SUMMARY The integration of biophysical data from multiple sources is critical for developing accurate structural models of large multiprotein systems and their regulators. Mass spectrometry (MS) can be used to measure the insertion location for a wide range of topographically sensitive chemical probes, and such insertion data provide a rich, but disparate set of modeling restraints. We have developed a software platform that integrates the analysis of label-based MS data with protein modeling activities (Mass Spec Studio). Analysis packages can mine any labeling data from any mass spectrometer in a proteomics-grade manner, and link labeling methods with data-directed protein interaction modeling using HADDOCK. Support is provided for hydrogen/ deuterium exchange (HX) and covalent labeling chemistries, including novel acquisition strategies such as targeted HX-tandem MS (MS2) and data-independent HX-MS2. The latter permits the modeling of highly complex systems, which we demonstrate by the analysis of microtubule interactions.
Background: Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) experiments implemented to characterize protein interaction and protein folding generate large quantities of data. Organizing, processing and visualizing data requires an automated solution, particularly when accommodating new tandem mass spectrometry modes for H/DX measurement. We sought to develop software that offers flexibility in defining workflows so as to support exploratory treatments of H/DX-MS data, with a particular focus on the analysis of very large protein systems and the mining of tandem mass spectrometry data.
Hydrogen-deuterium exchange mass spectrometry is an important method for protein structure-function analysis. The bottom-up approach uses protein digestion to localize deuteration to higher resolution, and the essential measurement involves centroid mass determinations on a very large set of peptides. In the course of evaluating systems for various projects, we established two (HDX-MS) platforms that consisted of a FT-MS and a highresolution QTOF mass spectrometer, each with matched front-end fluidic systems. Digests of proteins spanning a 20 -110 kDa range were deuterated to equilibrium, and figures-of-merit for a typical bottom-up (HDX-MS) experiment were compared for each platform. The Orbitrap Velos identified 64% more peptides than the 5600 QTOF, with a 42% overlap between the two systems, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the QTOF, depending on the Orbitrap resolution setting. However, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate, because of destructive interference arising from mismatches in elemental mass defects. This is shown through the analysis of the peptides common to both platforms, where deuteration values can be as low as 35% of the expected values, depending on FT-MS resolution, peptide length and charge state. Hydrogen-deuterium exchange mass spectrometry (HDX-MS)1 provides a powerful means to study the link between protein structure and function (1). The method involves a chemical process in which labile hydrogens within a protein are exchanged with hydrogen from bulk water. When D 2 O is used in place of H 2 O, a mass shift results at every point of exchange, but it is the backbone amide hydrogens that offer exchange rates on a measurable timescale (2, 3). Measuring an amide hydrogen exchange rate can provide access to conformational dynamics, stability, and the interaction characteristics in that location of structure (4, 5). H/D exchange rates have be used to explore mechanisms of protein folding (6), determine the allosteric impact of post-translational modifications and ligand binding (7, 8), define truncation points for enhancing crystallization success (9), and they have also found a role in mapping interactions between proteins (10). Applications have stepped outside of primary research to include the characterization of protein drugs for stability and similarity testing (11-13). The capacity to provide such information has attracted increased attention from regulatory bodies and is generating a push for standardizing HDX methods. Mass spectrometers are very effective tools for measuring exchange rates, from whole proteins down to the individual amide levels. Classical methods of rate measurement have used NMR (3), but mass spectrometry offers all the advantages of speed, sensitivity and scale that have made the tool so useful in proteomics. Measurements at the peptide level provide an important intermediate resolution. As with bottom-up proteomics, r...
Advancedcontrol rooms will use advancedhuman-systeminterface(HSI) technologies thatmay have significant implications for plant safety in that they will affect the operator's overall role in the system, the method of informationpresentation,andthe ways in which operatorsinteractwith the system. The U.S. Nuclear Regulatory Commission (NRC) reviews the HSI aspects of control rooms to ensure that they are designed to good humanfactors engineeringprinciplesand that operatorperformance and reliability are appropriately supported to protect public health and safety. The principal guidance availableto the NRC, however, was developedmore than ten years ago, well before these technological changes. Accordingly, the human factorsguidance needs to be updatedto serve as the basis for NRC review of these advanced designs. The purpose of this project was to develop a general approach to advanced H$1 review and the human factors guidelines to support NRC safety reviews of advanced systems. This two-volume report provides the results of the project. Volume 1 describes the development of the Advanced HSI Design Review Guideline (DRG) including (1) its theoretical and technicalfoundation, (2) a general model for the review of advanced HSIs, (3) guideline development in both hard-copyand computer-basedversions, and (4) the tests and evaluationsperformed to develop and validate the DRG. Volume 1 also includes a discussion of the gaps in available guidance and a methodology for addressingthem. Volume 2 providesthe guidelinesto be used for advancedHSIreview and the proceduresfor their use.
This study was conducted to determine the speed and accuracy of form recognition as a function of the size of target forms for various amounts of detail resolution. The stimulus forms were generated by filling in, on a statistical basis, some of the cells of a 90,000-cell matrix. The subjects were shown a “briefing target” and instructed to locate that target on a display containing numerous other forms. The significant finding is that both search time and errors remain invariant until the visual angle subtense of the targets falls below 12 min; at values below 12 min performance deteriorates. This effect is independent of the range of resolutions investigated. The implications of these findings to equipment design are discussed.
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