Signal Attrition in Whole Cell Crosslinking Mass Spectrometry

Crosslinking 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 crosslinking reaction, estimated to be ∼0.1% based on relative ion abundance in the mass spectrometer. Here, using a new two-step crosslinker installation process, we demonstrate that the yield of the chemical reaction is not the limiting factor in sampling the interactome. Low crosslink detection levels persist even when crosslink yields approach 30% of total peptide. We show that crosslinked 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 crosslinked peptides, which is likely exacerbated by low-sensitivity database search tools and compounded by ion suppression.