Mining whole sample mass spectrometry proteomics data for biomarkers: an overview

In this paper we aim to provide a concise overview of designing and conducting an MS proteomics experiment in such a way as to allow statistical analysis that may lead to the discovery of novel biomarkers. We provide a summary of the various stages that make up such an experiment, highlighting the need for experimental goals to be decided upon in advance. We discuss issues in experimental design at the sample collection stage, and good practise for standardising protocols within the proteomics laboratory. We then describe approaches to the data mining stage of the experiment, including the processing steps that transform a raw mass spectrum into a useable form. We propose a permutation-based procedure for determining the significance of reported error rates. Finally, because of its general advantages in speed and cost, we suggest that MS proteomics may be a good candidate for an early primary screening approach to disease diagnosis, identifying areas of risk and making referrals for more specific tests without necessarily making a diagnosis in its own right. Our discussion is illustrated with examples drawn from experiments on bovine blood serum conducted in the Centre for Proteomic Research (CPR) at Southampton University

Advances in structure elucidation of small molecules using mass spectrometry

The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successive chapter discusses mass spectral fragmentation pathways, biotransformation reactions and drug metabolism studies, the mass spectral simulation and generation of in silico mass spectra, expert systems for mass spectral interpretation, and the use of computational chemistry to explain gas-phase phenomena. A single chapter discusses data handling for hyphenated approaches including mass spectral deconvolution for clean mass spectra, cheminformatics approaches and structure retention relationships, and retention index predictions for gas and liquid chromatography. The last section reviews the current state of electronic data sharing of mass spectra and discusses the importance of software development for the advancement of structure elucidation of small molecules

Investigation and development of analytical techniques for trace level detection of drugs, explosives and their precursors

The present dissertation investigates drugs, explosives and their precursors with different analytical techniques and performs improvements, extensions and evaluations for these techniques. This is done for laboratory as well as on-site analytical techniques.Die vorliegende Dissertation beschäftigt sich mit der Untersuchung von Drogen, Sprengstoffen und deren Vorstufen mittels verschiedener analytischer Techniken. Dabei werden die verwendeten Techniken evaluiert, verbessert und erweitert. Die Untersuchungen erfolgen sowohl für Labor- als auch für Vorort-Techniken

Comparative metabolomics in primates reveals the effects of diet and gene regulatory variation on metabolic divergence.

Human diets differ from those of non-human primates. Among few obvious differences, humans consume more meat than most non-human primates and regularly cook their food. It is hypothesized that a dietary shift during human evolution has been accompanied by molecular adaptations in metabolic pathways. Consistent with this notion, comparative studies of gene expression levels in primates have found that the regulation of genes with metabolic functions tend to evolve rapidly in the human lineage. The metabolic consequences of these regulatory differences, however, remained unknown. To address this gap, we performed a comparative study using a combination of gene expression and metabolomic profiling in livers from humans, chimpanzees, and rhesus macaques. We show that dietary differences between species have a strong effect on metabolic concentrations. In addition, we found that differences in metabolic concentration across species are correlated with inter-species differences in the expression of the corresponding enzymes, which control the same metabolic reaction. We identified a number of metabolic compounds with lineage-specific profiles, including examples of human-species metabolic differences that may be directly related to dietary differences

Apportionment of primary and secondary organic aerosols in Southern California during the 2005 Study of Organic Aerosols in Riverside (SOAR-1)

Ambient sampling was conducted in Riverside, California during the 2005 Study of Organic Aerosols in Riverside to characterize the composition and sources of organic aerosol using a variety of state-of-the-art instrumentation and source apportionment techniques. The secondary organic aerosol (SOA) mass is estimated by elemental carbon and carbon monoxide tracer methods, water soluble organic carbon content, chemical mass balance of organic molecular markers, and positive matrix factorization of high-resolution aerosol mass spectrometer data. Estimates obtained from each of these methods indicate that the organic fraction in ambient aerosol is overwhelmingly secondary in nature during a period of several weeks with moderate ozone concentrations and that SOA is the single largest component of PM1 aerosol in Riverside. Average SOA/OA contributions of 70−90% were observed during midday periods, whereas minimum SOA contributions of ~45% were observed during peak morning traffic periods. These results are contrary to previous estimates of SOA throughout the Los Angeles Basin which reported that, other than during severe photochemical smog episodes, SOA was lower than primary OA. Possible reasons for these differences are discussed

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context

Investigations of biological interactions by hydrogen deuterium exchange Fourier transform ion cyclotron mass spectrometry: novel methods, automated analysis and data reduction

textHydrogen/deuterium (H/D) exchange is used to investigate biological interactions by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). For the first time, a series of oligonucleotides of varying length are interrogated by negative mode gas phase H/D exchange. Data presented describes the reactivity of these oligonucleotides and correlates reactivity to functional moieties of the model compound. The results of the study are subjected to center of mass analysis, a technique that uses the high mass resolving power of FT-ICR MS to facilitate an isotope-counting / abundance-weighted algorithm to determine deuterium incorporation. A maximum entropy method produces the relative reaction rates for each of the model compounds. Curve fitting of rates reveal >90% correlation between the areas of individual rate curves and available hydrogens. Observed data is consistent with literature reported reaction mechanisms. This work describes the successful implementation of the thorough high resolution analysis of spectra by Horn (THRASH) algorithm for the analysis of electron capture dissociation (ECD) spectra. Speed improvements in THRASH arise from optimized libraries and use of modern processors. The nonergodic nature of ECD alleviates deuterium scrambling and affords improved localization of exchange data. An ECD spectrum of ubiquitin is subjected to automated THRASH analysis resulting in 85% sequence coverage. Automated data analysis is extended to the batch processing of entire high performance liquid chromatography (HPLC) FT-ICR MS experiments. Center of mass calculations are determined by combining the data from multiple scans to maximize signal. Data is output as a single spreadsheet. Results of automated and manual data processing are compared. Complete analysis of a H/D exchange study is completed in two hours, rather than the two months required for manual analysis. Results based on this method are demonstrated for biological interactions of HIV capsid and Nop5-fibrilliarin complex. New FT-ICR hardware increases data station throughput and reduces the amount of back exchange in liquid phase studies. The improved data station shows a three-fold improvement in scan speed and collects scans every 1.25 s. No significant loss of performance results from increased scan rates. Faster scan rates result in better chromatographic resolution and decreased spectral complexity.Chemistry and BiochemistryChemistr

Characterization and quantifi cation of endogenous fatty acid nitroalkene metabolites in human urine

The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca- 9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 + 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl β-oxidation metabolites. High resolution mass determinations and co-elution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with HgCl2. This reaction increases the level of free nitroalkene fraction >10-fold and displays a KD of 7.5x10-6 M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive non-invasive index of metabolic and inflammatory status.Fil: Salvatore, Sonia Rosana. University of Pittsburgh; Estados UnidosFil: Vitturi, Dario A.. University of Pittsburgh; Estados UnidosFil: Baker, Paul R. S.. University of Pittsburgh; Estados UnidosFil: Bonacci, Gustavo Roberto. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Koenitzer, Jeffrey R.. University of Pittsburgh; Estados UnidosFil: Woodcock, Steven R.. University of Pittsburgh; Estados UnidosFil: Freeman, Bruce A.. University of Pittsburgh; Estados UnidosFil: Schopfer, Francisco J.. University of Pittsburgh; Estados Unido