Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical Proteomics.

IF 6.1 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Molecular & Cellular Proteomics Pub Date : 2024-07-01 Epub Date: 2024-05-21 DOI:10.1016/j.mcpro.2024.100790
Anders H Kverneland, Florian Harking, Joel Mario Vej-Nielsen, Magnus Huusfeldt, Dorte B Bekker-Jensen, Inge Marie Svane, Nicolai Bache, Jesper V Olsen
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Abstract

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified ∼8000 protein groups and ∼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.

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集成样品消化和 Evotip 装载的全自动工作流程,实现了高通量临床蛋白质组学。
蛋白质鉴定和定量是发现生物标志物的重要工具。随着现代质谱仪灵敏度和速度的提高,样品制备仍然是研究大型群体的瓶颈。为了解决这个问题,我们在 Opentrons OT-2 (OT-2)机器人上准备并评估了一个简单高效的工作流程,它以全自动的方式将样品消化、清理和装载到 Evotips 上,可在 6 小时内处理多达 192 个样品。使用 Evosep One 进行 11.5 分钟的主动液相色谱梯度分析,并使用 Orbitrap Astral 质谱仪进行窄窗数据独立采集,每天可提供 100 个样品的处理量。我们的研究结果表明,这种高灵敏度的工作流程在低样品输入量的情况下也能获得可重复性和稳定性。工作流程经过优化,样品起始量极少,从而降低了样品制备所需的试剂成本,这在分析大型生物队列时至关重要。在消化工作流程的基础上,我们使用磁性 Ti-IMAC 珠加入了自动磷肽富集步骤。这样,蛋白质组和磷酸蛋白组样品的制备就能在一个步骤内实现全自动、高灵敏度。利用集成消化和 Evotip 装载工作流程,我们评估了癌症免疫疗法对转移性黑色素瘤患者血浆蛋白质组的影响。
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来源期刊
Molecular & Cellular Proteomics
Molecular & Cellular Proteomics 生物-生化研究方法
CiteScore
11.50
自引率
4.30%
发文量
131
审稿时长
84 days
期刊介绍: The mission of MCP is to foster the development and applications of proteomics in both basic and translational research. MCP will publish manuscripts that report significant new biological or clinical discoveries underpinned by proteomic observations across all kingdoms of life. Manuscripts must define the biological roles played by the proteins investigated or their mechanisms of action. The journal also emphasizes articles that describe innovative new computational methods and technological advancements that will enable future discoveries. Manuscripts describing such approaches do not have to include a solution to a biological problem, but must demonstrate that the technology works as described, is reproducible and is appropriate to uncover yet unknown protein/proteome function or properties using relevant model systems or publicly available data. Scope: -Fundamental studies in biology, including integrative "omics" studies, that provide mechanistic insights -Novel experimental and computational technologies -Proteogenomic data integration and analysis that enable greater understanding of physiology and disease processes -Pathway and network analyses of signaling that focus on the roles of post-translational modifications -Studies of proteome dynamics and quality controls, and their roles in disease -Studies of evolutionary processes effecting proteome dynamics, quality and regulation -Chemical proteomics, including mechanisms of drug action -Proteomics of the immune system and antigen presentation/recognition -Microbiome proteomics, host-microbe and host-pathogen interactions, and their roles in health and disease -Clinical and translational studies of human diseases -Metabolomics to understand functional connections between genes, proteins and phenotypes
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