtained from iTRAQ experiments. We demonstrate how these factors can be incorporated into a statistical model for use in evaluating differential protein expression and highlight the benefits of using analysis of variance to quantify fold change. We demonstrate the models utility based on an analysis of iTRAQ data derived from a spike-in study.

Quantification of Protein Expression Changes in the Aging Left Ventricle of Rattus norvegicus

As the heart ages, electrophysiological and biochemical changes can occur, and the ventricle in many cases loses distensibility, impairing diastolic function. How the proteomic signature of the aged ventricle is unique in comparison to young hearts is still under active investigation. We have undertaken a quantitative proteomics study of aging left ventricles (LVs) utilizing the isobaric Tagging for Relative and Absolute Quantification (iTRAQ) methodology. Differential protein expression was observed for 117 proteins including proteins involved in cell signaling, the immune response, structural proteins, and proteins mediating responses to oxidative stress. For many of these proteins, this is the first report of an association with the aged myocardium. Additionally, two proteins of unknown function were identified. This work serves as the basis for making future comparisons of the aged left ventricle proteome to that of left ventricles obtained from other models of disease and heart failure.

iQuantitator: A tool for protein expression inference using iTRAQ

BackgroundIsobaric Tags for Relative and Absolute Quantitation (iTRAQ™) [Applied Biosystems] have seen increased application in differential protein expression analysis. To facilitate the growing need to analyze iTRAQ data, especially for cases involving multiple iTRAQ experiments, we have developed a modeling approach, statistical methods, and tools for estimating the relative changes in protein expression under various treatments and experimental conditions.ResultsThis modeling approach provides a unified analysis of data from multiple iTRAQ experiments and links the observed quantity (reporter ion peak area) to the experiment design and the calculated quantity of interest (treatment-dependent protein and peptide fold change) through an additive model under log transformation. Others have demonstrated, through a case study, this modeling approach and noted the computational challenges of parameter inference in the unbalanced data set typical of multiple iTRAQ experiments. Here we present the development of an inference approach, based on hierarchical regression with batching of regression coefficients and Markov Chain Monte Carlo (MCMC) methods that overcomes some of these challenges. In addition to our discussion of the underlying method, we also present our implementation of the software, simulation results, experimental results, and sample output from the resulting analysis report.ConclusioniQuantitators process-based modeling approach overcomes limitations in current methods and allows for application in a variety of experimental designs. Additionally, hypertext-linked documents produced by the tool aid in the interpretation and exploration of results.

AKAP Signaling in Reinstated Cocaine Seeking Revealed by iTRAQ Proteomic Analysis

To identify candidate proteins in the nucleus accumbens (NAc) as potential pharmacotherapeutic targets for treating cocaine addition, an 8-plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was performed using NAc tissue obtained from rats trained to self-administer cocaine followed by extinction training. Compared with yoked-saline controls, 42 proteins in a postsynaptic density (PSD)-enriched subfraction of the NAc from cocaine-trained animals were identified as significantly changed. Among proteins of interest whose levels were identified as increased was AKAP79/150, the rat ortholog of human AKAP5, a PSD scaffolding protein that localizes signaling molecules to the synapse. Functional downregulation of AKAP79/150 by microinjecting a cell-permeable synthetic AKAP (A-kinase anchor protein) peptide into the NAc to disrupt AKAP-dependent signaling revealed that inhibition of AKAP signaling impaired the reinstatement of cocaine seeking. Reinstatement of cocaine seeking is thought to require upregulated surface expression of AMPA glutamate receptors, and the inhibitory AKAP peptide reduced the PSD content of protein kinase A (PKA) as well as surface expression of GluR1 in NAc. However, reduced surface expression was not associated with changes in PKA phosphorylation of GluR1. This series of experiments demonstrates that proteomic analysis provides a useful tool for identifying proteins that can regulate cocaine relapse and that AKAP proteins may contribute to relapse vulnerability by promoting increased surface expression of AMPA receptors in the NAc.

Improved methods for the mathematically controlled comparison of biochemical systems

The method of mathematically controlled comparison provides a structured approach for the comparison of alternative biochemical pathways with respect to selected functional effectiveness measures. Under this approach, alternative implementations of a biochemical pathway are modeled mathematically, forced to be equivalent through the application of selected constraints, and compared with respect to selected functional effectiveness measures. While the method has been applied successfully in a variety of studies, we offer recommendations for improvements to the method that (1) relax requirements for definition of constraints sufficient to remove all degrees of freedom in forming the equivalent alternative, (2) facilitate generalization of the results thus avoiding the need to condition those findings on the selected constraints, and (3) provide additional insights into the effect of selected constraints on the functional effectiveness measures. We present improvements to the method and related statistical models, apply the method to a previously conducted comparison of network regulation in the immune system, and compare our results to those previously reported.

