Proteome-Wide Structural Probing of Low-Abundant Protein Interactions by Cross-Linking Mass Spectrometry

Proteome-wide cross-linking studies have spurred great interest as they facilitate structural probing of protein interactions in living cells and organisms. However, current studies have a bias for high-abundant proteins. In this study we demonstrate both experimentally and by a kinetic model that this bias is also caused by the propensity of cross-links to preferentially form on high abundant proteins and not by the inability to detect cross-links due to limitations in current technology. We further show, by using both an in vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that parameters optimized toward a pseudo first order kinetics model result in a significant increase in the detection of lower-abundant proteins on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide cross-linking studies and demonstrates how to address a larger part of the proteome by cross-linking.