RAVE

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Contents

Introduction

Mass spectrometry-based chemical probing methods are an elegant and well-established approach to empirical determination of structural details for macromolecules. Rigorous analysis is hampered by the complexity and volume of data to process and interpret. An extensively user-interfaced software suite (RAVE™) has been developed to assist various aspects of these processes. A viewer capable of full analysis on old experiment data will be kept available at no charge to support progress using this powerful technology. The full-featured version will soon be available for a small fee, and will support the construction of new experiments.


The Experiment

To perform these experiments, highly purified protein is exposed to a sidechain-specific probing reagent, and aliquots are collected with the reaction stopped at various time points.

time course illustration

These samples are then exhaustively digested with amino-acid specific endoproteases and subjected to time-of-flight mass spectrometry, to separate and quantify by molecular weight. Accumulating reaction products are observed in mass spectra as peaks that increase in intensity at a molecular weight representing the fragment mass plus that of the anticipated chemical modification.


The Workspace

A typical RAVE workspace

A screen capture is shown of a typical RAVE/SPADE working environment, where the RAVE application was launched from the SPADE control panel (far left) onto an open MolecularViewer (center). Only the ProjectManager is initially loaded onto the MolecularViewer, which allows definition of project parameters and import of spectral files. Once files are loaded, RAVE launches a SpectralViewer (far right), processes the data, and extracts quantitative peak-fragment associations from which chemical modification rates can be deduced.


The Spectral Viewer

The Spectral Viewer

The SpectralViewer shows the zero time point at the top location, followed by subsequent spectral observations of the reaction timepoints. A click-able map of the whole spectrum is visualized below the spectra, to allow users to quickly move around the interesting features of the experiment. As various features of the spectra are scrolled over, different information panels react and present useful information to the user, including a plot that shows peak-quantity shifts that occur through the time course.


The Rate Viewer

The RAVE application window

Intensity plots (bottom right) are extracted by peak height or area, and then associated with fragments of corresponding molecular weight from a theoretical digest of the target proteins. The Project Manager is shown in greater detail.


Planning Experiments

RAVE condition suggestion window

A reactivity map shows all of the interesting proteolytic fragments and where they intersect with reactive residues in the main chain. Fragments can be clicked to scroll the Spectral Viewer to center the spectra on the fragment's molecular weight.

Many other feature details are listed in the Documentation subdirectory of the main SPADE folder.


Thanks to

RAVE was produced by Deacon Sweeney as thesis work for the Biomedical Sciences PhD program at Wright State University, with support from the labs of Gerald Alter and Michael Raymer.


Copyright © 2001-2009 Deacon Sweeney. All rights reserved.

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