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Quantitative targeted proteomics by combining microfluidics with electron microscopy

Giss, Dominic. Quantitative targeted proteomics by combining microfluidics with electron microscopy. 2014, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11141

Abstract

Nearly all cellular functions are dictated by proteins, thus the proteome defines the functional capacity of a cell. Protein interactions form and dissociate in order to perform specific cellular functions. These dynamic interactions are subject to stochastic fluctuations causing cell-to-cell variability within a population. The investigation of dynamic and heterogeneous multimolecular protein complexes is a hallmark of experimental systems biology. However, this aim puts demanding requirements on analytical methods used since these should provide single molecule and single cell resolution. Due to the low copy number of proteins from individual cells, the lack of amplification techniques for proteins and the small sample volumes, single-cell proteomics still challenges existing biophysical and biochemical methods and requires novel and complementary approaches.
This thesis is part of a project that seeks for a novel approach to visual proteomics that aims for the qualitative and quantitative analysis of the entire proteome from a single eukaryotic cell by transmission electron microscopy (TEM), also termed as 'single-cell visual proteomics'. However, the identification of all the molecular components constituting the crude cell lysate is difficult and suggests to follow a targeted proteomics approach where isolation and separation techniques are applied in combination with TEM to make protein identification comprehensible.
During the course of this thesis, a novel approach to targeted proteomics was developed and evaluated, combining microfluidic techniques with magnetic beads and photocleavable composites. The proposed method enables rapid and specific isolation of different protein complexes from a few thousand cells under close to physiological condition. The isolation method yields samples of high purity, in particular for a one-step purification method. Subsequent single particle analyses of negative stain TEM images provide averaged projection structures of the isolated target proteins. During the isolation process, the target protein complexes are immobilized and immuno-labelling techniques using electron-dense markers can be applied. This procedure allows protein-binding partners constituting a complex to be detected. Hence, the approach can provide initial information on structure and composition of weakly interacting protein complexes formed in vivo without applying time consuming traditional large-scale methods for protein expression and purification followed by complex hybrid techniques for analysis.
In addition, initial experiments showed that quantitative information on protein abundances can be extracted upon combining the isolation method with semi-automatic image acquisition and analysis procedures used for TEM investigations. Thereby the suitability of single particle TEM for protein quantification is reported for the first time. Consequently, the fundamentals of 'quantitative TEM' were elucidated and a method for reliable and efficient concentration measurements proposed. The results imply that picomolar to nanomolar ranged concentrations, typically hard to assess with traditional absorbance-based methods, can be reliably measured. Interestingly, this technique uses standard equipment readily available in many laboratories.
Advisors:Stahlberg, Henning
Committee Members:Lim, Roderick
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Stahlberg)
Item Type:Thesis
Thesis no:11141
Bibsysno:Link to catalogue
Number of Pages:99 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:57
Deposited On:04 Mar 2015 14:55

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