Development of miniaturized bioanalytical methods for the qualitative and quantitative analysis of proteins and proteomes by means of digital microfluidics and nanoLC-MS
Constant technological advances in analytics enable proteomics research on an increasingly smaller scale, which inevitably requires suitable downscaling of sample preparation workflows as well. The overall goals of this thesis were to investigate and develop promising sample preparation methods for handling low-input biological samples, and adapt these methods to become suitable for different concepts of proteomics.
The first subproject primarily served the purpose of being an introduction to digital microfluidics (DMF) in general, while simultaneously providing the invaluable opportunity to test an oil-based DMF device in early access with regard to a multitude of different aspects relevant for potential use in nanoproteomics.
Next, the knowledge and experience acquired on the theory and practical use of DMF, as well as the preferred instrumental setup for LC-MS/MS, were applied in a series of Bottom-Up proteomics (BUP) experiments, using single specimens of the model organism Caenorhabditis elegans (C. elegans) as the preferred sample material. The aim was to pair a highly efficient DMF-based sample preparation with LC-MS conditions optimized for low-input proteomics to reproducibly achieve an unprecedented number of protein identifications from single nematodes. Once in place, the workflow was extended to also allow label-free quantification. Compared to an identical in-tube sample preparation, the DMF workflow led to a markedly higher number of inferred proteins considered differentially abundant between two groups.
Once a DMF-based workflow, viable both qualitatively and quantitatively, had been successfully established for BUP, the next goal was to transfer this mode of sample preparation to Top-Down (TDP) and eventually Middle-Down proteomics (MDP), and thus generate independent yet comparable quantitative datasets for single C. elegans according to three different approaches in proteomics (BUP, TDP and MDP).Vorschau
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