Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2000 Aug;9(8):1503–1518. doi: 10.1110/ps.9.8.1503

Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts.

K L Bennett 1, M Kussmann 1, P Björk 1, M Godzwon 1, M Mikkelsen 1, P Sørensen 1, P Roepstorff 1
PMCID: PMC2144725  PMID: 10975572

Abstract

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.

Full Text

The Full Text of this article is available as a PDF (863.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Artemyev N. O., Mills J. S., Thornburg K. R., Knapp D. R., Schey K. L., Hamm H. E. A site on transducin alpha-subunit of interaction with the polycationic region of cGMP phosphodiesterase inhibitory subunit. J Biol Chem. 1993 Nov 5;268(31):23611–23615. [PubMed] [Google Scholar]
  2. Ayed A., Krutchinsky A. N., Ens W., Standing K. G., Duckworth H. W. Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 1998;12(7):339–344. doi: 10.1002/(SICI)1097-0231(19980415)12:7<339::AID-RCM163>3.0.CO;2-6. [DOI] [PubMed] [Google Scholar]
  3. Davies G. E., Stark G. R. Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc Natl Acad Sci U S A. 1970 Jul;66(3):651–656. doi: 10.1073/pnas.66.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Farmer T. B., Caprioli R. M. Determination of protein-protein interactions by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom. 1998 Aug;33(8):697–704. doi: 10.1002/(SICI)1096-9888(199808)33:8<697::AID-JMS711>3.0.CO;2-H. [DOI] [PubMed] [Google Scholar]
  5. Gobom J., Nordhoff E., Mirgorodskaya E., Ekman R., Roepstorff P. Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom. 1999 Feb;34(2):105–116. doi: 10.1002/(SICI)1096-9888(199902)34:2<105::AID-JMS768>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
  6. Greenfield E. A., Nguyen K. A., Kuchroo V. K. CD28/B7 costimulation: a review. Crit Rev Immunol. 1998;18(5):389–418. doi: 10.1615/critrevimmunol.v18.i5.10. [DOI] [PubMed] [Google Scholar]
  7. Haniu M., Horan T., Arakawa T., Le J., Katta V., Rohde M. F. Extracellular domain of granulocyte-colony stimulating factor receptor. Interaction with its ligand and identification of a domain in close proximity of ligand-binding region. Arch Biochem Biophys. 1995 Dec 20;324(2):344–356. doi: 10.1006/abbi.1995.0047. [DOI] [PubMed] [Google Scholar]
  8. Hegyi G., Mák M., Kim E., Elzinga M., Muhlrad A., Reisler E. Intrastrand cross-linked actin between Gln-41 and Cys-374. I. Mapping of sites cross-linked in F-actin by N-(4-azido-2-nitrophenyl) putrescine. Biochemistry. 1998 Dec 22;37(51):17784–17792. doi: 10.1021/bi981285j. [DOI] [PubMed] [Google Scholar]
  9. Helin J., Caldentey J., Kalkkinen N., Bamford D. H. Analysis of the multimeric state of proteins by matrix assisted laser desorption/ionization mass spectrometry after cross-linking with glutaraldehyde. Rapid Commun Mass Spectrom. 1999;13(3):185–190. doi: 10.1002/(SICI)1097-0231(19990215)13:3<185::AID-RCM481>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  10. Jensen R. B., Lurz R., Gerdes K. Mechanism of DNA segregation in prokaryotes: replicon pairing by parC of plasmid R1. Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8550–8555. doi: 10.1073/pnas.95.15.8550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jung S. M., Moroi M. Crosslinking of platelet glycoprotein Ib by N-succinimidyl(4-azidophenyldithio)propionate and 3,3'-dithiobis(sulfosuccinimidyl propionate). Biochim Biophys Acta. 1983 Dec 13;761(2):152–162. doi: 10.1016/0304-4165(83)90224-6. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Leffak I. M. Decreased protein staining after chemical crosslinking. Anal Biochem. 1983 Nov;135(1):95–101. doi: 10.1016/0003-2697(83)90735-2. [DOI] [PubMed] [Google Scholar]
  14. Lomant A. J., Fairbanks G. Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate). J Mol Biol. 1976 Jun 14;104(1):243–261. doi: 10.1016/0022-2836(76)90011-5. [DOI] [PubMed] [Google Scholar]
