Genomics:GTL

Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.

Genomics:GTL Program Projects

Oak Ridge National Laboratory and Pacific Northwest National Laboratory

10

Investigating Gas Phase Dissociation Pathways of Crosslinked Peptides: Application to Protein Complex Determination

Sara P. Gaucher* (spgauch@sandia.gov), Masood Z. Hadi, and Malin M. Young

Sandia National Laboratories, Livermore, CA

Chemical crosslinking is an important tool for probing protein structure¹ and protein-protein interactions.²-3 The approach usually involves crosslinking of specific amino acids within a folded protein or protein complex, enzymatic digestion of the crosslinked protein(s), and identification of the resulting crosslinked peptides by liquid chromatography/mass spectrometry (LC/MS). In this manner, distance constraints are obtained for residues that must be in close proximity to one another in the native structure or complex. As the complexity of the system under study increases, for example, a large multi-protein complex, simply measuring the mass of a crosslinked species will not always be sufficient to determine the identity of the crosslinked peptides. In such a case, tandem mass spectrometry (MS/MS) could provide the required information if the data can be properly interpreted. In MS/MS, a species of interest is isolated in the gas phase and allowed to undergo collision induced dissociation (CID). Because the gas-phase dissociation pathways of peptides have been well studied, methods are established for determining peptide sequence by MS/MS. However, although crosslinked peptides dissociate through some of the same pathways as isolated peptides, the additional dissociation pathways available to the former have not been studied in detail. Software such as MS2Assign⁴ has been written to assist in the interpretation of MS/MS from crosslinked peptide species, but it would be greatly enhanced by a more thorough understanding of how these species dissociate. We are thus systematically investigating the dissociation pathways open to crosslinked peptide species. A series of polyalanine and polyglycine model peptides have been synthesized containing one or two lysine residues to generate defined inter- and intra-molecular crosslinked species, respectively. Each peptide contains 11 total residues, and one arginine residue is present at the carboxy terminus to mimic species generated by tryptic digestion. The peptides have been allowed to react with a series of commonly used crosslinkers such as DSS, DSG, and DST. The tandem mass spectra acquired for these crosslinked species are being examined as a function of crosslinker identity, site(s) of crosslinking, and precursor charge state. Results from these model studies and observations from actual experimental systems are being incorporated into the MS2Assign software to enhance our ability to effectively use chemical crosslinking in protein complex determination.

This project is funded by the DOE Genomics:GTL Program Project lead by ORNL/PNNL.

References

1. Young, MM; Tang, N; Hempel, JC; Oshiro, CM; Taylor, EW; Kuntz, ID; Gibson, BW; Dollinger, G. “High throughput protein fold identification by using experimental constraints derived from intramolecular crosslinks and mass spectrometry.” PNAS 2000, 97, 5802-5806.
2. Lanman, J; Lam, TT; Barnes, S; Sakalian, M; Emmett, MR; Marshall, AG; Prevelige, PE. “Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry.” J. Mol. Biol. 2003, 325, 759-772.
3. Rappsilber, J.; Siniossoglou, S; Hurt, EC; Mann, M. “A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry.” Anal. Chem. 2000, 72, 267-275.
4. Schilling, B; Row, RH; Gibson, BW; Guo, X; Young, MM. “MS2Assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides.” J. Am. Soc. Mass Spectrom. 2003, 14, 834-850.

* Presenting author