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Substrate profiling of post-translational modifying enzymes enabled by synthetic peptide chemistry and mass spectrometry

  • Author(s): Olson, Nicole Cooley
  • Advisor(s): Craik, Charles S
  • et al.
Abstract

Post-translational modifying (PTM) enzymes constitute 5% of the human genome but most have yet to be fully characterized.(Walsh, Garneau-Tsodikova, & Gatto, 2005) Elucidating the biological functions of PTM enzymes requires tools that allow for profiling their activities in biological systems. Over the course of my graduate studies I furthered the development of a mass spectrometry-based technology, which enabled the study of a variety of post-translational modifying enzymes. The technology that I expanded, known as Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), employs a 228-member physico-chemically diverse library of rationally designed peptide substrates that have maximum sequence diversity within a short 14-mer peptide.(O’Donoghue et al., 2012) I employed this technology in order to study proteases from Mycobacterium, detect modifications from an O-GlcNAc transferase and methyltransferase, and established the use of this method for the characterization of kinases.

The more than 500 protein kinases comprising the human kinome catalyze hundreds of thousands of phosphorylation events to regulate a diversity of cellular functions; however, the substrate specificity has been annotated for only a fraction of these kinases. I established that this method can be deployed with as low as 10 nM kinase to determine activity against S/T/Y-containing peptides; additionally, label-free quantitation was used to ascertain catalytic efficiency values for individual peptide substrates in the multiplex assay. Using this profiling approach, I developed quantitative motifs for a selection of kinases from each branch of the kinome. To increase the information content of the quantitative kinase motifs, a sub-library approach was used to expand the testable sequence space within a peptide library of approximately 100 members. I applied this method to address disputed aspects of the HIV-1 Tat – positive transcription elongation factor b (P-TEFb) interaction as well as to characterize several kinases without known substrates. The sensitivity of this approach is highlighted by its ability to detect phosphorylation events from microgram quantities of immunoprecipitated material, which will allow for wider applicability of this method.

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