  15. Loo J. A. Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom Rev. 1997 Jan-Feb;16(1):1–23. doi: 10.1002/(SICI)1098-2787(1997)16:1<1::AID-MAS1>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  16. Machold J., Utkin Y., Kirsch D., Kaufmann R., Tsetlin V., Hucho F. Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7282–7286. doi: 10.1073/pnas.92.16.7282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Metzler W. J., Bajorath J., Fenderson W., Shaw S. Y., Constantine K. L., Naemura J., Leytze G., Peach R. J., Lavoie T. B., Mueller L. Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28. Nat Struct Biol. 1997 Jul;4(7):527–531. doi: 10.1038/nsb0797-527. [DOI] [PubMed] [Google Scholar]
  18. Mills J. S., Miettinen H. M., Barnidge D., Vlases M. J., Wimer-Mackin S., Dratz E. A., Sunner J., Jesaitis A. J. Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry. J Biol Chem. 1998 Apr 24;273(17):10428–10435. doi: 10.1074/jbc.273.17.10428. [DOI] [PubMed] [Google Scholar]
  19. Neubauer G., King A., Rappsilber J., Calvio C., Watson M., Ajuh P., Sleeman J., Lamond A., Mann M. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat Genet. 1998 Sep;20(1):46–50. doi: 10.1038/1700. [DOI] [PubMed] [Google Scholar]
  20. Ngai S. M., Sönnichsen F. D., Hodges R. S. Photochemical cross-linking between native rabbit skeletal troponin C and benzoylbenzoyl-troponin I inhibitory peptide, residues 104-115. J Biol Chem. 1994 Jan 21;269(3):2165–2172. [PubMed] [Google Scholar]
  21. Patterson S. D., Katta V. Prompt fragmentation of disulfide-linked peptides during matrix-assisted laser desorption ionization mass spectrometry. Anal Chem. 1994 Nov 1;66(21):3727–3732. doi: 10.1021/ac00093a030. [DOI] [PubMed] [Google Scholar]
  22. Roepstorff P., Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 1984 Nov;11(11):601–601. doi: 10.1002/bms.1200111109. [DOI] [PubMed] [Google Scholar]
  23. Rossi V., Gaboriaud C., Lacroix M., Ulrich J., Fontecilla-Camps J. C., Gagnon J., Arlaud G. J. Structure of the catalytic region of human complement protease C1s: study by chemical cross-linking and three-dimensional homology modeling. Biochemistry. 1995 Jun 6;34(22):7311–7321. doi: 10.1021/bi00022a004. [DOI] [PubMed] [Google Scholar]
  24. Scaloni A., Miraglia N., Orrù S., Amodeo P., Motta A., Marino G., Pucci P. Topology of the calmodulin-melittin complex. J Mol Biol. 1998 Apr 10;277(4):945–958. doi: 10.1006/jmbi.1998.1629. [DOI] [PubMed] [Google Scholar]
  25. Shevchenko A., Wilm M., Vorm O., Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996 Mar 1;68(5):850–858. doi: 10.1021/ac950914h. [DOI] [PubMed] [Google Scholar]
  26. Yang T., Horejsh D. R., Mahan K. J., Zaluzec E. J., Watson T. J., Gage D. A. Mapping cross-linking sites in modified proteins with mass spectrometry: an application to cross-linked hemoglobins. Anal Biochem. 1996 Nov 1;242(1):55–63. doi: 10.1006/abio.1996.0427. [DOI] [PubMed] [Google Scholar]
  27. Yu Z., Friso G., Miranda J. J., Patel M. J., Lo-Tseng T., Moore E. G., Burlingame A. L. Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques. Protein Sci. 1997 Dec;6(12):2568–2577. doi: 10.1002/pro.5560061209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zappacosta F., Ingallinella P., Scaloni A., Pessi A., Bianchi E., Sollazzo M., Tramontano A., Marino G., Pucci P. Surface topology of Minibody by selective chemical modifications and mass spectrometry. Protein Sci. 1997 Sep;6(9):1901–1909. doi: 10.1002/pro.5560060911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. van der Merwe P. A., Bodian D. L., Daenke S., Linsley P., Davis S. J. CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics. J Exp Med. 1997 Feb 3;185(3):393–403. doi: 10.1084/jem.185.3.393. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES