Dataset Information

A Quantitative Chemoproteomic Platform to Monitor Selenocysteine Reactivity within a Complex Proteome.

ABSTRACT: Mammalian selenocysteine (Sec)-containing proteins, selenoproteins, are important to (patho)physiological processes, including redox homeostasis. Sec residues have been recalcitrant to mass spectrometry-based chemoproteomic methods that enrich for reactive cysteine (Cys) residues with electrophilic chemical probes, despite confirmed reactivity of Sec with these electrophiles. Highly abundant Cys peptides likely suppress low-abundant Sec peptides. By exploiting the decreased pKa of Sec relative to Cys, we have developed a chemoproteomic platform that relies on low pH (pH 5.75) electrophile labeling, reducing Cys reactivity and enhancing identification of Sec-containing peptides across mouse tissues and cell lines. The utility of this Sec-profiling platform is underscored by evaluation of the selectivity of auranofin, an inhibitor of the selenoprotein, thioredoxin reductase, against both reactive Cys- and Sec-containing proteins. Platform limitations pertain to the non-physiological low-pH conditions that could perturb protein structure and function. Future work necessitates the discovery of Sec-selective electrophiles that function at physiological pH.

SUBMITTER: Bak DW

PROVIDER: S-EPMC6510024 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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A Quantitative Chemoproteomic Platform to Monitor Selenocysteine Reactivity within a Complex Proteome.

Bak Daniel W DW Gao Jinjun J Wang Chu C Weerapana Eranthie E

Cell chemical biology 20180705 9

Mammalian selenocysteine (Sec)-containing proteins, selenoproteins, are important to (patho)physiological processes, including redox homeostasis. Sec residues have been recalcitrant to mass spectrometry-based chemoproteomic methods that enrich for reactive cysteine (Cys) residues with electrophilic chemical probes, despite confirmed reactivity of Sec with these electrophiles. Highly abundant Cys peptides likely suppress low-abundant Sec peptides. By exploiting the decreased pK<sub>a</sub> of Sec ...[more]

PMID: 29983274

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Project description:The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access quantitative database that provides the most efficient extraction conditions of 2065 unique mammalian MPs. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to capture membrane 'nano-scoops' containing a target MP efficiently and interface with structural, functional, and other bioanalytical approaches. We demonstrate an example of this by combining our extraction platform with single-molecule TIRF imaging to demonstrate how it can enable rapid determination of homo-oligomeric states of target MPs in native cell membranes.

Dataset Information

A Quantitative Chemoproteomic Platform to Monitor Selenocysteine Reactivity within a Complex Proteome.

Publications

A Quantitative Chemoproteomic Platform to Monitor Selenocysteine Reactivity within a Complex Proteome.

Similar Datasets

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