Cu-Catalyzed Azide-Alkyne-Thiol Reaction Forms Ubiquitous Background in Chemical Proteomic Studies.

We report here a Cu-catalyzed azide-alkyne-thiol reaction forming thiotriazoles as the major byproduct under widely used bio-orthogonal protein labeling "click" conditions. The development of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) had a tremendous impact on many biological discoveries. However, the considered chemoselectivity of CuAAC is hampered by the high reactivity of cysteine free thiols, yielding thiotriazole protein conjugates. The reaction byproducts generate false-positive protein hits in functional proteomic studies. The reported detail investigation of conjugates between chemical probes containing terminal alkynes, azide tags, and cell lysates reveals the formation of thiotriazoles, which can be readily detected by in-gel fluorescence scanning or after peptide and protein enrichment by mass spectrometry-based proteomics. In protein level identification and quantification experiments, the produced fluorescent bands or enriched proteins may not result from the important enzymatically driven reaction and can be falsely assigned as hits. This study provides a complete list of the most common background proteins. The knowledge of this previously overlooked reactivity now leads to the introduction of modified CuAAC conditions, which avoids the undesired product formation, diminishes the background, and hence improves the signal-to-noise ratio.

Transforming Chemical Proteomics Enrichment into a High-Throughput Method Using an SP2E Workflow.

Protein post-translational modifications (PTMs) play a critical role in the regulation of protein catalytic activity, localization, and protein-protein interactions. Attachment of PTMs onto proteins significantly diversifies their structure and function, resulting in proteoforms. However, the sole identification of post-translationally modified proteins, which are often cell type and disease-specific, is still a highly challenging task. Substoichiometric amounts and modifications of low abundant proteins necessitate the purification or enrichment of the modified proteins. Although the introduction of mass spectrometry-based chemical proteomic strategies has enabled the screening of protein PTMs with increased throughput, sample preparation remains highly time-consuming and tedious. Here, we report an optimized workflow for the enrichment of PTM proteins in a 96-well plate format, which could be extended to robotic automation. This platform allows us to significantly lower the input of total protein, which opens up the opportunity to screen specialized and difficult-to-culture cell lines in a high-throughput manner. The presented SP2E protocol is robust and time- and cost-effective, as well as suitable for large-scale screening of proteoforms. The application of the SP2E protocol will thus enable the characterization of proteoforms in various processes such as neurodevelopment, neurodegeneration, and cancer. This may contribute to an overall acceleration of the recently launched Human Proteoform Project.

Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation.

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.