Chemical and enzymatic modification of proteins is a well-established treatment technique for probing the conformational properties of these macromolecules. Investigators have recently extended the approach to probe many sites on a protein's structure in parallel manner, such that conformational properties of a target protein can be inferred. The modern approach uses mass spectrometry to quantify reactant loss and product formation. Rigorous analysis is challenging due to the high volume of mass spectrometric data that must be processed and interpreted.
An extensively interactive software suite has been developed to assist various aspects of the analytical protocol. The software offers a variety of tools for data processing of mass spectra, including collation, normalization, calibration, trough definition, baseline removal and peak identification. Peak areas are quantified and associated with specific peptide residues. MS data can then be visualized as time course plots of spectral intensity for data at selected peaks. A variety of quality control tools present users with the means for critical analysis of spectral results.
Results of data extraction performed with the software described here are compared with analyses done by hand. The results demonstrate the computer-aided analyses to match the accuracy of manual analyses, and to exceed the consistency of those efforts. The time required for this analysis is substantially shortened, enhancing the practicality of using modification data to analyze and select physiologically relevant structures for proteins and protein complexes.
The results of an experiment comparing the conformation of Replication Protein A with and without ssDNA are extracted and analyzed, illustrating the utility of the software. The results show that the expected ssDNA binding sites are protected from enzymatic cleavage when in the presence of ligand. In addition, three helices forming the interface between the subunits of the trimeric protein appear to be exposed upon binding ssDNA. This observation supports the notion of a "hinge" in the protein that provides a means for straightening upon binding ligand. Results implicate four other regions of the protein in novel interactions, and a model that might explain these interactions is discussed.