“…4a), thereby greatly expanding the cysteine redoxome 12,[42][43][44][45] . Nevertheless, the chemoselectivity of oxoform-specific probes has long been controversial, even for those classic ones (i.e., dimedone for SOH) 46,47 . To address this controversy in an unbiased fashion, we used pChem to re-analyze the redox proteomic datasets generated in our laboratory.…”
Chemoproteomics has emerged as a key technology to expand the functional space in complex proteomes for probing fundamental biology and for discovering new small molecule-based therapies. Here we report a modification-centric computational tool termed pChem to provide a streamlined pipeline for unbiased performance assessment of chemoproteomic probes. The pipeline starts with an experimental setting for isotopically coding probe-derived modifications (PDMs) that can be automatically recognized by pChem, with masses accurately calculated and sites precisely localized. Further, pChem exports on-demand reports by scoring the profiling efficiency, modification-homogeneity and proteome-wide residue selectivity of a tested probe. The performance and robustness of pChem were benchmarked by applying it to eighteen bioorthogonal probes. Of note, the analyses reveal that the formation of unexpected PDMs can be driven by endogenous reactive metabolites (e.g., bioactive aldehydes and glutathione). Together, pChem is a powerful and user-friendly tool that aims to facilitate the development of probes for the ever-growing field of chemoproteomics.Chemical probe coupled with mass spectrometry (MS)-based proteomics, herein termed chemoproteomics, offers versatile tools to globally profile protein features and to systematically interrogate the mode of action of small molecules in a native biological system 1 . For instance, bioorthogonal probes surrogating endogenous metabolites (e.g., sugars and lipids) enable the proteome-wide mapping of post-translational modifications (PTMs) on specific amino acid residues 2 . In addition, various activity-based protein profiling (ABPP) probes have been developed by targeting amino acid residues including cysteine 3, 4 , lysine 5 , tyrosine 6 , methionine 7, 8 , histidine 9 , aspartate and glutamate 10,11 , as well as their PTM forms [12][13][14] , which greatly expand the chemical space in complex proteomes for probing fundamental biology and for discovering new small molecule-based therapies.Nonetheless, the development of an efficient and selective probe for chemoproteomics can still be challenging. It is particularly difficult to unbiasedly assess its chemoselectivity at a proteome-wide scale, since a chemical probe displaying selectivity well-characterized in vitro would possibly generate unexpected modifications owing to potential cross-reactivity in complex biological systems. In addition, unforeseeable probe-derived modifications (PDMs) may be yielded during sample preparation or in-source MS fragmentation, thereby causing inhomogeneous modifications on the same sites and
“…4a), thereby greatly expanding the cysteine redoxome 12,[42][43][44][45] . Nevertheless, the chemoselectivity of oxoform-specific probes has long been controversial, even for those classic ones (i.e., dimedone for SOH) 46,47 . To address this controversy in an unbiased fashion, we used pChem to re-analyze the redox proteomic datasets generated in our laboratory.…”
Chemoproteomics has emerged as a key technology to expand the functional space in complex proteomes for probing fundamental biology and for discovering new small molecule-based therapies. Here we report a modification-centric computational tool termed pChem to provide a streamlined pipeline for unbiased performance assessment of chemoproteomic probes. The pipeline starts with an experimental setting for isotopically coding probe-derived modifications (PDMs) that can be automatically recognized by pChem, with masses accurately calculated and sites precisely localized. Further, pChem exports on-demand reports by scoring the profiling efficiency, modification-homogeneity and proteome-wide residue selectivity of a tested probe. The performance and robustness of pChem were benchmarked by applying it to eighteen bioorthogonal probes. Of note, the analyses reveal that the formation of unexpected PDMs can be driven by endogenous reactive metabolites (e.g., bioactive aldehydes and glutathione). Together, pChem is a powerful and user-friendly tool that aims to facilitate the development of probes for the ever-growing field of chemoproteomics.Chemical probe coupled with mass spectrometry (MS)-based proteomics, herein termed chemoproteomics, offers versatile tools to globally profile protein features and to systematically interrogate the mode of action of small molecules in a native biological system 1 . For instance, bioorthogonal probes surrogating endogenous metabolites (e.g., sugars and lipids) enable the proteome-wide mapping of post-translational modifications (PTMs) on specific amino acid residues 2 . In addition, various activity-based protein profiling (ABPP) probes have been developed by targeting amino acid residues including cysteine 3, 4 , lysine 5 , tyrosine 6 , methionine 7, 8 , histidine 9 , aspartate and glutamate 10,11 , as well as their PTM forms [12][13][14] , which greatly expand the chemical space in complex proteomes for probing fundamental biology and for discovering new small molecule-based therapies.Nonetheless, the development of an efficient and selective probe for chemoproteomics can still be challenging. It is particularly difficult to unbiasedly assess its chemoselectivity at a proteome-wide scale, since a chemical probe displaying selectivity well-characterized in vitro would possibly generate unexpected modifications owing to potential cross-reactivity in complex biological systems. In addition, unforeseeable probe-derived modifications (PDMs) may be yielded during sample preparation or in-source MS fragmentation, thereby causing inhomogeneous modifications on the same sites and
