Protein chemistry, combined with modern mass spectrometric techniques, is an emerging technology for the structural analysis of proteins and protein complexes. It can be used to overcome the limitations of the current structural biology technologies like X-Ray crystallography and NMR regarding protein amount and concentration. Furthermore, these approaches are mainly restricted to in-vitro systems and certain protein classes.
The Proteomics Centre focuses on the characterization of changes in protein conformation and protein-protein interactions by combining crosslinking and/or hydrogen/deuterium exchange (HDX) and mass spectrometry. The analysis of protein-ligand interactions are based on photoaffinity labelling.
HDX Combined with Top-down MS for Elucidating Protein Tertiary Structure and Changes in Protein Conformation
The analysis of intact proteins through top-down proteomics would be ideal for using the HDX approaches to characterize the tertiary structure and conformational changes in proteins since the major limitation for HDX experiments is back-exchange of H/D which can occur during the proteolysis. Conversely the foremost challenge of top-down MS experiments is H/D “scrambling” during MS sequencing which can lead to incorrect results. However, combining the advantages of FTICR-MS with novel sequencing mechanism like Electron Capture Dissociation (ECD) that minimizes H/D scrambling enables top-down experiments for HDX, as Dr. Borchers has recently demonstrated in a paper in JACS.
- Pan, J., Han, J., Borchers, C.H., Konermann L. Characterizing Short-Lived Protein Folding Intermediates by Top-Down Hydrogen Exchange Mass Spectrometry. Analytical Chemistry, (2010).
- Han, J. and Borchers, C.H.Top-down analysis of recombinant histone H3 and its methylated analogs by ESI/FTICR mass spectrometry. Proteomics, (2010)
- Pan J., Han, J., Borchers, C.H., Konermann, L. Hydrogen/Deuterium Exchange Mass Spectrometry with Top-Down Electron Capture Dissociation for Characterizing Structural Transitions of a 17 kDa Protein. J Am Chem Soc. 131(35) :12801-8. (2009)
- Pan, J, Han, J, Borchers, C.H, Konermann, L. Electron Capture Dissociation of Electrosprayed Protein Ions for Spatially-Resolved Hydrogen Exchange Measurements. J American Chem Soc. 130(35): 11574-5. (2008)
- Ouvry-Patat, S.A., Torres, M.P., Quek, H., Gelfand, C.A., Schroeder, G.K., Han, J., Elliott, M., Dryhurst, D., Ausio, J., Wolfenden, R., Borchers, C.H. Free Flow Electrophoresis for Top-Down Proteomics by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Proteomics. 8:2798-2808. (2008)
- Borchers, C.H.,, Thapar, R., Petrotchenko, E.V., Torres M.P., Speir, S.P., Easterling M., Dominski, Z., Marzluff, W.F., Combined Top-down and Bottom-up Proteomics Identifies a Phosphorylation Site in Stem-Loop Binding Proteins That Contributes to High-affinity RNA Binding. PNAS. 103(9): 3094-3099. (2006)
Chemical Crosslinking Combined with MS Approaches for Characterizing Protein-Protein Interactions
The concept of chemical crosslinking followed by mass spectrometric characterization of crosslinked peptides (also called “crosslinks”) is well-proven and straight forward; however, there are numerous challenges inherent in this approach. First, the combinatorial nature of the crosslinks creates an intrinsic problem of identification, if identification of the crosslinked peptides is done by mass alone. Second, mass spectrometric sequencing of crosslinked peptides often results in low quality or difficult to interpret MS/MS spectra because of insufficient or complex fragmentation. The relatively low number of crosslinks compared to non-crosslinker-containing peptides that are obtained by proteolysis of the crosslinked protein complex, results in further complications.
Several recent developments help address these issues. Mass spectrometers capable of determining molecular weight with high mass accuracy, such as FTICR-MS, reduces the number of potential crosslinks which can be assigned to a specific mass. A further advance is the development of isotopically-coded crosslinkers and proteolytic isotopic labelling of the crosslinks which provide a specific mass spectrometric “signature” for crosslinks. This has been crucial for the easy MS detection of crosslinks. Dr. Borchers has recently reported the development of isotopically-coded chemically cleavable crosslinkers which allow discrimination between the crosslink types (dead-end, intrapeptide, or interpeptide), and facilitates subsequent MS/MS sequencing of the individual crosslinked peptides.
To advance the applicability of the MS-based Structural Proteomics approach, the Proteomics Centre has custom synthesized a panel of novel crosslinkers, which are isotopically-coded, cleavable, and affinity-tagged to facilitate specific separation, detection, and identification. In addition, we are developing novel techniques and software packages for automated data analysis thereby simplifying this process and making MS-based structural proteomics more accessible to a broader audience. The customized crosslinkers, and software packages are available through Creative Molecules.
- Petrotchenko, E.V., Serpa J., and Borchers, C.H. An isotopically-coded CID-cleavable biotinylated crosslinker for structural proteomics. Mol Cell Proteomics. (2010)
- Petrotchenko, E.V. and Borchers, C.H. Crosslinking combined with mass spectrometry for structural proteomics. Mass Spec Rev. (2010)
- Petrotchenko, E., Serpa J., Borchers, C.H. Use of a combination of isotopically-coded crosslinkers and isotopically-coded N-terminal modification reagents for selective identification of inter-peptide crosslinks. Anal Chem. 82(3): 817-23. (2010)
- Petrotchenko, E.V. and Borchers, C.H. ICC-CLASS: Isotopically-Coded Cleavable Cross-Linking Analysis Software Suite. BMC Bioinformatics. 11(1): 64. (2010)
- Petrotchenko, E.V., Xiao K, Cable J, Chen Y, Dokholyan, N.V., Borchers, C.H. BiPS, a photo-cleavable, isotopically-coded, fluorescent crosslinker for structural proteomics. Mol Cell Proteomics. 8:273-286. (2008)
- Petrotchenko, E.V. and Borchers, C.H., Cross-Linking as a Tool Examine Protein Complexes: Examples of Crosslinking Strategies and Computational Modeling. In: Mass Spectrometry Analysis For Protein-Protein Interactions and Dynamics, Chance, M., (ed.) John Willey & Sons, Inc. Chapter 8, 157-168. (2008)
- Petrotchenko, E.V., Olkhovik, V.K., Borchers, C.H. Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes. Mol Cell Proteomics. 4: 1167-1179. (2005)
- Wine, R.N., Dial, J.M., Tomer, K.B., Borchers, C.H., Identification of Components of Protein Complexes Using a Fluorescent Photo-Cross-Linker and Mass Spectrometry. Anal Chem. 74: 1939-1945. (2002)
- Borchers, C., Tomer, K.B., Characterization of the Non-Covalent Complex of HIV-gp120 with its Cellular Receptor CD4 by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Biochemistry. 38: 11734-11740. (1999)