Structural Biology by Mass Spectrometry: 3D Proteomics of Supramolecular Assemblies
28/02/2012 - Room C209/10 at 14:00 - External Seminar
(The University of Edinburgh)
Current structural biology methods leave an information gap in the mid-resolution range at which protein interactions or conformation changes are defined at domain or sub-domain level. Mass spectrometry in conjunction with cross-linking is providing exactly this information. We have applied our tools to complexes up to 670 kDa in size, endogenous, tagged complexes and even whole cell lysates. We have furthermore analysed conformation changes in solution using stable isotope labelling for quantitative analyses.
We have transformed cross-linking/mass spectrometry from an expert approach to routine application by establishing an integrated workflow through having: (1) developed an enrichment strategy for cross-linked peptides based on charge; (2) characterised in detail the fragmentation behaviour of cross-linked peptides in a high resolution mass spectrometer; (3) derived lessons from this for a search algorithm that does not require isotope-labelled cross-linkers and overcomes the n2 problem of database searching for cross-links; and (4) written user friendly web-based search software that includes a revolutionary spectrum viewer for match evaluation with implications reaching beyond this field and a cross-link map viewer for fast hypothesis generation that expands the current visualisation concepts of protein network viewers such as used in STRING.
We believe that cross-linking/mass spectrometry is now ready for deployment into structural and molecular biology laboratories for routine application.