“…However, the formation of a (cyclic) sulfenamide will face a huge hurdle, because under normal circumstances, an amide (p K a ~17) is a very poor nucleophile and a sulfenic acid has a very short lifetime. Although the sulfenamide modification is rarely observed in proteins, a cyclic sulfenamide structure in protein tyrosine phosphatase 1B (PTP1B) was characterized crystallographically [ 32 , 33 ], which draws questions as to whether the target of C-nucleophile probes is actually the sulfenamide [ 34 , 35 ]. In fact, the formation of PTP1B sulfenamide is facilitated by two key factors, including a decreased p K a (increased nucleophilicity) of the Ser216 amide nitrogen due to hydrogen bonding, and steric shielding of the Cys215 sulfenic acid which becomes inaccessible by small-molecule nucleophiles, e.g., dimedone [ 36 ].…”
Section: Resultsmentioning
confidence: 99%
“…These findings further indicate that the pocket harboring PTP1B-SN is not exposed for nucleophilic attack, regardless of the probe's reactivity towards sulfenic acids. Apart from PTP1B, the cyclic sulfenamide modification has proved to be exceedingly rare [ 38 ], having only been identified previously in recombinant OhrR [ 39 ] or an AhpC mutant when analyzed in the gas phase by MS [ 25 ]. Fig.…”
Cysteine sulfenic acids (Cys-SOH) are pivotal modifications in thiol-based redox signaling and central intermediates
en route
to disulfide and sulfinic acid states. A core mission in our lab is to develop bioorthogonal chemical tools with the potential to answer mechanistic questions involving cysteine oxidation. Our group, among others, has contributed to the development of nucleophilic chemical probes for detecting sulfenic acids in living cells. Recently, another class of Cys-SOH probes based on strained alkene and alkyne electrophiles has emerged. However, the use of different models of sulfenic acid and methodologies, has confounded clear comparison of these probes with respect to chemical reactivity, kinetics, and selectivity. Here, we perform a parallel evaluation of nucleophilic and electrophilic chemical probes for Cys-SOH. Among the key findings, we demonstrate that a probe for Cys-SOH based on the norbornene scaffold does not react with any of the validated sulfenic acid models in this study. Furthermore, we show that purported cross-reactivity of dimedone-like probes with electrophiles, like aldehydes and cyclic sulfenamides, is a not meaningful in a biological setting. In summary, nucleophilic probes remain the most viable tools for bioorthogonal detection of Cys-SOH.
“…However, the formation of a (cyclic) sulfenamide will face a huge hurdle, because under normal circumstances, an amide (pK a ~ 17) is a very poor nucleophile and a sulfenic acid has a very short lifetime. Although the sulfenamide modification is rarely observed in proteins, a cyclic sulfenamide structure in protein tyrosine phosphatase 1B (PTP1B) was characterized crystallographically [32,33], which draws questions as to whether the target of 1,3-diketone derived C-nucleophiles is actually the sulfenamide [34,35]. In fact, the formation of PTP1B sulfenamide is facilitated by two key factors, including a decreased pK a (increased nucleophilicity) of the Ser215 amide nitrogen due to hydrogen bonding, and steric shielding of the Cys216 sulfenic acid which becomes inaccessible by small-molecule nucleophiles, e.g., dimedone [36].…”
Section: Selectivity Of 13-diketone-based Nucleophilic Probes In Biological Contextmentioning
S-sulfenylation of cysteine thiols (Cys-SOH) is a regulatory posttranslational modification in redox signaling and an important intermediate to other cysteine chemotypes. Owing to the dual chemical nature of the sulfur in sulfenic acid, both nucleophilic and electrophilic chemical probes have been developed to react with and detect Cys-SOH; however, the efficiency of existing probes has not been evaluated in a side-by-side comparison. Here, we employ small-molecule and protein models of Cys-SOH and compare the chemical probe reactivity. These data clearly show that 1,3-diketone-based nucleophilic probes react more efficiently with sulfenic acid as compared to strained alkene/alkyne electrophilic probes. Kinetic experiments that rigorously address the selectivity of the 1,3-diketone-based probes are also reported. Consideration of these data alongside relative cellular abundance, indicates that biological electrophiles, including cyclic sulfenamides, aldehydes, disulfides and hydrogen peroxide, are not meaningful targets of 1,3-diketone-based nucleophilic probes, which still remain the most viable tools for the bioorthogonal detection of Cys-SOH.
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