Senior Researcher, currently on leave

Keiryn L. Bennett
Mass Spectrometry and Proteomics

Proteomics is the study of expression, localization, function, and interaction of proteomes. Keiryn Bennett has a broad range of research interests predominantly focused on proteins and post-translational modifications (PTMs) that are involved in different aspects of immunology/infection and cancer. She collaborates closely with the group of Jacques Colinge on the development of algorithms and analysis tools for interpretation of MS-generated data, with other CeMM groups, and with colleagues at the adjacent Medical University of Vienna.

Her work focuses on the application of mass spectrometry to understand complex biological systems and the molecular mechanisms underlying disease states and includes:

  1. Protein-protein interactions to generate protein interactomes.
  2. Drug-protein interactions.
  3. Quantitative expression proteomics for the relative and comparative quantitative analysis of global proteomes.
  4. Monitoring the role of PTMs in cellular signalling pathways.

Interaction Proteomics

Affinity purification is a generic protocol that enables the enrichment and isolation of non-covalent protein complexes under near-physiological conditions. Chemical proteomics is a powerful mass spectrometry–based affinity chromatography approach for proteome-wide identification of small molecule–protein interactions. Focus is centred on:  

  • miniaturization of affinity-purified protein complexes and the drug pulldown technology to reduce the quantity of input material to the level of clinical samples
  • standardization of the affinity purification procedure at the level of tagged-protein expression
  • transcription factor and organellar protein complexes and interactomes
  • profiling target proteins from human tumour tissue subsequent to drug enrichment for post-translational modification

Quantitative Proteomics

iTRAQ and TMT are a multiplexed set of reagents (4-, 6- or 8-plex) used for the quantitative analysis of peptides in a digest mixture. A chemical reaction occurs between the reagent and the N-termini of peptides plus the ε-amino side chains of lysine residues. Isobaric mass labels are thereby covalently bound to the peptides. The reagents are differentially-isotopically-labelled and thus, all derivatized peptides are isobaric and chromatographically-indistinguishable. Following peptide fragmentation, the derivatized peptides yield low-molecular-mass reporter ions that can be used to quantitate the individual peptide components of the multiplex set. Coupled to reversed-phase reversed-phase two-dimensional gel-free proteomics, her team routinely applied this technology to a range of projects including:

  • perturbation or stimulation of cellular systems by, e.g., drugs, infective agents, mutated proteins, exogenous stimuli
  • comparative analyses of sub-cellular proteomes, e.g., chromatin, membrane; drug pulldowns and affinity-purified protein complexes

Dynamics of Post-translation Modifications

Modification of proteins is a key regulatory event in a living cell. Complex biological processes such as cell cycle, cell growth, cell differentiation and metabolism are orchestrated and tightly controlled by often reversible events that modulate protein activity, stability, interaction and localisation. Identification and detailed investigation of modified forms of proteins is a prerequisite for understanding the dynamics of complex biological systems. There are many techniques available for selective enrichment of modified peptides, however, the approaches adopted by Keiryn Bennett and collaborators are:

  • global phosphopeptide enrichment via Fe(III)-NTA
  • antibody-based enrichment of modified peptides, e.g., phoshotyrosine, acetyl-lysine, and extension to other post-translational modifications

Biosketch

Keiryn Bennett obtained her PhD in protein mass spectrometry at the University of Wollongong, Australia. She was a post-doctoral fellow at the Australian Proteome Analysis Facility (APAF), Macquarie University, and in the laboratory of Peter Roepstorff in Odense, Denmark. This was followed with Matthias Mann at MDS Proteomics, Denmark. Keiryn joined the laboratory of Giulio Superti-Furga in 2004 and established the mass spectrometry and proteomic research group; and since 2009, she has an independent group leader position.

Selected Papers

Chamrad I, Rix U, Stukalov A, et al. A miniaturized chemical proteomic approach for target profiling of clinical kinase inhibitors in tumor biopsies. J Proteome Res. 2013;12(9):4005-4017. (abstract)

Giambruno R, Grebien F, Stukalov A, et al. Affinity purification strategies for proteomic analysis of transcription factor complexes. J Proteome Res. 2013;12(9):4018-4027. (abstract)

Rudashevskaya EL, Sacco R, Kratochwill K, et al. A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry. Nat Protoc. 2013;8(1):75-97. (abstract)

Rudashevskaya EL, Breitwieser FP, Huber ML, Colinge J, Muller AC, Bennett KL. Multiple and sequential data acquisition method: an improved method for fragmentation and detection of cross-linked peptides on a hybrid linear trap quadrupole orbitrap velos mass spectrometer. Anal Chem. 2013;85(3):1454-1461. (abstract)