Development of chemical reporters for glutarylation and HMGylation

Abstract

Protein post-translational modifications (PTMs) are covalent chemical modifications that occur after protein translation. As such, PTMs have greatly expanded the diversity and complexity of proteomes. To understand this vital regulation system, study towards PTM has received intense attention in recent years. With the rapid advancements in mass-spectrometry technology, several new PTMs have been identified recently (e.g. lysine N-malonylation (2012), lysine N-succinylation (2012), and lysine N-glutarylation (2014)). To better understand those newly identified PTM substrates and discover potentially uncovered new PTMs, a chemical proteomics approach is proposed.

To identify the substrates of a new PTM, namely, HMGylation, a chemical reporter HMG-AM-yne (2.1) is synthesized in 8 steps with 9.2% overall yield (Chapter 2). Meanwhile, to achieve a better understanding of the cell proteome-wide biodistribution of glutarylation substrates and its biological function, another reporter Glut-AM-yne (3.1) is synthesized in 6 steps with 9.3% overall yield (Chapter 3). To verify the existence of HMGylation and glutarylation on histones, standard peptides with HMGylation and glutarylation (H3K9 Glut and H3K9 HMG) are chemically synthesized by solid-phase peptide synthesis (SPPS) and the building block for peptide synthesis Fmoc-Glut-lys-tBu (4.1) and Fmoc-HMG-lys-all (4.21) are synthesized. To validate the labeling outcome of chemical reporter and map PTM sites, another two building blocks Fmoc-Glut-yne-lys-all (4.23) and Fmoc-HMG-yne-lys-all (4.25) are synthesized for H3K9 Glut-yne/HMG-yne standard peptide synthesis, as described in Chapter 4.