Main content

Host-pathogen interactions

Mycobacterium tuberculosis (Mtb) is an ancient intracellular pathogen that has co-evolved with its human host and, as such, we expect a deep level of integration and interaction with host systems in order to manipulate the cellular defenses and avoid destruction. Identifying which host proteins and protein complexes come into physical contact with bacterial proteins is crucial for a comprehensive understanding of how Mtb rewires the host’s cellular machinery during the course of infection. Recent pioneering studies in viral host-pathogen protein-protein interactions have proved two key points that are relevant, (i) the scope of manipulation of host defenses by a pathogen via protein-protein interactions can be remarkably broad, (ii) technologies in mass spectrometry-based interaction proteomics have matured to a point sufficient to approach this key problem in the biology of infectious disease. We are attempting to employ advanced methods in quantitative interaction proteomics to characterize the host-pathogen protein-protein interaction landscape in Mtb.


Quantitative Interaction Proteomics

Protein complexes and protein interaction networks are essential mediators of most biological functions. Complexes supporting transient functions such as signal transduction processes are frequently subject to dynamic remodeling. To better understand inherently dynamic biological processes, methods to reliably quantify temporal changes of protein interaction networks are essential. We are using affinity purification combined with quantitative mass spectrometry to study dynamic reorganisation of protein complexes and protein interaction networks. In particular, we have studied signaling complexes which are remodeled in response to changes in phosphorylation state. We have recently demonstrated the capabilities of SWATH mass spectrometry to study the dynamics of the 14-3-3β scaffold protein interactome after stimulation of the insulin-PI3K-AKT pathway (Collins, BC et al. (2013) Nat Methods).


Quantitaitve proteomics of microbial pathogens

Previous work from the Aebersold group has shown that by utilizing a comprehensive proteome assay library the majority of the Mtb proteome can be quantified in a single inejction using SWATH MS (Schubert, OS, et al. (2015), Cell Host & Microbe). We are now employing this method to routinely quantify 2500-3000 proteins from in vitro culture experiments to examine the effect of genomic diversity in Mtb on its quantitative proteome, and to address questions relating to drug resistance mechanisms as part of the TbX project.


SWATH mass spectrometry

SWATH MS is a recently described implementation of data independent acquisition (DIA) mass spectrometetry which cycles through fixed precursor isolation windows using a quadrupole-time-of-flight mass spectrometer achieving essentially complete peptide fragment ion coverage for detectable precursors in the tryptic peptides mass range (Gillet LC et al. (2012) Mol Cell Proteomics). An essential feature that distinguishes SWATH MS from other DIA strategies is the use of prior knowledge regarding fragmentation and chromatographic behavior of target peptides. This information is used for scoring signal groups extracted from SWATH MS datasets to identify and quantify peptides automatically and at large scale (Röst HL et al. (2014) Nat Biotech, SWATH MS provides SRM-like performance in terms of quantitative accuracy, data completeness and dynamic range without specifying target peptides prior to data acquisition. Further, and unlike SRM, SWATH MS can quantify an unlimited number of target peptides as long as they have been previously observed by shotgun MS. We are actively pursuing advances in data acquisition and analysis strategies to improve the performance of SWATH MS as an enabling tool in quantitative proteomics. In particular, we have been pursuing a project to compare the performance of SWATH MS protein quantification across labs.

Page URL:
Fri Apr 28 20:02:00 CEST 2017
© 2017 Eidgenössische Technische Hochschule Zürich