17th International Mass Spectrometry Conference :: Prague, 2006
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|Session:||Protein Conformation and Structure Biology|
|Presentation date:||Thu, Aug 31, 2006|
|Presentation time:||14:30 – 16:00|
Martin Sadilek1, Oleksandr Kalyuzhniy1, Catalin Doneanu1, Jan Seebacher2, Michael H. Gelb1, Ruedi Aebersold3, Dmitry M. Shayakmetov11 University of Washington, Seattle, United States
Correspondence address: Martin Sadilek, University of Washington, Chemistry, Box 351700, Seattle, 98195-1700 United States.
Keywords: Computer Program; Cross-linking; Labeling, Isotope; Virus.
Novel aspect: Analysis of protein interaction using stable isotope labeled cross-linkers and new software tools.
Adenoviruses (Ad) are efficient vehicles for gene delivery. However, major obstacles to clinical use include innate immune response and Ad sequestration by non-target tissues. The factors and mechanisms underlying these processes are poorly understood.
A/ Adenovirus Host Factors Identification
Recombinant Ad5 and Ad35 fiber knob domains were expressed in E. coli and purified. Proteins were conjugated to agarose beads and incubated in mouse plasma. Eluted interacting proteins were digested and subjected to LC/ESI-MS/MS analysis. The low concentration blood factor proteins were tested for interaction with the knob domains using slot blot analysis with purified plasma proteins immobilized on a nitrocellulose membrane.
11 plasma proteins were identified with high confidence. From all these knob-binding plasma proteins only the coagulation factor IX (FIX) and C4 binding protein lead to high level liver cell infection.
B/ Interaction of FIX with adenovirus protein
To better understand the structural basis of Ad fiber knob domain interaction with FIX, we analyzed the efficiency of FIX-Ad knob association using dynamic light scattering. The data suggest that interaction between hydrophilic amino acids in FIX and Ad fiber knob domain, rather than hydrophobic amino acids, plays the major role in complex formation.
To further identify the precise areas of protein-protein interaction, we chemically cross-linked FIX with purified recombinant Ad5 or Ad9 fiber knob domains, enzymatically digested and analyzed digests by tandem mass spectrometry.
We have used stable isotope labeled cross-linkers to generate pairs of peptide signal in the mass spectrum to detect the cross-linked peptides. In addition, by using of 16O/18O water during digestion one can distinguish between true cross-links and the peptide modifications. The samples were analyzed using a MALDI TOF-TOF and nanoLC/ESI-QTOF mass spectrometry.
The integrated software tools iXLINK, doXLINK, and XLinkViewer were developed to assist the workflow of sample analysis. The software generates a list of possible cross-links and compares it with the peaks from the MALDI MS spectra. It provides a list of ions for subsequent MS/MS analysis to confirm the sequence and to locate the cross-linked residues.
Analysis of the cross-linked Ad fiber knob-FIX complex using tandem mass spectrometry revealed several candidate peptides participating in the cross-linking reaction. These peptides define the interface of Ad fiber knob-FIX interaction. To confirm mass spectrometry analysis data, we are currently generating a panel of Ad fiber knobs possessing amino acid mutations within the putative protein interface area.
This mass spectrometric approach may be widely applied to study interactions of virus proteins with host factors in vitro and in vivo and ultimately allow for the construction of safe and efficient adenovirus vectors for therapeutic application.