Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

Post-translational control of proteins through covalent attachment of ubiquitin plays important roles in all eukaryotic cell functions. The ubiquitin system in humans consists of 2 E1, 35 E2 and >600 E3 ubiquitin ligases as well as hundreds of deubiquitylases, which reverse ubiquitin attachment. Moreover, there are hundreds of proteins with ubiquitin-binding domains that bind one of the eight possible polyubiquitin chains. Dysfunction of the ubiquitin system is associated with many diseases such as cancer, autoimmunity and neurodegeneration, demonstrating the importance of ubiquitylation. Therefore, enzymes of the ubiquitin system are considered highly attractive drug targets. In recent years, mass spectrometry (MS)-based techniques have become increasingly important in the deciphering of the ubiquitin system. This short review addresses the state-of-the-art MS techniques for the identification of ubiquitylated proteins and their ubiquitylation sites. We also discuss the identification and quantitation of ubiquitin chain topologies and highlight how the activity of enzymes in the ubiquitin pathway can be measured. Finally, we present current MS tools that can be used for drug discovery in the ubiquitin space.

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we...

Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)—collectively Parkinson’s disease, Parkinson’s disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a “pathological package” capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein–ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.

BMDMs are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from BM are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 cell-conditioned media (LCCM) and how it affects the BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a 2-wk period, identifying 2,193 proteins. Whereas M-CSF is very abundant in LCCM, we identified several other immune-regulatory proteins such as macrophage migration inhibitory factor (MIF), osteopontin, and chemokines such as Ccl2 and Ccl7 at surprisingly high abundance levels. We therefore further characterised the proteomes of BMDMs after differentiation with M-CSF, M-CSF + MIF, or LCCM, respectively. Interestingly, macrophages differentiated with LCCM induced a stronger anti-inflammatory M1 phenotype that those differentiated with M-CSF. This resource will be valuable to all researchers using LCCM for the differentiation of BMDMs.

A fast and robust sample preparation method for MALDI TOF MS analysis of whole mammalian cells and phenotypic profiling of mESCs.

<p>Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of <a>new medicines. Here, we report a screening platform that combines ‘direct-to-biology’ high-throughput chemistry (D2B-HTC) with photoreactive covalent fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates. Screening the HTC-PhABit library with </a>carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn²⁺ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design-make-test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.</p>

Bone marrow-derived macrophages (BMDMs) are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from bone marrow are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 cell conditioned media (LCCM) and how it affects BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a two-week period, identifying 2,193 proteins. While M-CSF is very abundant in LCCM, we identified several other immune-regulatory proteins such as macrophage migration inhibitory factor (MIF), osteopontin and chemokines such as Ccl2 and Ccl7 at surprisingly high abundance levels. We therefore further characterised the proteomes of BMDMs after differentiation with M-CSF, M-CSF + MIF or LCCM, respectively. While MIF has no significant effect on the BMDM proteome, LCCM induced a slightly pre-activated phenotype and the expression of a number of known innate immune proteins in macrophages. Interestingly, LCCM induced higher expression of CD11b, while BMDMs differentiated with M-CSF alone showed higher expression of CD11c. This resource will be valuable to all researchers using LCCM for the differentiation of BMDMs.