用于新合成蛋白质的快速、稳健和可重复富集的自动化高通量方法,Journal of Proteome Research

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用于新合成蛋白质的快速、稳健和可重复富集的自动化高通量方法
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2021-12-03 , DOI: 10.1021/acs.jproteome.1c00743
David Vargas-Diaz _{1,

2} , Maarten Altelaar _{1,

2}

Affiliation

开发了一种用于自动富集新合成蛋白质 (NSP) 的高通量方法，这些蛋白质通过用“可点击”类似物叠氮高丙氨酸 (AHA) 取代蛋氨酸进行代谢标记。含有二苯并环辛炔 (DBCO) 基团的合适缀合物允许通过与 AHA 的快速 1 小时点击化学反应来特定选择 NSP。通过自动移液平台，将样品加载到链霉亲和素试剂盒中，通过缀合物中包含的生物素诱饵选择性地结合 NSP。富集的蛋白质通过结合物中 4,4-二甲基-2,6-二氧亚环己基 (Dde) 基团的可重复化学裂解而洗脱，从而提高了选择性。NSP 可以在同一个孔板中收集和消化，随后可以加载生成的肽进行自动净化，然后进行质谱分析。所提出的自动化方法允许在 3 小时内对 96 孔板中的样品进行稳健和有效的富集。我们开发的富集方法经过全面评估，然后应用于黑色素瘤 A375 细胞分泌组的蛋白质组学分析，在用细胞因子干扰素 α (IFN-α) 和 γ (IFN-γ) 处理后，对 283 和 263 种蛋白质进行量化，分别揭示了复杂的肿瘤生长支持和抑制作用。

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Automated High-Throughput Method for the Fast, Robust, and Reproducible Enrichment of Newly Synthesized Proteins

A high-throughput method was developed for the automated enrichment of newly synthesized proteins (NSPs), which are labeled metabolically by substituting methionine with the “click-able” analogue azidohomoalanine (AHA). A suitable conjugate containing a dibenzocyclooctyne (DBCO) group allows the specific selection of NSPs by a fast 1 h click chemistry-based reaction with AHA. Through an automated pipetting platform, the samples are loaded into streptavidin cartridges for the selective binding of the NSPs by means of a biotin bait contained in the conjugate. The enriched proteins are eluted by a reproducible chemical cleavage of the 4,4-dimethyl-2,6-dioxocyclohexylidene (Dde) group in the conjugate, which increases selectivity. The NSPs can be collected and digested in the same well plate, and the resulting peptides can be subsequently loaded for automated cleanup, followed by mass spectrometry analysis. The proposed automated method allows for the robust and effective enrichment of samples in 96-well plates in a period of 3 h. Our developed enrichment method was comprehensively evaluated and then applied to the proteomics analysis of the melanoma A375 cell secretome, after treatment with the cytokines interferon α (IFN-α) and γ (IFN-γ), resulting in the quantification of 283 and 263 proteins, respectively, revealing intricate tumor growth-supportive and -suppressive effects.

更新日期：2022-01-07

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