Tandem mass spectrometry: a novel approach for metabolic flux analysis

The goal of metabolic flux analysis (MFA) is the accurate estimation of intracellular fluxes in metabolic networks. Here, we introduce a new method for MFA based on tandem mass spectrometry (MS) and stable-isotope tracer experiments. We demonstrate that tandem MS provides more labeling information than can be obtained from traditional full scan MS analysis and allows estimation of fluxes with better precision. We present a modeling framework that takes full advantage of the additional labeling information obtained from tandem MS for MFA. We show that tandem MS data can be computed for any network model, any compound and any tandem MS fragmentation using linear mapping of isotopomers. The inherent advantages of tandem MS were illustrated in two network models using simulated and literature data. Application of tandem MS increased the observability of the models and improved the precision of estimated fluxes by 2- to 5-fold compared to traditional MS analysis.     

swissPIT: a novel approach for pipelined analysis of mass spectrometry data

The identification and characterization of peptides from tandem mass spectrometry (MS/MS) data represents a critical aspect of proteomics. Today, tandem MS analysis is often performed by only using a single identification program achieving identification rates between 10-50% (Elias and Gygi, 2007). Beside the development of new analysis tools, recent publications describe also the pipelining of different search programs to increase the identification rate (Hartler et al., 2007; Keller et al., 2005). The Swiss Protein Identification Toolbox (swissPIT) follows this approach, but goes a step further by providing the user an expandable multi-tool platform capable of executing workflows to analyze tandem MS-based data. One of the major problems in proteomics is the absent of standardized workflows to analyze the produced data. This includes the pre-processing part as well as the final identification of peptides and proteins. The main idea of swissPIT is not only the usage of different identification tool in parallel, but also the meaningful concatenation of different identification strategies at the same time. The swissPIT is open source software but we also provide a user-friendly web platform, which demonstrates the capabilities of our software and which is available at http://swisspit.cscs.ch upon request for account.     

A novel approach to a bifunctional photosensitizer for tumor imaging and phototherapy

Optical imaging has attracted a great attention for studying molecular recognitions because minute fluorescent tracers can be detected in homogeneous and heterogeneous media with existing laboratory instruments. In our preliminary study, a clinically relevant photosensitizer (HPPH, a chlorophyll-a analog) was linked with a cyanine dye (with required photophysical characteristics but limited tumor selectivity), and the resulting conjugate was found to be an efficient tumor imaging (fluorescence imaging) and photosensitizing agent. Compared to HPPH, the presence of the cyanine dye moiety in the conjugate produced a significantly higher uptake in tumor than skin. At a therapeutic/imaging dose, the conjugate did not show any significant skin phototoxicity, a major drawback associated with most of the porphyrin-based photosensitizers. These results suggest that tumor-avid porphyrin-based compounds can be used as "vehicles" to deliver the desired fluorescent agent(s) to tumor. The development of tumor imaging or improved photodynamic therapy agent(s) by itself represents an important step, but a dual function agent (fluorescence imaging and photodynamic therapy) provides the potential for tumor detection and targeted photodynamic therapy, combining two modalities into a single cost-effective "see and treat" approach.     

Tube-gel digestion: a novel proteomic approach for high throughput analysis of membrane proteins

This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl beta-d-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. Enhanced digestions, i.e. improved number and intensity of detected peptides, are also demonstrated for digestion-resistant proteins such as myoglobin, ubiquitin, and bacteriorhodopsin. An additional advantage of the Tube-Gel digestion protocol is that, even without electrophoresis separation, it allows high throughput analysis of complex protein mixtures when coupled with LC-MS/MS. The protocol was used to analyze a complex membrane protein mixture prepared from prostate cancer cells. The protocol involves only a single digestion and 2.5 h of LC-MS/MS analysis and identified 178 membrane proteins. In comparison, the same membrane fraction was resolved by SDS-PAGE, and 20 gel slices were excised and individually digested and analyzed by LC-MS/MS. The more elaborate effort demanded more than 50 h of LC-MS/MS analysis and identified 268 proteins. The new Tube-Gel digestion protocol is an alternative method for high throughput analysis of membrane proteins.     

Suicide seasonality: complex demodulation as a novel approach in epidemiologic analysis

Background

Seasonality of suicides is well-known and nearly ubiquitous, but recent evidence showed inconsistent patterns of decreasing or increasing seasonality in different countries. Furthermore, strength of seasonality was hypothesized to be associated with suicide prevalence. This study aimed at pointing out methodological difficulties in examining changes in suicide seasonality.

Methododology/Principal Findings

The present study examines the hypothesis of decreasing seasonality with a superior method that allows continuous modeling of seasonality. Suicides in Austria (1970–2008, N = 67,741) were analyzed with complex demodulation, a local (point-in-time specific) version of harmonic analysis. This avoids the need to arbitrarily split the time series, as is common practice in the field of suicide seasonality research, and facilitates incorporating the association with suicide prevalence. Regression models were used to assess time trends and association of amplitude and absolute suicide numbers. Results showed that strength of seasonality was associated with absolute suicide numbers, and that strength of seasonality was stable during the study period when this association was taken into account.

