Leiden University Scholarly Publications

Li, N. (2016)

Activity-based proteasome profiling

Doctoral Thesis

The work described in this thesis is mainly focusing on setting up and application of a quantitative activity‐based proteasome profiling method. Chapter 1 provides a general introduction on the ubiquitin proteasome system (UPS) and activity‐based proteasome profiling. Chapter 2 is a literature review of some new achievements in the activity‐based protein profiling field in the recent years, focusing on application in biochemistry, molecular and cellular biology, medicinal chemistry, pathology, physiology and pharmacology research. Chapter 3 is a protocol for performing quantitative activity‐based proteasome profiling experiments. In the protocol, both high throughput fluorescent ABPP and biotinylated probe plus LC/MS approaches are described. Chapter 4 is a brief technical report about bioorthogonal chemistry in ABPP. The commonly used secondary azide group is compared with a primary azide group in proteasome ABPs performing Cu(I) catalyzed azide‐alkyne cycloaddition and...Show moreThe work described in this thesis is mainly focusing on setting up and application of a quantitative activity‐based proteasome profiling method. Chapter 1 provides a general introduction on the ubiquitin proteasome system (UPS) and activity‐based proteasome profiling. Chapter 2 is a literature review of some new achievements in the activity‐based protein profiling field in the recent years, focusing on application in biochemistry, molecular and cellular biology, medicinal chemistry, pathology, physiology and pharmacology research. Chapter 3 is a protocol for performing quantitative activity‐based proteasome profiling experiments. In the protocol, both high throughput fluorescent ABPP and biotinylated probe plus LC/MS approaches are described. Chapter 4 is a brief technical report about bioorthogonal chemistry in ABPP. The commonly used secondary azide group is compared with a primary azide group in proteasome ABPs performing Cu(I) catalyzed azide‐alkyne cycloaddition and Staudinger‐Bertozzi reaction under native/denatured protein conditions Chapter 5 is focusing on the application of quantitative activity‐based proteasome profiling in the prognosis of cancer therapeutics. A combination of ABPP and global proteomics is performed to elucidate the bortezomib sensitivity and resistance mechanisms in leukemia and solid tumor cells. Chapter 6 describes the characterization of the newly discovered proteasome subunit β5t by ABPP and LC/MS proteomics. The subunit is proven to be catalytically active. A hydrophilic Thr residue on the P2 position of the proteasome inhibitor improves the inhibitory efficiency of β5t, which indicates it might prefer to cleave hydrophilic peptides. Chapter 7 describes the identification of O‐GlcNAcylation modifications on the ubiquitin receptor protein hHR23B and characterization of how the sugar moiety influences the conformation and functions of the protein.Show less

Preteasome profiling

Ubiquitin proteasome system

Protein

LC-MS/MS

All authors

Li, N.

Supervisor

Overkleeft, H.S.

Co-supervisor

Florea, B.I.

Committee

Brouwer, J.; Driessen, C.; Ovaa, H.; Stelt, M. van der

Qualification

Doctor (dr.)

Awarding Institution

Leiden Institute of Chemistry, Faculty of Science, Leiden University

Date

2016-12-16