To Cleave or Not To Cleave in XL-MS?

Cross-linking mass spectrometry (XL-MS) is an efficient technique for uncovering structural features and interactions of the in-solution state of the proteins under investigation. Distance constraints obtained by this technique are highly complementary to classical structural biology approaches like X-ray crystallography and cryo-EM and have successfully been leveraged to shed light on protein structures of increasing size and complexity. To accomplish this, small reagents are used that typically incorporate two amine reactive moieties connected by a spacer arm and that can be applied in solution to protein structures of any size. Over the years, many reagents initially developed for different applications were adopted, and others were specifically developed for XL-MS. This has resulted in a vast array of options, making it difficult to make the right choice for specific experiments. Here, we delve into the previous decade of published XL-MS literature to uncover which workflows have been predominantly applied. We focus on application papers as these represent proof that biologically valid results can be extracted. This ignores some more recent approaches that did not have sufficient time to become more widely applied, for which we supply a separate discussion. From our selection, we extract information on the types of samples, cross-linking reagent, prefractionation, instruments, and data analysis, to highlight widely used workflows. All of the results are summarized in an easy-to-use flow chart defined by selection points resulting from our analysis. Although potentially biased by our own experiences, we expect this overview to be useful for novices stepping into this rapidly expanding field.