PNNL

Abstract

The analysis of protein interactions in biological systems represents a significant challenge for today's technology. Chemical cross-linking provides the potential to impart new chemical bonds in a complex system that result in mass changes in the analysis of a set of tryptic peptides. However, system complexity and cross-linking product heterogeneity have precluded widespread chemical cross-linking use for large-scale identification of protein-protein interactions. The development of mass spectrometry identifiable cross-linkers called Protein Interaction Reporters (PIRs) has enabled on-cell chemical cross-linking experiments with product type differentiation. However, the complex datasets resultant from PIR experiments demand new informatics capabilities to allow interpretation. This manuscript details our efforts to develop such capabilities and describes the program X-links which allows PIR product type differentiation. Furthermore, we also present the results from Monte Carlo simulation of PIRtype experiments to provide false positive identification rate estimates for the PIR product type identification through observed precursor and released peptide masses. Our simulations also provide peptide identification calculations based on accurate masses and database complexity that can provide an estimation of false positive rates for peptide identification. Overall, the calculations show a low rate of false positive identification of PIR product types due to random mass matching of approximately 12% with 10 ppm mass measurement accuracy. In addition, consideration of a reduced database resulting from stage 1 analysis of Shewanella oneidensis MR-1 containing 367 proteins resulted in a significant reduction of expected identification false positive identification rate estimation compared to that from the entire Shewanella oneidensis MR-1 proteome.