Immuno-Competitive Capture Mass Spectrometry, a novel unbiased approach to study endogenous protein-protein interactions

DSpace Repository


Dateien:

URI: http://hdl.handle.net/10900/109751
http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1097512
http://dx.doi.org/10.15496/publikation-51127
Dokumentart: Dissertation
Date: 2020-11-23
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biochemie
Advisor: Rothbauer, Ulrich (Prof. Dr.)
Day of Oral Examination: 2020-10-26
DDC Classifikation: 570 - Life sciences; biology
Keywords: Proteine
License: Publishing license including print on demand
Order a printed copy: Print-on-Demand
Show full item record

Abstract:

Protein-protein interactions (PPIs) are controlling the majority of biological functions and are the main driver of cellular processes observed in normal as well as pathological conditions. Such a level of controlling is only possible via a high degree of complexity; i.e. a massive number of protein-protein interactions (in the range of couple hundreds of thousands), a variety of physical and structural properties and their reversibility. Moreover, binding affinities can span from micro-molar to high pico-molar level and some proteins are acting as “hubs” by having multiple partners. This sophisticated organization and regulation of PPIs explains why their study is so challenging. No single approaches can capture the full picture and there is an urgent need for innovative platforms to study and analyze PPIs. In this thesis, a novel platform named Immuno-Competitive Capture Mass Spectrometry (ICC-MS) was developed to screen in an unbiased fashion intracellular PPIs. ICC-MS was designed to reach higher specificity compared to classical affinity purification mass spectrometry by introducing a competition step between free and capturing antibody prior to immunoprecipitation. This antibody-based label-free quantitative approach was then combined with a rigorous statistical analysis to extract the cellular interactome of proteins of interest while filtering out non-specifically binding proteins. ICC-MS was first applied to elucidate hepatitis C viral non-structural protein 5A interactome in human hepatoma cells revealing LATS kinases as potential important regulators of viral infection. The study of Glypican-2 and HtrA1 interacting partners further confirmed the ability of ICC-MS to deliver a limited number of highly confident interacting proteins being promising candidates for functional validation. Interestingly, ICC-MS can also be adapted to study interactions formed between proteins and oligonucleotides (Oligo-Competitive Capture Mass Spectrometry or OCC-MS). While it contributed to a better understanding of the mode of action of an SMN2 splicing modifier, the approach could not elucidate the role of protein interactions in antisense oligonucleotides toxicity. Taken together, this innovative approach is suitable to improve the comprehensiveness and accuracy of current protein-protein interactions databases in term of true biological interactome representation.

This item appears in the following Collection(s)