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.