用于光亲和标记目标参与和解旋的端到端通量化学蛋白质组学。

IF 3.8 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Journal of Proteome Research Pub Date : 2024-11-01 Epub Date: 2024-10-07 DOI:10.1021/acs.jproteome.4c00442
Sheldon T Cheung, Yongkang Kim, Ji-Hoon Cho, Kristoffer R Brandvold, Brahma Ghosh, Amanda M Del Rosario, Harris Bell-Temin
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引用次数: 0

摘要

事实证明,光亲和标记(PAL)方法有助于对生理相关系统中的蛋白质-配体结合事件进行无偏解构。然而,与其他化学蛋白质组工作流程一样,这些方法在很多方面受到耗时的样品处理和数据采集技术的限制。在这里,我们介绍了一种解决这一难题的方法,即创新性地采用基于羧酸珠的蛋白质净化程序来去除多余的小分子污染物,并将其与基于平板的蛋白质组样品处理相结合,作为一种半自动化解决方案。通过无标记、数据独立采集(DIA)技术对样品进行分析,每个样品的工作流程时间比目前的标准做法有了显著改善。利用三种已知靶点的 PAL 配体((+)-JQ-1、来那度胺和达沙替尼)进行的实验表明,灵活设计具有多种变量的实验非常有用。数据显示,该工作流程能以出色的蛋白质信号-背景富集灵敏度对已知靶标和推定靶标进行可靠的鉴定和排序。这种处理和分析复杂样本的端到端统一吞吐量策略能极大地促进高效的药物发现工作,并为化学蛋白质组学领域带来新的机遇。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
End-to-End Throughput Chemical Proteomics for Photoaffinity Labeling Target Engagement and Deconvolution.

Photoaffinity labeling (PAL) methodologies have proven to be instrumental for the unbiased deconvolution of protein-ligand binding events in physiologically relevant systems. However, like other chemical proteomic workflows, they are limited in many ways by time-intensive sample manipulations and data acquisition techniques. Here, we describe an approach to address this challenge through the innovation of a carboxylate bead-based protein cleanup procedure to remove excess small-molecule contaminants and couple it to plate-based, proteomic sample processing as a semiautomated solution. The analysis of samples via label-free, data-independent acquisition (DIA) techniques led to significant improvements on a workflow time per sample basis over current standard practices. Experiments utilizing three established PAL ligands with known targets, (+)-JQ-1, lenalidomide, and dasatinib, demonstrated the utility of having the flexibility to design experiments with a myriad of variables. Data revealed that this workflow can enable the confident identification and rank ordering of known and putative targets with outstanding protein signal-to-background enrichment sensitivity. This unified end-to-end throughput strategy for processing and analyzing these complex samples could greatly facilitate efficient drug discovery efforts and open up new opportunities in the chemical proteomics field.

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来源期刊
Journal of Proteome Research
Journal of Proteome Research 生物-生化研究方法
CiteScore
9.00
自引率
4.50%
发文量
251
审稿时长
3 months
期刊介绍: Journal of Proteome Research publishes content encompassing all aspects of global protein analysis and function, including the dynamic aspects of genomics, spatio-temporal proteomics, metabonomics and metabolomics, clinical and agricultural proteomics, as well as advances in methodology including bioinformatics. The theme and emphasis is on a multidisciplinary approach to the life sciences through the synergy between the different types of "omics".
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