Conclusion/Significance

Continuous modeling of suicide seasonality with complex demodulation avoids spurious findings that can result when time series are segmented and analyzed piecewise or when the association with suicide prevalence is disregarded.     

A novel fluorescence microscopic approach to quantitatively analyse protein-induced membrane remodelling

Membrane remodelling or the bending and rupture of the lipid bilayer occurs during diverse cellular processes such as cell division, synaptic transmission, vesicular transport, organelle biogenesis and sporulation. These activities are brought about by the localized change in membrane curvature, which in turn causes lipid-packing stress, of a planar lipid bilayer by proteins. For instance, vesicular transport processes are typically characterized by the cooperative recruitment of proteins that induce budding of a planar membrane and catalyse fission of the necks of membrane buds to release vesicles. The analysis of such membrane remodelling reactions has traditionally been restricted to electron microscopy–based approaches or force spectroscopic analysis of membrane tethers pulled from liposome-based model membrane systems. Our recent work has demonstrated the facile creation of tubular model membrane systems of supported membrane tubes (SMrTs), which mimic late-stage intermediates of typical vesicular transport reactions. This review addresses the nature of such an assay system and a fluorescence-intensity-based analysis of changes in tube dimensions that is indicative of the membrane remodelling capacity of proteins.     

A novel approach for oxidation analysis of therapeutic proteins

Measuring and monitoring of protein oxidation modifications is important for biopharmaceutical process development and stability assessment during long-term storage. Currently available methods for biomolecules oxidation analysis use time-consuming peptide mapping analysis. Therefore, it is desirable to develop high-throughput methods for advanced process control of protein oxidation. Here, we present a novel approach by which oxidative protein modifications are monitored by an indirect potentiometric method. The method is based on adding an electron mediator, which enhances electron transfer (ET) between all redox species and the electrode surface. Specifically, the procedure involves measuring the sharp change in the open circuit potential (OCP) for the mediator system (redox couple) as a result of its interaction with the oxidized protein species in the solution. Application of Pt and Ag/AgCl microelectrodes allowed for a high-sensitivity protein oxidation analysis. We found that the Ru(NH₃)₆^2+/3+ redox couple is suitable for measuring the total oxidation of a wide range of therapeutic proteins between 1.1 and 13.6%. Accuracy determined by comparing with the known percentage oxidation of the reference standard showed that percentage oxidation determined for each sample was within ±20% of the expected percentage oxidation determined by mass spectrometry.     

Binding matrix: a novel approach for binding site recognition

Recognition of protein-DNA binding sites in genomic sequences is a crucial step for discovering biological functions of genomic sequences. Explosive growth in availability of sequence information has resulted in a demand for binding site detection methods with high specificity. The motivation of the work presented here is to address this demand by a systematic approach based on Maximum Likelihood Estimation. A general framework is developed in which a large class of binding site detection methods can be described in a uniform and consistent way. Protein-DNA binding is determined by binding energy, which is an approximately linear function within the space of sequence words. All matrix based binding word detectors can be regarded as different linear classifiers which attempt to estimate the linear separation implied by the binding energy function. The standard approaches of consensus sequences and profile matrices are described using this framework. A maximum likelihood approach for determining this linear separation leads to a novel matrix type, called the binding matrix. The binding matrix is the most specific matrix based classifier which is consistent with the input set of known binding words. It achieves significant improvements in specificity compared to other matrices. This is demonstrated using 95 sets of experimentally determined binding words provided by the TRANSFAC database.     

Simultaneous PET-MRI: a new approach for functional and morphological imaging

Noninvasive imaging at the molecular level is an emerging field in biomedical research. This paper introduces a new technology synergizing two leading imaging methodologies: positron emission tomography (PET) and magnetic resonance imaging (MRI). Although the value of PET lies in its high-sensitivity tracking of biomarkers in vivo, it lacks resolving morphology. MRI has lower sensitivity, but produces high soft-tissue contrast and provides spectroscopic information and functional MRI (fMRI). We have developed a three-dimensional animal PET scanner that is built into a 7-T MRI. Our evaluations show that both modalities preserve their functionality, even when operated isochronously. With this combined imaging system, we simultaneously acquired functional and morphological PET-MRI data from living mice. PET-MRI provides a powerful tool for studying biology and pathology in preclinical research and has great potential for clinical applications. Combining fMRI and spectroscopy with PET paves the way for a new perspective in molecular imaging.     

Application of MALDI-TOF MS to lysine-producing Corynebacterium glutamicum: a novel approach for metabolic flux analysis.

In the present work, a novel comprehensive approach of (13)C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 microL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-(13)C glucose or with mixtures of (13)C6/(12)C6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.     

