Sulfoxide-Containing Mass Spectrometry (MS)-Cleavable Cross-Linkers

Cross-linking mass spectrometry is an emergent technology for interactomics and structural biology1. Chemical cross-linking enables the capture of protein-protein interactions (PPIs) in native cellular environments, and the identification of cross-linked peptides permits the determination of both identity and connectivity of PPIs. In addition, the resulting cross-links can be utilized as distance constraints for various applications, ranging from structure validation and integrative modeling to de novo structure prediction. The development of sulfoxide-containing MS-cleavable cross-linkers enables fast, simplified and accurate identification of cross-linked peptides to significantly advance targeted and proteome-wide XL-MS studies for PPI mapping in vitro and in vivo.

Table 1. Sulfoxide-containing MS-cleavable Homobifunctional Cross-linkers

Advantages of MS-cleavable cross-linkers:

Fast, Simplified and Accurate Identification of Cross-linked Peptides

These homobifunctional sulfoxide-containing MS-cleavable cross-linkers possess two symmetric C-S bonds that can be preferentially cleaved in the gas phase using collision-induced dissociation (CID) during tandem mass spectrometry (MS/MS or MS2). This results in the physical separation of a cross-link to yield unique peptide fragment pairs with a defined mass relationship1-7. These characteristic and predictable MS2 fragment ion pairs are then subjected to MS3 analysis for simplified and unambiguous identification of cross-linked peptides by conventional database searching tools. The MSn-based workflow provides fast and accurate cross-link identification at various scales including systems-wide analysis. In addition, these MS-cleavable cross-linkers provide the flexibility of allowing MS2-based and MS2-MS3-combined workflows for cross-link identification by implementing higher-energy collisional dissociation (HCD) and/or electron transfer dissociation (ETD)1.

Quantitative XL-MS Analysis to Define Interaction and Structural Dynamics

The isotope-coded cross-linkers (d0/d10-DMDSSO) can be used for pair-wise comparison of conformational changes of protein complexes1, 8. All of the listed cross-linkers can be coupled with isobaric labeling reagents (e.g. TMT) for multiplexed quantitative XL-MS analyses1,9. In addition, parallel reaction monitoring (PRM) can be employed for targeted quantitative XL-MS analyses10.

Combinatory XL-MS Analysis to Expand PPI Coverage

Cross-linkers with distinct cross-linking chemistries (e.g. DSSO, DHSO, and BMSO) can be integrated for combinatory XL-MS analyses to expand PPI coverage and facilitate structure modeling of protein complexes10.

A Wide-range of Applications for Mapping PPIs in Vitro and in Vivo1

These reagents have been successfully applied to map PPIs at the proteome-wide scale and elucidate architectures of protein complexes in vivo and in vitro.

  • Cross-linking of proteins and protein complexes
  • Cross-linking of subcellular organelles and cell lysates
  • Cross-linking of intact cells and tissues

Materials
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References

1.
Yu C, Huang L. 2018. Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology. Anal. Chem.. 90(1):144-165. http://dx.doi.org/10.1021/acs.analchem.7b04431
2.
Kao A, Chiu C, Vellucci D, Yang Y, Patel VR, Guan S, Randall A, Baldi P, Rychnovsky SD, Huang L. 2011. Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes. Mol Cell Proteomics. 10(1):M110.002212. http://dx.doi.org/10.1074/mcp.m110.002212
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Yu C, Kandur W, Kao A, Rychnovsky S, Huang L. 2014. Developing New Isotope-Coded Mass Spectrometry-Cleavable Cross-Linkers for Elucidating Protein Structures. Anal. Chem.. 86(4):2099-2106. http://dx.doi.org/10.1021/ac403636b
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Kaake RM, Wang X, Burke A, Yu C, Kandur W, Yang Y, Novtisky EJ, Second T, Duan J, Kao A, et al. 2014. A Newin VivoCross-linking Mass Spectrometry Platform to Define Protein?Protein Interactions in Living Cells. Mol Cell Proteomics. 13(12):3533-3543. http://dx.doi.org/10.1074/mcp.m114.042630
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Burke AM, Kandur W, Novitsky EJ, Kaake RM, Yu C, Kao A, Vellucci D, Huang L, Rychnovsky SD. Synthesis of two new enrichable and MS-cleavable cross-linkers to define protein?protein interactions by mass spectrometry. Org. Biomol. Chem.. 13(17):5030-5037. http://dx.doi.org/10.1039/c5ob00488h
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Gutierrez CB, Yu C, Novitsky EJ, Huszagh AS, Rychnovsky SD, Huang L. 2016. Developing an Acidic Residue Reactive and Sulfoxide-Containing MS-Cleavable Homobifunctional Cross-Linker for Probing Protein?Protein Interactions. Anal. Chem.. 88(16):8315-8322. http://dx.doi.org/10.1021/acs.analchem.6b02240
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Gutierrez CB, Block SA, Yu C, Soohoo SM, Huszagh AS, Rychnovsky SD, Huang L. 2018. Development of a Novel Sulfoxide-Containing MS-Cleavable Homobifunctional Cysteine-Reactive Cross-Linker for Studying Protein?Protein Interactions. Anal. Chem.. 90(12):7600-7607. http://dx.doi.org/10.1021/acs.analchem.8b01287
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Yu C, Mao H, Novitsky EJ, Tang X, Rychnovsky SD, Zheng N, Huang L. 2015. Gln40 deamidation blocks structural reconfiguration and activation of SCF ubiquitin ligase complex by Nedd8. Nat Commun. 6(1): http://dx.doi.org/10.1038/ncomms10053
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Yu C, Huszagh A, Viner R, Novitsky EJ, Rychnovsky SD, Huang L. 2016. Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes. Anal. Chem.. 88(20):10301-10308. http://dx.doi.org/10.1021/acs.analchem.6b03148
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Gutierrez C, Chemmama IE, Mao H, Yu C, Echeverria I, Block SA, Rychnovsky SD, Zheng N, Sali A, Huang L. 2020. Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling. Proc Natl Acad Sci USA. 117(8):4088-4098. http://dx.doi.org/10.1073/pnas.1915542117