Signal Attrition in Whole Cell Cross-Linking Mass Spectrometry

Cross-linking mass spectrometry (XL-MS) could replace traditional techniques for sampling the cellular interactome, such as affinity pulldown MS. It generates superior interaction data that can be used to better model cellular structure and function. However, the sampling depth of current XL-MS techniques is disappointing. Poor sampling is often blamed on a low-yielding cross-linking reaction, estimated to be ≤0.1% based on relative ion abundance in the mass spectrometer. Here, using a new two-step cross-linker installation process, we demonstrate that the yield of the chemical reaction is not the limiting factor in sampling the interactome. Low cross-link detection levels persist even when cross-link yields approach 30% of total peptide. We show that cross-linked peptides are not preferentially lost during sample workup; they enter the mass spectrometer at least as efficiently as linear peptides. Low detection rates arise mostly from severe signal splitting that reduces the S/N of cross-linked peptides, which is likely exacerbated by low-sensitivity database search tools and compounded by ion suppression.