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Evaluation of fully functionalized diazirine tags for chemical proteomic applications

ABSTRACT: The use of photo-affinity labeling (PAL) reagents for the mapping of noncovalent small molecule-protein interactions has become widespread. Recently, several ‘fully-functionalized’ (FF) chemical tags have been developed wherein a photoactivatable capture group, an enrichment handle, and a functional group for synthetic conjugation to a molecule of interest are integrated into a single modular tag. Diazirine-based FF tags in particular are increasingly employed in chemical proteomic investigations, however, despite routine usage, their relative utility has not been established. Here, we systematically evaluate several diazirine-containing FF tags, including a terminal diazirine analog developed herein, for chemical proteomic investigations. Specifically, we compared the general reactivity of five diazirine tags and assessed their impact on the profiles of various small molecules, including fragments and known inhibitors revealing that such tags can have profound effects on the proteomic profiles of chemical probes. Our findings should be informative for chemical probe design, PAL-reagent development, and chemical proteomic investigations.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Louis Conway

LAB HEAD: Christopher G Parker

PROVIDER: PXD025652 | Pride | 2021-05-18

REPOSITORIES: Pride

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Project description:Numerous reagents have been developed to enable chemical proteomic analysis of small molecule-protein interactomes. However, the performance of these reagents has not been systematically evaluated and compared. Herein, we report our efforts to conduct a parallel assessment of two widely-used chemically-cleavable linkers equipped with dialkoxydiphenylsilane (DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular cysteinome using iodoacetamide alkyne probe demonstrated a significant discrepancy between the experimental results obtained through the application of each of the reagents. To better understand the source of observed discrepancy, a mass tolerant database search strategy using MSFragger software was performed. This resulted in identifying a previously unreported artifactual modification on the residual mass of the azobenzene linker. Furthermore, we conducted a comparative analysis of enrichment modes using both cleavable linkers. This effort determined that enrichment of proteolytic digests yielded a far greater number of identified cysteine residues than the enrichment conducted prior to protein digest. Inspired by recent studies where multiplexed quantitative labeling strategies were applied to cleavable biotin linkers, we combined this further optimized protocol using the DADPS cleavable linker with tandem mass tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase inhibitor dacomitinib against the cysteinome of an epidermoid cancer cell line. Our analysis resulted in the detection and quantification of over 10,000 unique cysteine residues, a nearly 3-fold increase over previous studies that used cleavable biotin linkers for enrichment. Critically, cysteine residues corresponding to proteins directly as well as indirectly modulated by dacomitinib treatment were identified. Overall, our study suggests that the dialkoxydiphenylsilane linker could be broadly applied wherever chemically cleavable linkers are required for chemical proteomic characterization of cellular proteomes.

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Evaluation of fully functionalized diazirine tags for chemical proteomic applications

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