Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics.,ACS Chemical Biology

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Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-05-02 , DOI: 10.1021/acschembio.0c00040
Cecil J Howard ₁ , Brendan M Floyd ₂ , Angela M Bardo ₂ , Jagannath Swaminathan ₂ , Edward M Marcotte ₂ , Eric V Anslyn ₁

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The field of proteomics has expanded recently with more sensitive techniques for the bulk measurement of peptides as well as single-molecule techniques. One limiting factor for some of these methods is the need for multiple chemical derivatizations and highly pure proteins free of contaminants. We demonstrate a solid-phase capture-release strategy suitable for the proteolysis, purification, and subsequent chemical modification of peptides. We use this resin on an HEK293T cell lysate and perform one-pot proteolysis, capture, and derivatization to survey peptide capture biases from over 40 000 unique peptides from a cellular proteome. We also show that this capture can be reversed in a traceless manner, such that it is amenable for single-molecule proteomics techniques. With this technique, we perform a fluorescent labeling and C-terminal derivatization on a peptide and subject it to fluorosequencing, demonstrating that washing the resin is sufficient to remove excess dyes and other reagents prior to single-molecule protein sequencing.

中文翻译：

固相肽的捕获和释放，用于大分子和单分子蛋白质组学。

蛋白质组学领域最近通过用于肽的批量测量的更灵敏的技术以及单分子技术得以扩展。这些方法中的一些限制因素是需要多种化学衍生作用和无杂质的高纯度蛋白质。我们展示了适用于肽的蛋白水解，纯化和后续化学修饰的固相捕获-释放策略。我们在HEK293T细胞裂解物上使用这种树脂，并进行一锅蛋白水解，捕获和衍生化，以调查来自细胞蛋白质组的40 000多种独特肽的肽捕获偏倚。我们还表明，这种捕获可以以无痕方式逆转，因此适用于单分子蛋白质组学技术。用这种技术

更新日期：2020-06-19

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