Development of tools for single molecule peptide fluorosequencing

Date

2021-09-16

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Hinson, Caroline Marie

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Abstract

Fluorosequencing, technology that combines selective fluorophore labeling of amino acid residues, total-internal reflection fluorescence (TIRF) microscopy, and Edman degradation, is a novel technology that has been developed to sequence peptides. Unique fluorophores are covalently attached to amino acid residues and through TIRF microscopy are traced through Edman degradation cycles that remove one amino acid at a time. By determining which Edman cycles fluorescence is lost sequence information can be obtained. This has been shown to be a useful tool for determining the sequence of peptides in a highly parallel manner. As each molecule is individually monitored, this single molecule method is useful for the discovery of low abundance peptides and single cell proteomics. New tools are needed to improve sequencing and label new amino acid residues in selective fashion with fluorophores. Single molecule peptide fluorosequencing has unique requirements. Surfaces need to selectively attach peptides by their C-terminus but resist non-specifically bound fluorophores throughout an entire fluorosequencing experiment. Developing a surface that would be suitable for fluorosequencing is detailed in Chapter 2. Preparing peptides for fluorosequencing also has many challenges. Labeling amino acid residues with fluorophores requires multiple steps and purification that can erode the number of peptides in a sample with each step. This can be overcome with a solid support that captures peptides by their N-termini and enables chemical labeling without the loss of sample (Chapter 3).
Additional labeling schemes, that are selective and high yield, are needed to expand the number of amino acid residues capable of being sequenced by fluorosequencing. Two-step labeling opens new possibilities for labeling of amino acids without fluorophore degradation. This is demonstrated in the two-step labeling of glutamic acid with dithiol-aldehyde labeling (Chapter 4). A fluorophore linker with a 1,3-dithiol is synthesized for the labeling of aldehydes, which was done in an efficient manner by deprotecting the aldehyde and labeling in one step. Another two-step labeling scheme was developed for labeling the locations of glycosylation on peptides. By exploiting the susceptibility of glycosylations to β-elimination over phosphorylation, glycosylations were selectively labeled in the presence of phosphorylations and fluorosequenced (Chapter 5).

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