基于柱開關(guān)循環(huán)尺寸排阻色譜、微酶反應(yīng)器和μRPLC-ESI-MSMS的集成蛋白質(zhì)分析平臺

2025-02-05

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應(yīng)用領(lǐng)域蛋白/抗體/蛋白質(zhì)組/蛋白/重組蛋白/多肽/胰島素/GLP-1
樣品蛋白質(zhì)和肽
項目分離分析
參考標準/文獻H. Yuan et al. / J. Chromatogr. A 1216 (2009) 7478–7482

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摘要

Integrated protein analysis platform based on column switch recycling size exclusion chromatography, microenzymatic reactor and μRPLC–ESI-MSMS
基于柱開關(guān)循環(huán)尺寸排阻色譜、微酶反應(yīng)器和μRPLC-ESI-MSMS的集成蛋白質(zhì)分析平臺

摘要

An integrated platform with the combination of proteins and peptides separation was established via the unit of on-line proteins digestion, by which proteins were in sequence separated by column switch recycling size exclusion chromatography (csrSEC), on-line digested by an immobilized trypsin microre actor, trapped and desalted by two parallel C8 precolumns, separated by RPLC with the linear gradient of organic modifier concentration, and identified by ESI-MS/MS. A 6-protein mixture, with Mr ranging from 10 kDa to 80 kDa, was used to evaluate the performance of the integrated platform, and all proteins were identified with sequence coverage over 5.67%. Our experimental results demonstrate that such an integrated platform is of advantages such as good time compatibility, high peak capacity, and facile automation, which might be a promising approach for proteome study.

通過在線蛋白質(zhì)消化單元建立了一個結(jié)合蛋白質(zhì)和肽分離的集成平臺，通過該平臺，蛋白質(zhì)通過柱開關(guān)循環(huán)尺寸排阻色譜（csrSEC）按順序分離，通過固定化胰蛋白酶微反應(yīng)器在線消化，通過兩個平行的C8預(yù)柱捕獲和脫鹽，通過有機修飾劑濃度線性梯度的RPLC分離，并通過ESI-MS/MS鑒定。使用Mr范圍為10 kDa至80 kDa的6蛋白混合物來評估集成平臺的性能，所有蛋白質(zhì)的序列覆蓋率均超過5.67%。我們的實驗結(jié)果表明，這種集成平臺具有時間兼容性好、峰值容量高、易于自動化等優(yōu)點，可能是蛋白質(zhì)組學研究的一種有前景的方法。

SEC SRT-150 ?-5 μm and SEC nanofilm-250 ?-5 μm particles were purchased from Sepax Technologies (Newark, Delaware, USA)

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