CySP3-96 enables scalable, streamlined, and low-cost sample preparation for cysteine chemoproteomic applications.

Cysteine chemoproteomic screening platforms are widely utilized for chemical probe and drug discovery campaigns. Chemoproteomic compound screens, which use a mass spectrometry-based proteomic readout, can interrogate the structure activity relationship (SAR) for thousands of proteins in parallel across the proteome. The versatility of chemoproteomic screens has been demonstrated across electrophilic, nucleophilic, and reversible classes of molecules.

Chemoproteogenomic stratification of the missense variant cysteinome.

Cancer genomes are rife with genetic variants; one key outcome of this variation is widespread gain-of-cysteine mutations. These acquired cysteines can be both driver mutations and sites targeted by precision therapies. However, despite their ubiquity, nearly all acquired cysteines remain unidentified via chemoproteomics; identification is a critical step to enable functional analysis, including assessment of potential druggability and susceptibility to oxidation.

Delineating cysteine-reactive compound modulation of cellular proteostasis processes.

Covalent modulators and covalent degrader molecules have emerged as drug modalities with tremendous therapeutic potential. Toward realizing this potential, mass spectrometry-based chemoproteomic screens have generated proteome-wide maps of potential druggable cysteine residues. However, beyond these direct cysteine-target maps, the full scope of direct and indirect activities of these molecules on cellular processes and how such activities contribute to reported modes of action, such as degrader activity, remains to be fully understood.

Generating cysteine-trypsin cleavage sites with 2-chloroacetamidine capping.

An electrophilic arginine mimetic, 2-chloroacetamidine (CAM), was deployed to enable trypsin-mediated proteolysis at cysteine residues and to enhance mass spectrometry-based proteomic detection of cysteine-containing peptides. Illustrating the value of the CAM-capping strategy, proteogenomic analysis using a two-stage false discovery rate (FDR) search revealed >50% enhanced coverage of missense variants, when compared to established workflows.

Pervasive aggregation and depletion of host and viral proteins in response to cysteine-reactive electrophilic compounds.

Protein homeostasis is tightly regulated, with damaged or misfolded proteins quickly eliminated by the proteasome and autophagosome pathways. By co-opting these processes, targeted protein degradation technologies enable pharmacological manipulation of protein abundance. Recently, cysteine-reactive molecules have been added to the degrader toolbox, which offer the benefit of unlocking the therapeutic potential of 'undruggable' protein targets.

Solid-Phase Compatible Silane-Based Cleavable Linker Enables Custom Isobaric Quantitative Chemoproteomics.

Mass spectrometry-based chemoproteomics has emerged as an enabling technology for functional biology and drug discovery. To address limitations of established chemoproteomics workflows, including cumbersome reagent synthesis and low throughput sample preparation, here, we established the silane-based cleavable isotopically labeled proteomics (sCIP) method. The sCIP method is enabled by a high yielding and scalable route to dialkoxydiphenylsilane fluorenylmethyloxycarbonyl (DADPS-Fmoc)-protected amino acid building blocks, which enable the facile synthesis of customizable, isotopically labeled, and chemically cleavable biotin capture reagents.

Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase.

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein.

Predicting and validating a model of suppressor of IKKepsilon through biophysical characterization.

Suppressor of IKKepsilon (SIKE) is a 207 residue protein that is implicated in the TLR3-TANK-binding kinase-1-mediated response to viral infection. SIKE's function in this pathway is unknown, but SIKE forms interactions with two distinct cytoskeletal proteins, α-actinin and tubulin, and SIKE knockout reduces cell migration. As structure informs function and in the absence of solved structural homologs, our studies were directed toward creating a structural model of SIKE through biochemical and biophysical characterization to probe and interrogate SIKE function.