Two-dimensional fluorescence spectroscopy: a novel approach for controlling fed-batch cultivations

In industrial fed-batch cultivations it is often necessary to control substrate concentrations at a low level to prevent the production of overflow metabolites and thus optimize the biomass yield. A new method for on-line monitoring and fed-batch control based on fluorescence measurements has been developed. Via instantaneous in situ measurements and multivariate data analysis a chemometric model has been established, which enables the rapid detection of ethanol production at aerobic Saccharomyces cerevisiae fed-batch cultivations. The glucose feed rate is controlled by predicting the metabolic state directly from the fluorescence intensities. Thus, ethanol production could be avoided completely while increasing the biomass yield accordingly. The robust instrumentation is suitable for industrial applications.     

3D facial imaging: a novel approach for metabolic abnormalities risk profiling

The human face harbors a rich tapestry of complex phenotypic information spanning genetic, environmental, and physiological dimensions. While facial images excel in diagnosing genetic diseases, their untapped potential for predicting metabolic health presents an intriguing prospect. Metabolic Syndrome(Met S), marked by a constellation of metabolic abnormalities, poses a significant risk for various chronic diseases. Utilizing Face-Wide Association Studies(Fa WAS) on a discovery cohort of 2,621 Chinese individuals and a replication cohort of 2,188 Chinese individuals, we investigated the associations between facial features and Met S and its related conditions. Our findings highlight half of our investigated facial features strongly correlated with Met S risk, such as a slender forehead, a broader and shorter jawline, and fuller features around the temples-eye-cheek region, with notable genetic correlations(0.55–0.58) and influences from environmental factors like age, urban residency, and educational level. The developed face-based prediction model demonstrated significant predictive robustness, achieving an AUC of up to 0.87 for Met S and 0.89 for obesity in external validations, surpassing traditional 2D imaging techniques. Our model also aids in identifying subtypes within healthy populations, with a 2.07 to 2.40-fold increased risk of developing different metabolic disorders within the next five years. This paves the way for precise risk stratification of individuals who are ‘at risk'. Integrating 3D facial imaging for metabolic health predictions, our research introduces an innovative, non-invasive framework for health assessment and subtype identification, demonstrating high potential in personalized medicine and health monitoring.     

High-pressure hydrocephalus: a novel analytical modeling approach

Hydrocephalus is an abnormal accumulation of cerebrospinal fluid (CSF) within ventricles and subarachnoid space (SAS) as a result of disturbances in secretion or absorption procedures. It is believed that arachnoid villi cells, which are microscopic projections of pia-arachnoid mater that extend into venous channels in sagittal sinus, are the main sites for CSF absorption, but it is tempting to speculate that a significant portion of CSF is removed from the SAS by nasal lymphatic vessels around olfactory nerve. Thus, in this paper, we propose an analytical model of CSF-lymphatic-blood circulation, in which these two output pathways for CSF absorption have been considered. Mathematical relations governing the pressures in different interacting compartments of the brain are considered. In addition, for increasing the similarity of our model to the physiological conditions, the bulk flow mechanism, which is supposed to occur during CSF absorption, has been considered in our model. We used our model to simulate hydrocephalus. The results indicate that the lymphatic disorders have more considerable effect in decreasing CSF absorption, compared to the disturbances in arachnoid villi cells. Based on our modeling, we believe that disorders in lymphatic pathway may be a cause of high-pressure hydrocephalus. Surely experimental studies are required to validate our hypothesis.     

Mining human genome for novel purinergic P2Y receptors: a sequence analysis and molecular modeling approach

The purinergic P2Y receptors are G-protein coupled receptors (GPCRs) that control many physiological processes by mediating cellular responses to purines, pyrimidines and their analogues. They can be used as potential therapeutic targets in a variety of disease conditions. Therefore, it is critical to identify new members of this family of receptors from the human genome and characterize them for their role in health and disease. In the present work, molecular modeling was carried out for the 21 known P2Y receptors. Binding site analysis was done on the basis of docking and site-directed mutagenesis data. Thus, conserved features of P2Y receptors could be formulated. These features can be used to determine the purinergic nature of potential P2Y receptors in the human genome. We applied this knowledge to human genome GPCR sequences found by sensitive sequence search techniques and identified two orphan receptors, namely GPR34 and GP171 that have all the necessary conserved features of P2Y receptors.     

Raman and FTIR microscopic imaging of colon tissue: a comparative study

Colon tissue constitutes a valid model for the comparative analysis of soft tissue by Raman and Fourier transform infrared (FTIR) imaging because it contains four major tissue types such as muscle tissue, connective tissue, epithelium and nerve cells. Raman microscopic images were recorded in the mapping mode using 785 nm laser excitation and a step size of 10 μm from three regions within a thin section that encompassed mucus, mucosa, submucosa, and longitudinal and circular muscle layers. FTIR microscopic images that were composed of 4, 8 and 9 individual images of 4096 spectra each were recorded from the same regions using a FTIR spectrometer coupled to a microscope with a focal plane array detector. Furthermore, Raman microscopic images were recorded at a step size of 2.5 μm from three ganglia that belong to the myenteric plexus. The results are discussed with respect to lateral resolution, spectral resolution, acquisition time and sensitivity of both modalities. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)