Biological Chemistry Department, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
Molecular Biology Institute, University of California Los Angeles, Los Angeles, California.
Curr Protoc. 2022 Jul;2(7):e492. doi: 10.1002/cpz1.492.
Cysteine-directed chemoproteomic profiling methods yield high-throughput inventories of redox-sensitive and ligandable cysteine residues and therefore are enabling techniques for functional biology and drug discovery. However, the cumbersome nature of many sample preparation workflows, the requirements for large amounts of input material, and the modest yields of labeled peptides are limitations that hinder most chemoproteomics studies. Here, we report an optimized chemoproteomic sample-preparation workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhanced sample preparation (SP3) to improve the recovery of biotinylated peptides, even from small samples. We further tailor our SP3 method to specifically probe the redox proteome, which showcases the utility of the SP3 platform in multistep sample-preparation workflows. By implementing a customized workflow in the FragPipe computational pipeline, we achieve accurate MS1-based quantification, including for peptides containing multiple cysteine residues. Collectively these innovations enable enhanced high-throughput quantitative analysis of the cysteinome. This article includes detailed protocols for cysteine labeling with isotopically labeled iodoacetamide alkyne probes, biotinylation with CuAAC, sample cleanup with SP3, enrichment of cysteines with NeutrAvidin agarose beads, LC-FAIMS-MS/MS analysis, and FragPipe-IonQuant analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling of cysteines in human proteome and SP3-based sample cleanup Alternate Protocol 1: Labeling of cysteines in human proteome, SP3-based sample cleanup, and enrichment of cysteines for isoTOP-ABPP analysis Alternate Protocol 2: Labeling of cysteines in human proteome and SP3-based sample cleanup for redox proteome analysis Basic Protocol 2: Peptide-level cysteine enrichment Basic Protocol 3: LC-FAIMS-MS/MS analysis Basic Protocol 4: FragPipe data analysis.
半胱氨酸导向的化学蛋白质组学分析方法能够高通量地鉴定出氧化还原敏感和配体结合的半胱氨酸残基，因此是功能生物学和药物发现的重要技术手段。然而，许多样品制备工作流程繁琐、需要大量输入材料以及标记肽的产量适中，这些都是限制大多数化学蛋白质组学研究的因素。在这里，我们报告了一种优化的化学蛋白质组学样品制备工作流程，该流程结合了增强的肽标记与单步固相增强样品制备（SP3），以提高生物素化肽的回收率，即使是从小样本中也能得到提高。我们进一步调整我们的 SP3 方法，专门探测氧化还原蛋白质组，展示了 SP3 平台在多步样品制备工作流程中的实用性。通过在 FragPipe 计算管道中实现定制的工作流程，我们实现了基于 MS1 的准确定量，包括含有多个半胱氨酸残基的肽的定量。这些创新共同实现了对半胱氨酸组的增强高通量定量分析。本文包括详细的方案，用于使用同位素标记的碘乙酰胺炔烃探针对半胱氨酸进行标记、用 CuAAC 进行生物素化、用 SP3 进行样品净化、用 NeutrAvidin 琼脂糖珠对半胱氨酸进行富集、LC-FAIMS-MS/MS 分析以及 FragPipe-IonQuant 分析。 © 2022 Wiley Periodicals LLC. 基本方案 1：人蛋白质组中半胱氨酸的标记和基于 SP3 的样品净化 可选方案 1：人蛋白质组中半胱氨酸的标记、基于 SP3 的样品净化和 isoTOP-ABPP 分析对半胱氨酸的富集 可选方案 2：人蛋白质组中半胱氨酸的标记和基于 SP3 的样品净化用于氧化还原蛋白质组分析 基本方案 2：肽水平对半胱氨酸的富集 基本方案 3：LC-FAIMS-MS/MS 分析 基本方案 4：FragPipe 数据分析。
