Proteomics of oxidative stress-induced immunological disorders
Keywords
post-translational modifications, oxidative stress, protein complexes, mass spectrometry, proteomics, kinases, quantitative proteomics, immunology
Research interest
The Bennett group has a broad range of research interests that are predominantly focused on proteins, protein complexes and post-translational modifications (PTMs) involved in immunology/infection. These areas encompass the application of mass spectrometry to understand complex biological systems and the molecular mechanisms underlying disease states and include: (i) protein-protein interactions chemical crosslinking to generate protein interactomes; (ii) drug-protein interactions; (iii) quantitative expression proteomics for the relative and comparative quantitative analysis of global proteomes; and (iv) monitoring the role of PTMs in cellular signaling pathways. More specifically, our interests lie in the enrichment and isolation of non-covalent protein complexes under near-physiological conditions and monitoring the effects of perturbation and/or stimulation on protein complex rearrangement. We routinely apply quantitative gel-free proteomic approaches to assess the global proteome following such perturbation or stimulation of cellular systems by, e.g., drugs, infective agents, mutated proteins, exogenous stimuli; and (ii) perform comparative analyses of sub-proteomes, e.g., affinity-purified protein complexes, chromatin, drug pulldowns. As detailed investigation of modified forms of proteins is a prerequisite for understanding the dynamics of complex biological systems, another of our major research interests is in monitoring post-translation modifications. The approaches adopted by the group are: (i) global phosphopeptide enrichment via Fe(III)-NTA; (ii) antibody-based enrichment of modified peptides, e.g., phoshotyrosine, acetyl-lysine; and (iii) a two-step antibody- and chemically-based enrichment of oxidation-specific epitopes.
Collaborating research groups where PhD Students can perform their research stay
Henning Jacob Jessen, University of Zürich, Department of Chemistry, Winterthurerstrasse 190, 8057 Zürich, Switzerland
Albert Heck, Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands and Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
Know-how and infrastructure of the research group
Our group will bring to the DC expertise in tandem affinity purifications and affinity purification in general, chemical crosslinking of proteins and protein complexes, sample preparation of protein complexes ( crosslinking) for analysis by mass spectrometry, liquid chromatography mass spectrometry, relative quantitation of proteins via isobaric chemical labeling, antibody and chemical enrichment of post-translationally modified proteins, plus data analysis using internal and freeware tools. The group is equipped with three hybrid linear trap (LTQ) Orbitrap Velos mass spectrometers (ThermoFisher Scientific, Waltham, MA) and one quadrupole orbitrap (Q-Exactive) instrument. Each mass spectrometer is coupled to a nano-high performance liquid chromatography (HPLC) system (Agilent Technologies, Palo Alto, CA) for on-line liquid chromatography mass spectrometry (LCMS). An additional HPLC is available for peptide and/or protein fractionation. Laboratory infrastructure also includes a shared cell culture area and biochemistry laboratory, plus a separate space for sample preparation for subsequent mass spectrometric analysis. At present, the group consists of two Ph.D. students, one Staff Scientist, one Bioinformatician and one technician.