Next-Generation Protein Identification

Abstract

Proteins are the workhorses of the cell, as such, they form the basis of all living systems. In order to fully understand biological processes, the ability to identify and quantify the proteins in cells is crucial. Identification can be achieved by determining the amino acid sequence of proteins, since this sequence is unique for each protein. However, protein sequencing remains an enormous challenge. The dynamic range at which proteins can occur spans several orders of magnitude, and the need to identify all 20 different amino acids are only a few of the challenges that are currently preventing us from sequencing proteins. However, when realized, single-molecule protein sequencing will create the opportunity for single-cell proteomics and screening for on-site medical diagnostics. It will lead to a revolution in biophysics, biotechnology, and healthcare. Fluorescence techniques belong to one of the most commonly used techniques in biophysics and have brought about a deeper understanding of biological processes at the single-cell or single-molecule level. Furthermore, the field of DNA sequencing has demonstrated that the use of fluorescence approaches has enabled one of the main breakthroughs in DNA sequencing: the development of next-generation sequencers (NGS). These NGS greatly reduced the sequencing costs per human genome. It comes as no surprise that fluorescence approaches are being explored for protein sequencing as well. In this thesis, we pioneer with single-molecule FRET (fluorescence resonance energy transfer) and DNA nanotechnology approaches for the development of a protein identification platform.