P. Kielkowski

Chemoproteomics identifies a pyrimido[4,5-d]pyrimidine analogue as a tubulin-tyrosine ligase binder

Activity-based chemical proteomics uncovers unexpected covalent targets of E64d and reveals a role for cysteine cathepsins in PLD3 proteostasis

In neurons, treatment with E64d lead to about 50-fold PLD3 accumulation and dysregulation of its proteolytic cleavage, while there was only a minor overall change on the whole proteome level, which suggests that their activation might be responsible for decreased PLD3 levels in neurons of patients with Alzheimer’s diseases.

A Scalable and Robust Workflow for Cost-Effective Post-Translational Modifications Profiling by Chemical Proteomics

Mass spectrometry-based chemical proteomics is a powerful method to analyze proteins labelled by small molecules to identify protein targets of active compounds and to profile protein post-translational modifications. The throughput and high protein input for chemical proteomics workflows has been often a limiting factor for application of the technology for specialized and difficult to culture cell lines. The high protein input was necessary to gain significant difference of noise to signal ratio in proteomics readout. Here, we describe a general chemical proteomics workflow, which is performed in 96-well plate and necessitate only 25 μg of protein input to profile post-translationally modified proteins including abundant O-GlcNAcylated proteins as well as low abundant AMPylated proteins. The workflow integrates advances in Cu(I)-catalyzed azide-alkyne cycloaddition to minimize chemical side-reactivity of the ‘click reaction’ and data-independent acquisition mode during LC-MS/MS measurement. An iterative optimization of protein clean-up on carboxylate-coated paramagnetic beads led to significant saving of the beads usage and lowers the unspecific protein background that resulted in sensitivity gain.