Efficient and robust proteome-wide approaches for cross-linking mass spectrometry


Cross-linking mass spectrometry (XL-MS) has received considerable interest, owing to its potential to investigate protein–protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison with other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by the use of bifunctional cross-linking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by MS. XL-MS has received the required boost to tackle more-complex samples with recent advances in cross-linking chemistry with MS-cleavable or reporter-based cross-linkers and faster, more sensitive and more versatile MS platforms. This protocol provides a detailed description of our optimized conditions for a full-proteome native protein preparation followed by cross-linking using the gas-phase cleavable cross-linking reagent disuccinimidyl sulfoxide (DSSO). Following cross-linking, we demonstrate extensive sample fractionation and substantially simplified data analysis with XlinkX in Proteome Discoverer, as well as subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within ~10 d, including structural investigations.

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Fig. 1: Generic workflow for XL-MS experiments.
Fig. 2: Optimization of cross-linking reaction conditions by titration.
Fig. 3: SCX fractionation profile for whole PC9 cell lysate samples.
Fig. 4: Schematic representation of Proteome Discoverer workflows for identification of cross-linked peptides.
Fig. 5: Data overview.
Fig. 6: Mapping and application of identified crosslinks.

Data availability

Data are publicly available through PRIDE repository PXD008418.


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We thank all Heck-group members for their helpful contributions, and, in particular, F. Liu for her initial development in proteome-wide cross-linking, M. Damen and D. Hagemans for SCX support, and K. Dingess and K. Stecker for their help with the manuscript. From Thermo Fisher Scientific, we thank B. Delanghe, K. Fitzemeier and F. Berg for their collaboration on incorporating the XLinkX cross-link search engine into the Proteome Discoverer software and R. Viner for her collaborative work on DSSO cross-linking and support in mass spectrometry method development. We acknowledge financial support from the large-scale proteomics facility Proteins@Work (Project 184.032.201), embedded in the Netherlands Proteomics Centre and supported by the Netherlands Organization for Scientific Research (NWO). Additional support came from the European Union Horizon 2020 program FET-OPEN project MSmed, Project 686547.

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O.K., A.J.R.H. and R.A.S. conceived the study. O.K. prepared the cell extracts and acquired the MS data. O.K. and R.A.S. analyzed the data. B.S., S.P. and D.F. provided various optimizations of the protocol. All authors critically read and commented on the manuscript.

Corresponding author

Correspondence to Richard A. Scheltema.

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Key references using this protocol

Fagerlund, R. D. et al. Proc. Natl Acad. Sci. USA 114, E5122–E5128 (2017): http://www.pnas.org/content/114/26/E5122.long

Liu, F. et al. Nat. Commun. 8, 15473 (2017): https://www.nature.com/articles/ncomms15473

Benda, C. et al. Mol. Cell 56, 43–54 (2014): https://www.sciencedirect.com/science/article/pii/S1097276514007096?via%3Dihub

Fasci, D., van Ingen, H., Scheltema, R. A. & Heck, J. R. Mol. Cell Proteomics 17, 2018–2033 (2018). http://www.mcponline.org/content/17/2/216.long


Integrated supplementary information

Supplementary Figure 1 Setup of crosslinker properties in Proteome Discoverer.

Setting up DSSO as chemical modification. For calculating of the final modification mass, the NHS-esters must be subtracted from the initial mass of DSSO molecule. All possible reactive sites are exemplified. According to the DSSO fragmentation pathway, different fragments are formed: Alkene, Sulfenic acid and Thiol. Monolinks also must be set here, as shown for DSSO hydrolyzed and DSSO quenched with Tris. As connected fragments characteristic for DSSO, “Alkene” to “Sulfenic” acid and “Alkene” to “Thiol” are set.

Supplementary Figure 2 DisVis interaction prediction interface between LDH A and LDH B with defined distance restraint.

(a) Left view of LDH B with possible interaction interface of LDH A. (b) Right view. (c) Top view. (d) Bottom view.

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Klykov, O., Steigenberger, B., Pektaş, S. et al. Efficient and robust proteome-wide approaches for cross-linking mass spectrometry. Nat Protoc 13, 2964–2990 (2018). https://doi.org/10.1038/s41596-018-0074-x

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