Quantitative cross-linking/mass spectrometry (QCLMS/QXL-MS) probes structural changes of proteins in solution. This method has revealed induced conformational changes, composition shifts in conformational ensembles and changes in protein interactions. It illuminates different structural states of proteins or protein complexes by comparing which residue pairs can be cross-linked in these states. Cross-links provide information about structural changes that may be inaccessible by alternative technologies. Small local conformational changes affect relative abundances of nearby cross-links, whereas larger conformational changes cause new cross-links to be formed. Distinguishing between minor and major changes requires a robust analysis based on carefully selected replicates and, when using isotope-labeled cross-linkers, replicated analysis with a permutated isotope-labeling scheme. A label-free workflow allows for application of a wide range of cross-linking chemistries and enables parallel comparison of multiple conformations. In this protocol, we demonstrate both label-free and isotope-labeled cross-linker-based workflows using the cross-linker bis(sulfosuccinimidyl)suberate (BS3). The software XiSearch, developed by our group, is used to identify cross-linked residue pairs, although the workflow is not limited to this search software. The open-access software Skyline is used for automated quantitation. Note that additional manual correction greatly enhances quantitation accuracy. The protocol has been applied to purified multi-protein complexes but is not necessarily limited to that level of sample complexity. Optimizing the cross-linker/protein ratio and fractionating peptides increase the data density of quantified cross-links, and thus the resolution of QCLMS. The entire procedure takes ~1–3 weeks.
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This work was supported by the Wellcome Trust through a Senior Research Fellowship to J.R. (103139), a Centre Core Grant (203149) and an Instrument Grant (108504).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
O’Reilly, F. J. & Rappsilber, J. Nat. Struct. Mol. Biol. (2018): https://doi.org/10.1038/s41594-018-0147-0
Fischer, L., Chen, Z. A. & Rappsilber, J. J. Proteomics 88, 120–128 (2013): https://www.sciencedirect.com/science/article/pii/S1874391913001449?via%3Dihub
Chen, Z. A., Fischer, L., Cox, J. & Rappsilber, J. Mol. Cell. Proteomics 15, 2769–2778 (2016): http://www.mcponline.org/content/15/8/2769.long
Skyline is available at https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_ms1_filtering and XiSearch at https://github.com/Rappsilber-Laboratory/XiSearch.
Integrated supplementary information
Supplementary Figure 1 Example results of cross-linking titration experiments for proteins and protein complexes.
(a) The SDS-PAGE image shows the cross-linking titration results for the Pol II complex. The protein/cross-linker molar ratio of 1:1800 (marked with red frame) is optimal for CLMS analysis, since at this ratio, bands of free subunits disappears and a defined band with MW corresponding to monomeric Pol II complex forms, in addition, there is very little oligomeric complex forms. (b) The protein/cross-linker mass ratio of 1:2 is select for CLMS analysis for protein MECP2, the broader band of cross-linked MECP2 confirms the cross-linking reaction, and the different appearance of the band in comparison with the cross-linked protein using a higher ratio of cross-linker indicates that cross-linking reaction is not saturated at the selected condition. (c).The wild type (WT) and the mutant (Mu) versions of protein Tsr1 yield different products when cross-linked. The protein/cross-linker mass ratio of 1:1 is selected for QCLMS analysis, as at this ratio, both the WT and the mutant protein obtain reasonable amount of monomeric cross-linking products.
Supplementary Figure 2 Schematic demonstration of how to generate pseudo identification entries in the .ssl file.
(a) The extracted ion chromatogram (XIC) of the BS3- (light) and the BS3-d4- (heavy) version of a cross-linked peptide (the MS1 spectrum is shown below). The overlapped elution window of the light and the heavy versions of the cross-linked peptide allowed for applying the retention time of the identified MS2 event of the light version as the retention time of the pseudo identification event for the heavy version which not been actually identified. (b) An example on how to generate a pseudo identification entry of a BS3-d4 cross-linked peptide in an .ssl file, based on the MS2 information of the identified BS3 cross-linked version of this cross-linked peptide.
Supplementary Figure 3 Curved HPLC gradient applied for SCX-based enrichment of cross-linked peptides.
The curved gradient is shown on the left and the equation of the curved gradient is shown on the right.
Demonstration of how to define modifications in Skyline.
Transition settings that need to be defined when quantifying cross-linked peptides.
Demonstration of how to build a library in Skyline.
(a) An example of a correctly isolated chromatographic signal of a peptide. The elution peak is correctly isolated; the isotope distribution of the isolated signals is in good agreement (with the “idotp” close to 1) with the expected pattern calculated based on the sequence of the target peptide; the measured signals of isotope peaks are in good matches with the calculated m/z of isotope peaks of the target peptide. (b) One error on peptide signal picking is incorrectly isolated elution peak of the target peptide. (c) Skyline might pick wrong signals for a target peptide, the error can be indicated by poor agreement on isotope distribution between the expected and the measured pattern, showing as low “idotp”. (d) The mistakenly selected signal may share the same charge as the target peptide which might resulted in an “idotp” value close to one. Such signal picking error would only be revealed when compare isotope peaks of the isolated signal in the MS1 spectra with the m/z of expected isotope peaks of the target peptide.
Supplementary Figure 8 Mis-quantitation of a singlet cross-linked peptide as a consequence of overlapping signals of the light and the heavy versions.
The MS1 spectra show the signals of an example cross-linked peptide in both the forward-labeled (conformer 1+BS3/conformer II+BS3-d4) and the reverse-labeled (conformer I+BS3-d4/conformer II+BS3) analysis. The isotope peaks used for quantitation are highlighted (blue for the light version and pink for the heavy version of the peptide). The extended isotope envelope of the example peptides leads to an overlap between the signals of the light and the heavy versions. Therefore in the forward-labeled dataset, this conformer I-unique cross-linked peptide is mis-quantified as a “doublet” by the automated data process.
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Chen, Z.A., Rappsilber, J. Quantitative cross-linking/mass spectrometry to elucidate structural changes in proteins and their complexes. Nat Protoc 14, 171–201 (2019). https://doi.org/10.1038/s41596-018-0089-3
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