To Cleave or Not To Cleave in XL-MS?

Abstract

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.

Cross-linking/mass spectrometry at the crossroads

“…Clearly, amine-reactive N -hydroxysuccinimide (NHS) esters targeting lysine residues, but also serines, threonines, and tyrosines, are the cross-linkers overwhelmingly used to date. As far as the nature of the cross-linker itself is concerned, i.e., whether cleavable versus non-cleavable, approximately 78% of all studies currently make use of non-cleavable cross-linkers, such as disuccinimidylsuberate (DSS) and bis(sulfosuccinimidyl)suberate (BS 3 ) [ 12 ]. Both cross-linkers differ only in a sulfonic acid group that is incorporated into BS 3 to enhance water solubility and to bridge a distance of 11.4 Å, resulting in Cα-Cα distances of ~ 27 Å. MS-cleavable cross-linkers, such as disuccinimidyl sulfoxide (DSSO) and disuccinimidyldibutyric urea (DSBU) (Table 1 ), are increasingly being used as they allow targeted identification of the cross-linked product based on characteristic fragments generated during MS/MS experiments.…”

Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass-spectrometry

“…XL-MS has a particular utility to capture protein-protein interactions in solution by measuring spatial distance restraints, mirroring structural conformations of intact proteins. Concomitantly, a wide range of chemical cross-linkers have been explored so far, often relying on similar chemical principles (Sinz, 2003;Steigenberger et al, 2020). Most used cross-linkers are small, homobifunctional reagents, with two reactive moieties capable of covalently binding two nearby amino acids.…”

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

“…protein sequences and post-translational modifications) are typically not apparent with a technique like cryo-EM but are readily accessible by structural proteomics (3). At the same time, spatial information within and between proteins can be obtained by the use of cross-linking mass spectrometry (XL-MS) (4)(5)(6)(7)(8).…”