Network Modeling Reveals Steps in Angiotensin Peptide Processing

New insights into the intrarenal renin–angiotensin (Ang) system have modified our traditional view of the system. However, many finer details of this network of peptides and associated peptidases remain unclear. We hypothesized that a computational systems biology approach, applied to peptidomic data, could help to unravel the network of enzymatic conversions. We built and refined a Bayesian network model and a dynamic systems model starting from a skeleton created with established elements of the renin–Ang system and further developed it with archived matrix-assisted laser desorption ionization-time of flight mass spectra from experiments conducted in mouse podocytes exposed to exogenous Ang substrates. The model-building process suggested previously unrecognized steps, 3 of which were confirmed in vitro, including the conversion of Ang(2–10) to Ang(2–7) by neprilysin, Ang(1–9) to Ang(2–9), and Ang(1–7) to Ang(2–7) by aminopeptidase A. These data suggest a wider role of neprilysin and aminopeptidase A in glomerular formation of bioactive Ang peptides and shunting their formation. Other steps were also suggested by the model, and supporting evidence for those steps was evaluated using model-comparison methods. Our results demonstrate that systems biology methods applied to peptidomic data are effective in identifying novel steps in the Ang peptide processing network, and these findings improve our understanding of the glomerular renin–Ang system.

Revealing functionally coherent subsets using a spectral clustering and an information integration approach

BackgroundContemporary high-throughput analyses often produce lengthy lists of genes or proteins. It is desirable to divide the genes into functionally coherent subsets for further investigation, by integrating heterogeneous information regarding the genes. Here we report a principled approach for managing and integrating multiple data sources within the framework of graph-spectrum analysis in order to identify coherent gene subsets.ResultsWe investigated several approaches to integrate information derived from different sources that reflect distinct aspects of gene functional relationships including: functional annotations of genes in the form of the Gene Ontology, co-mentioning of genes in the literature, and shared transcription factor binding sites among genes. Given a list of genes, we construct a graph containing the genes in each information space; then the graphs were kernel transformed so they could be integrated; finally functionally coherent subsets were identified using a spectral clustering algorithm. In a series of simulation experiments, known functionally coherent gene sets were mixed and recovered using our approach.ConclusionsThe results indicate that spectral clustering approaches are capable of recovering coherent gene modules even under noisy conditions, and that information integration serves to further enhance this capability. When applied to a real-world data set, our methods revealed biologically sensible modules, and highlighted the importance of information integration. The implementation of the statistical model is provided under the GNU general public license, as an installable Python module, at: http://code.google.com/p/spectralmix.

Reduction of seizure occurrence from exposure to auditory stimulation in individuals with neurological handicaps: a randomized controlled trial.

Background The purpose of this work was to determine in a clinical trial the efficacy of reducing or preventing seizures in patients with neurological handicaps through sustained cortical activation evoked by passive exposure to a specific auditory stimulus (particular music). The specific type of stimulation had been determined in previous studies to evoke anti-epileptiform/anti-seizure brain activity. Methods The study was conducted at the Thad E. Saleeby Center in Harstville, South Carolina, which is a permanent residence for individuals with heterogeneous neurological impairments, many with epilepsy. We investigated the ability to reduce or prevent seizures in subjects through cortical stimulation from sustained passive nightly exposure to a specific auditory stimulus (music) in a three-year randomized controlled study. In year 1, baseline seizure rates were established. In year 2, subjects were randomly assigned to treatment and control groups. Treatment group subjects were exposed during sleeping hours to specific music at regular intervals. Control subjects received no music exposure and were maintained on regular anti-seizure medication. In year 3, music treatment was terminated and seizure rates followed. We found a significant treatment effect (p = 0.024) during the treatment phase persisting through the follow-up phase (p = 0.002). Subjects exposed to treatment exhibited a significant 24% decrease in seizures during the treatment phase, and a 33% decrease persisting through the follow-up phase. Twenty-four percent of treatment subjects exhibited a complete absence of seizures during treatment. Conclusion/Significance Exposure to specific auditory stimuli (i.e. music) can significantly reduce seizures in subjects with a range of epilepsy and seizure types, in some cases achieving a complete cessation of seizures. These results are consistent with previous work showing reductions in epileptiform activity from particular music exposure and offers potential for achieving a non-invasive, non-pharmacologic treatment of epilepsy. Trial Registration Clinicaltrials.gov NCT01459692

Matrix‐assisted laser desorption/ionization tissue profiling of secretoneurin in the nucleus accumbens shell from cocaine‐sensitized rats

Proteins in the nucleus accumbens mediate many cocaine-induced behaviors. In an effort to measure changes in nucleus accumbens protein expression as potential biomarkers for addiction, coronal tissue sections were obtained from rats that developed behavioral sensitization after daily administration of cocaine, or from daily saline-treated controls. The tissue sections were subjected to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) profiling and tissue imaging. For profiling experiments, brain sections were manually spotted with matrix over the nucleus accumbens, a brain region known to regulate cocaine sensitization. Summed mass spectra (10,000 laser shots, grid) were acquired and spectra were aligned to reference peaks. Using bioinformatics tools, eight spectral features were found to be altered by cocaine treatment. Based on additional sequencing experiments with MALDI tandem MS and database searches of measured masses, secretoneurin (m/z 3653) was identified as having an increased expression. In addition, the distribution of m/z 3653 in the nucleus accumbens was determined by MALDI tissue imaging, and the increased expression of its precursor protein, secretogranin II, was verified by immunoblotting.

Imbalanced expression of Vcan mRNA splice form proteins alters heart morphology and cellular protein profiles.

The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan (tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan (tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.