Chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Despite widespread adoption and considerable advances in both sample-preparation workflows and MS instrumentation, chemoproteomics experiments still typically only identify a small fraction of all cysteines encoded by the human genome. Here, we develop an optimized sample-preparation workflow that combines enhanced peptide labeling with singlepot, solid-phase-enhanced sample-preparation (SP3) to improve the recovery of biotinylated peptides, even from small sample sizes. By combining this improved workflow with on-line highfield asymmetric waveform ion mobility spectrometry (FAIMS) separation of labeled peptides, we achieve unprecedented coverage of > 14000 unique cysteines in a single-shot 70 min experiment. Showcasing the wide utility of the SP3-FAIMS chemoproteomic method, we find that it is also compatible with competitive small-molecule screening by isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). In aggregate, our analysis of 18 samples from seven cell lines identified 34225 unique cysteines using only~28 h of instrument time. The comprehensive spectral library and improved coverage provided by the SP3-FAIMS chemoproteomics method will provide the technical foundation for future studies aimed at deciphering the functions and druggability of the human cysteineome.
BackgroundSex differences in obesity and related diseases are well established. Gonadal hormones are a major determinant of these sex differences. However, sex differences in body size and composition are evident prior to exposure to gonadal hormones, providing evidence for gonadal-independent contributions attributable to the XX or XY sex chromosome complement. Large-scale genetic studies have revealed male/female differences in the genetic architecture of adipose tissue amount and anatomical distribution. However, these studies have typically neglected the X and Y chromosomes.Scope of the reviewHere we discuss how the sex chromosome complement may influence obesity, lipid levels, and inflammation. Human sex chromosome anomalies such as Klinefelter syndrome (XXY), as well as mouse models with engineered alterations in sex chromosome complement, support an important role for sex chromosomes in obesity and metabolism. In particular, the Four Core Genotypes mouse model—consisting of XX mice with either ovaries or testes, and XY mice with either ovaries or testes—has revealed an effect of X chromosome dosage on adiposity, hyperlipidemia, and inflammation irrespective of male or female gonads. Mechanisms may include enhanced expression of genes that escape X chromosome inactivation.Major conclusionsAlthough less well studied than effects of gonadal hormones, sex chromosomes exert independent and interactive effects on adiposity, lipid metabolism, and inflammation. In particular, the presence of two X chromosomes has been associated with increased adiposity and dyslipidemia in mouse models and in XXY men. The enhanced expression of genes that escape X chromosome inactivation may contribute, but more work is required.
The integration of proteomic, transcriptomic, and genetic variant annotation data will improve our understanding of genotypephenotype associations. Due, in part, to challenges associated with accurate inter-database mapping, such multi-omic studies have not extended to chemoproteomics, a method that measures the intrinsic reactivity and potential "druggability" of nucleophilic amino acid side chains. Here, we evaluated mapping approaches to match chemoproteomic-detected cysteine and lysine residues with their genetic coordinates. Our analysis revealed that database update cycles and reliance on stable identifiers can lead to pervasive misidentification of labeled residues. Enabled by this examination of mapping strategies, we then integrated our chemoproteomics data with computational methods for predicting genetic variant pathogenicity, which revealed that codons of highly reactive cysteines are enriched for genetic variants that are predicted to be more deleterious and allowed us to identify and functionally characterize a new damaging residue in the cysteine protease caspase-8. Our study provides a roadmap for more precise inter-database mapping and points to untapped opportunities to improve the predictive power of pathogenicity scores and to advance prioritization of putative druggable sites.
Cysteine chemoproteomics studies provide proteome-wide portraits of the ligandability or potential ‘druggability’ of thousands of cysteine residues. Consequently, these studies are enabling resources for closing the druggability gap, namely achieving pharmacological manipulation of ~99% of the human proteome that remains untargeted by FDA approved small molecules. Recent interactive dataset repositories, such as OxiMouse and SLCABPP, have enabled users to interface more readily with cysteine chemoproteomics studies1,2. However, these databases remain limited to single studies and therefore do not provide a mechanism to perform cross-study analyses. Here we report CysDB as a curated community-wide repository of cysteine chemoproteomics data that incorporates high coverage data derived from nine studies generated by the Backus, Cravatt, Gygi, Wang, and Yang research groups. CysDB is a SQL relational database that is publicly available at https://backuslab.shinyapps.io/cysdb/ and features chemoproteomic measures of identification, hyperreactivity, and ligandability for 62,888 cysteines (24% of all cysteines the human proteome). The CysDB web application also includes annotations of functionality (UniProtKB/Swiss-Prot, Pfam, Panther), known druggability (FDA approved targets, DrugBank, ChEMBL), disease-relevance and genetic variation (ClinVar, Cancer Gene Census, Online Mendelian Inheritance in Man), and structural features (Protein Data Bank). Showcasing the utility of CysDB, here we report the discovery and enrichment of ligandable cysteines in undruggable classes of proteins, the observation that a subset of cysteines showed marked preference for specific classes of electrophiles (chloroacetamide vs acrylamide), and that ligandable cysteines are present in numerous undrugged disease-relevant proteins. Most importantly, we have designed CysDB for the incorporation of new datasets and features to support the continued growth of the druggable cysteineome.
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