Disparate proteome reactivity profiles of carbon electrophiles

Abstract

Insights into the proteome reactivity of electrophiles are crucial for designing activity-based probes for enzymes lacking cognate affinity labels. Here, we show that different classes of carbon electrophiles exhibit markedly distinct amino acid labeling profiles in proteomes, ranging from selective reactivity with cysteine to adducts with several amino acids. These data thus specify electrophilic chemotypes with restricted and permissive reactivity profiles to guide the tailored design of next-generation functional proteomics probes.The field of activity-based protein profiling (ABPP) applies reactive chemical probes to profile the functional state of enzymes in native proteomes1. Original ABPP probes incorporated well-defined affinity labels as reactive groups to target enzyme classes such as the serine2 and cysteine3 hydrolases. Many enzymes, however, do not possess cognate affinity labels, and the design of ABPP probes for these proteins remains challenging. Structural insights into the substrate-binding pocket of enzyme classes can reveal nucleophilic residues for targeting with appropriate electrophiles. Recent work in the design of protein kinase probes positioned α-fluoromethyl ketone and acyl-phosphate electrophiles within an adenosine triphosphate (ATP) scaffold to exploit the nucleophilicity of proximal cysteine4 and lysine5 residues respectively. Differentiating among electrophilic chemotypes that show restricted and permissive amino acid reactivity profiles should streamline such endeavors to design ABPP probes for a wide range of enzyme classes.A variety of electrophiles are available for incorporation into ABPP probes. The proteome reactivity profiles of iodoacetamide and maleimide reactive groups have been extensively investigated 6 . Here, we expand on these studies by investigating the reactivity of a panel of carbon electrophiles (Fig. 1a), comprising a phenylsulfonate ester (SE, 1), linear-(EP, 2) and spiro-epoxides (SP, 5), an α-chloroacetamide (CA, 3) and an α,β-unsaturated ketone (UK, 4) in complex proteomes. An alkyne was incorporated into these electrophilic frameworks to provide a click chemistry handle for gel and mass spectrometric analysis 7 . Application of these electrophiles to a soluble mouse proteome, followed by click chemistry with a rhodamine azide (Rh-N 3 ) reporter tag and visualization of labeled proteins by SDS-PAGE and in-gel fluorescence scanning, demonstrated that the panel of electrophiles exhibit a range of protein reactivities (see Supplementary Information Fig. 1). Highest reactivity was observed for the UK probe, which demonstrated substantial protein labeling at concentrations as low as 1 μM. The CA and SE electrophiles demonstrated moderate levels of reactivity, whereas, the EP and SP probes displayed little to no protein labeling even at concentrations up to 20 μM. We then examined in greater depth the protein and amino-acid labeling profiles for the three probes that displayed the highest levels of proteome reactivity (SE, CA and UK). To address this ...

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