Statistical model for large-scale peptide identification in databases from tandem mass spectra using SEQUEST

Recent technological advances have made multidimensional peptide separation techniques coupled with tandem mass spectrometry the method of choice for high-throughput identification of proteins. Due to these advances, the development of software tools for large-scale, fully automated, unambiguous peptide identification is highly necessary. In this work, we have used as a model the nuclear proteome from Jurkat cells and present a processing algorithm that allows accurate predictions of random matching distributions, based on the two SEQUEST scores Xcorr and DeltaCn. Our method permits a very simple and precise calculation of the probabilities associated with individual peptide assignments, as well as of the false discovery rate among the peptides identified in any experiment. A further mathematical analysis demonstrates that the score distributions are highly dependent on database size and precursor mass window and suggests that the probability associated with SEQUEST scores depends on the number of candidate peptide sequences available for the search. Our results highlight the importance of adjusting the filtering criteria to discriminate between correct and incorrect peptide sequences according to the circumstances of each particular experiment.     

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.     

Chiral ionic liquids as stationary phases in gas chromatography

Recently, it has been found that room-temperature ionic liquids can be used as stable, unusual selectivity stationary phases. They show "dual nature" properties, in that they separate nonpolar compounds as if they are nonpolar stationary phases and separate polar compounds as if they are polar stationary phases. Extending ionic liquids to the realm of chiral separations can be done in two ways: (1) a chiral selector can be dissolved in an achiral ionic liquid, or (2) the ionic liquid itself can be chiral. There is a single precedent for the first approach, but nothing has been reported for the second approach. In this work, we present the first enantiomeric separations using chiral ionic liquid stationary phases in gas chromatography. Compounds that have been separated using these ionic liquid chiral selectors include alcohols, diols, sulfoxides, epoxides, and acetylated amines. Because of the synthetic nature of these chiral selectors, the configuration of the stereogenic center can be controlled and altered for mechanistic studies and reversing enantiomeric retention.     

Thermosiphon-based PCR reactor: experiment and modeling

A self-actuated, flow-cycling polymerase chain reaction (PCR) reactor that takes advantage of buoyancy forces to continuously circulate reagents in a closed loop through various thermal zones has been constructed, tested, and modeled. The heating required for the PCR is advantageously used to induce fluid motion without the need for a pump. Flow velocities on the order of millimeters per second are readily attainable. In our preliminary prototype, we measured a cross-sectionally averaged velocity of 2.5 mm/s and a cycle time of 104 s. The flow velocity is nearly independent of the loop's length, making the device readily scalable. Successful amplifications of 700- and 305-bp fragments of Bacillus cereus genomic DNA have been demonstrated. Since the device does not require any moving parts, it is particularly suitable for miniature systems.     

Intrinsic properties of the optical coupling between axisymmetric Gaussian beams

On the basis of the overlap integral method, an approximate analytical model is derived to estimate the coupled optical power between axisymmetric Gaussian beams when transverse, axial, and angular misalignments simultaneously exist in three dimensions. Seven optical properties are derived from a detailed analysis of the model. Because the model is an approximate analytical solution to the overlap integral method, the existence of each property is also investigated by a numerical solution. Results show that all seven properties are intrinsic to the optical coupling phenomenon between Gaussian beams. Because numerous single-mode device-to-fiber coupling systems can be well described by use of Gaussian beams, the seven properties provide a solid basis to develop model-based algorithms for single-mode device-to-fiber alignment automation.     

Real-time finite element modeling for surgery simulation: an application to virtual suturing

Real-time finite element (FE) analysis can be used to represent complex deformable geometries in virtual environments. The need for accurate surgical simulation has spurred the development of many of the new real-time FE methodologies that enable haptic support and real-time deformation. These techniques are computationally intensive and it has proved to be a challenge to achieve the high modeling resolutions required to accurately represent complex anatomies. The authors present a new real-time methodology based on linear FE analysis that is appropriate for a wide range of surgical simulation applications. A methodology is proposed that is characterized by high model resolution, low preprocessing time, unrestricted multipoint surface contact, and adjustable boundary conditions. These features make the method ideal for modeling suturing, which is an element common to almost every surgical procedure. This paper describes constraints in the context of a Suturing Simulator currently being developed by the authors.     

Interactive volume rendering of thin thread structures within multivalued scientific data sets

We present a threads and halos representation for interactive volume rendering of vector-field structure and describe a number of additional components that combine to create effective visualizations of multivalued 3D scientific data. After filtering linear structures, such as flow lines, into a volume representation, we use a multilayer volume rendering approach to simultaneously display this derived volume along with other data values. We demonstrate the utility of threads and halos in clarifying depth relationships within dense renderings and we present results from two scientific applications: visualization of second-order tensor valued magnetic resonance imaging (MRI) data and simulated 3D fluid flow data. In both application areas, the interactivity of the visualizations proved to be important to the domain scientists. Finally, we describe a PC-based implementation of our framework along with domain specific transfer functions, including an exploratory data culling tool, that enable fast data exploration.     

Tools for channels: moving towards molecular calculations of gating and permeation in ion channel biophysics

Recent X-ray structures of voltage gated potassium channels provide an exciting opportunity to connect molecular structures with measured biological function. Two of the most important connections for these channels are: first, to the molecular basis behind selectivity and the associated free energy profile underlying ionic current flow and, second, to a true molecular understanding of the large-scale conformational transitions that underlie voltage dependent gating. But, existing computational tools need to be further developed to reach these goals. In this contribution to the symposia on sampling methods we outline our dynamic importance sampling method for sampling large-scale conformational transitions as well as our studies with non-equilibrium work events and equilibrium overlap sampling (OS) methods for sampling events related to the calculation of relative free energies.     

Esophageal perforation. An unusual presentation with a benign clinical course

A case of esophageal perforation occurring in a 75-year-old man is presented. The clinical presentation was benign and the diagnosis of esophageal perforation was made incidental to an upper gastrointestinal barium examination. Conservative management consisting of nasogastric suctioning, antibiotics and antacids was effective in minimizing the clinical course. This represents a rare instance of a successful nonsurgical approach to esophageal perforation.