High-throughput methods enable rapid experimentation and/or screening of thousands of samples simultaneously. Mass-spectrometry based methods are of particular interest since they provide a label-free way to detect all species present in a given reaction mixture. To circumvent sample preparation and purification—which is typically a slow process—the Mrksich group developed a high-throughput approach that relies on self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry, termed SAMDI-MS. Functionalized alkanethiolates form organized self-assembled monolayers (SAMs) on gold surfaces. Species of interest are captured onto the SAMs using a selective immobilization chemistry, followed by analysis of the monolayers by MALDI-TOF. This dissertation introduces innovative, impressive applications that expand the breadth of the SAMDI-MS technology. Chapter 2 delves into the development of a SAMDI-MS assay capable of measuring protein tyrosine phosphatase (PTP) activity in single cells. Stochastic fluctuations in cellular processes can result in high cell-to-cell variability, even in a genetically identical population. The development of single-cell analysis tools is instrumental in understanding and addressing this heterogeneity, yet current enzyme measurements on the single-cell scale lag behind their genetic counterparts and typically report on expression rather than activity. Leveraging the well-defined surface chemistry of self-assembled monolayers, we describe an approach to measure PTP enzyme activity in lysates derived from single cells, without purification. We demonstrate that our approach is capable of measuring activity and distribution of PTPs in an accessible high-throughput format. Chapter 3 continues the discussion, and describes in detail different efforts towards the development of the next generation of single-cell assays. Chapter 4 covers the high-throughput mapping of the reverse β-oxidation (rBOX) cycle in vitro using cell-free metabolic engineering and SAMDI-MS. The rBOX cycle is an engineered pathway that offers an energy-efficient synthesis for the production of fatty acids and their derivatives, operating with coenzyme A (CoA) intermediates. CoA intermediates are selectively bound onto the SAM surface via a native chemical ligation (NCL) reaction, thereby allowing for direct analysis by SAMDI-MS. This approach, together with the cell-free metabolic engineering platform, enables us to screen hundreds of enzyme variants and assay conditions to optimize the production of medium-chain length fatty acids.

Creator

• Hakim Moully, Elamar

DOI

• https://doi.org/10.21985/n2-3cf7-gj23

Subject

• Chemistry

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:15661
• etdadmin_upload_836023

Keyword

• SAMDI
• Biochemical Assays
• High-Throughput
• Single-Cell
• Mass Spectrometry

Date created

• 2021-01-01

Resource type

• Dissertation

Rights statement

• In Copyright