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Abstract
The convergence of reactive cysteine-targeted electrophilic fragments and chemoproteomics have dramatically accelerated the discovery of ligandable sites in the proteome. Our genome encodes 214,000 cysteine residues, at least 20% of which are estimated to be redox-active. Oxidation blunts sulfur reactivity toward electrophiles but opens the door to a new class of nucleophilic covalent ligands that target cysteinyl sulfenic acids, which are widespread post-translational modifications. Here we report a quantitative analysis of nucleophilic fragments screened against the human sulfenome. Ligands were discovered for >500 sulfenated cysteines in >400 proteins, including sites not targeted by electrophiles with the same scaffold. Among these were compounds that preferentially react with hepatoma-derived growth factor (HDGF)-related proteins (HRPs) one of which was able to block nuclear transport of this oncoprotein. Nucleophilic fragments provide a rich resource for chemical biology and drug discovery, where ligandability in the human proteome extends beyond protein thiols.
Supplementary materials
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Supplementary Data 1
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Supplementary Data 1. Quantitative chemoproteomic analysis of sulfenome reactivity in the human proteome. The protein lysates from A549 cells were labeled with 500 µM and 5 mM BTD, respectively, and subjected to tryptic digestion. The resulting BTD-modified peptides were further conjugated to light and heavy azido biotin reagents with a photocleavable linker (Az-UV-biotin), respectively, via copper catalyzed alkyne-azide cycloaddition reaction (CuAAC). The light and heavy labeled samples then were mixed equally in amount and subjected to streptavidin-based enrichment. After several washing steps, the modified peptides were selectively eluted from beads under 365 nm wavelength of UV light and subjected to LC-MS/MS-based proteomic analysis. Hyperreactive cysteines would be expected to label to completion at low probe concentrations and less reactive cysteines should show concentration-dependent increases in BTD labeling.
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Supplementary Data 2.
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Supplementary Data 2. Comparing nucleophilic fragments with electrophilic fragments showed distinct target profiles of electrophilc and nucleophilc fragments with the same scaffold.
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Supplementary Data 3
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Supplementary Data 3. Global profiling of sulfenic acid ligandability
in the human proteome. The protein lysates from MDA-MB-231 cells were firstly labeled with 0 or 2mM fragment, and then labeled with 5mM BTD, and finally subjected to tryptic digestion. The resulting BTD-modified peptides were further conjugated to light and heavy azido biotin reagents with a photocleavable linker (Az-UV-biotin), respectively, via copper catalyzed alkyne-azide cycloaddition reaction. The light and heavy labeled samples then were mixed equally in amount and subjected to streptavidin-based enrichment. After several washing steps, the modified peptides were selectively eluted from beads under 365 nm wavelength of UV light and subjected to LC-MS/MS-based proteomic analysis. For each cysteine detected, we calculated a control/fragment ratio (RH/L). Fragment liganded sulfenic acid would enabl less BTD-derived chemoselective conjugation, thereby rendering relatively high RH/L values.
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Supplementary Data 4
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Supplementary Data 4. Correlation of sulfenylome reactivity of each fragment with the corresponding structural features and kinetic parameters, including number of free NH , molecular weight (MW, ), CLogP value, the observed binding constant (kobs) and reaction rate constant (M-1S-1).
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Supplementary Data 5
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Supplementary Data 5. Function annotation of all liganded cysteines
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Supporting Information
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Supporting Information including detailed Methods and Supporting Figures.
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