RAVE

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.

The Experiment

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

These time points are digested with amino-acid specific proteases 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 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 SpectralViewer shows one timepoint per row, then a clickable whole-spectrum map and a noise spectrum. Minutes are in the labels on the right. Red is the unmodified fragment, green is the accumulating modified product. As features are scrolled over, the peaks present useful information to the user, including time course peak-quantity shift plots in the bottom right corner.

The Rate Viewer

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

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

There is an application to assist selecting combinations of enzymes and probes, based on the logic of these maps.

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

Copyright © 2001