Mono- and Intralink Filter (Mi-Filter) To Reduce False Identifications in Cross-Linking Mass Spectrometry Data

[Image: see text] Cross-linking mass spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein–protein interactions (PPIs) depends on the correct assessment of individual inter-protein cross-links. In this article, we describe a mono- and intralink filter (mi-filter) that is applicable to any kind of cross-linking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein cross-link has been identified within a given data set are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of cross-linking data ranging from individual protein complexes over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly reduces the number of false-positive identifications for inter-protein links in all these types of XL-MS data.