Department of Chemistry, University of Texas at Austin, 100 E. 24th Street, Austin, Texas 78712, United States.
Department of Molecular Biosciences, University of Texas at Austin, 2500 Speedway, Austin, Texas 78712, United States.
ACS Chem Biol. 2020 Jun 19;15(6):1401-1407. doi: 10.1021/acschembio.0c00040. Epub 2020 May 14.
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.
蛋白质组学领域最近随着更敏感的肽批量测量技术以及单分子技术而得到扩展。这些方法中的一个限制因素是需要多次化学衍生化和高度纯净的无污染物的蛋白质。我们展示了一种适合肽的酶解、纯化和随后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，并进行一锅法酶解、捕获和衍生化，以从细胞蛋白质组中的 40000 多个独特肽中检测肽捕获偏倚。我们还表明，这种捕获可以以无痕迹的方式逆转，因此适用于单分子蛋白质组学技术。通过这项技术，我们对肽进行荧光标记和 C 末端衍生化，并对其进行荧光测序，证明在进行单分子蛋白质测序之前，用洗涤树脂足以去除多余的染料和其他试剂。
