Two-dimensional liquid chromatography consisting of twelve second-dimension columns for comprehensive analysis of intact proteins

TALANTA

卷:182

Article

Ren, Jiangtao¹  Beckner, Matthew A.¹  Lynch, Kyle B.¹  Chen, Huang¹  Zhu, Zaifang¹  Yang, Yu¹  Chen, Apeng¹  Qiao, Zhenzhen²  Liu, Shaorong¹  Lu, Joann J.¹ 

[1] Univ Oklahoma, Dept Chem & Biochem, 101 Stephenson Pkwy, Norman, OK 73019 USA

[2] Univ Oklahoma, Dept Microbiol & Plant Biol, Norman, OK 73019 USA

关键词: High performance liquid chromatography;    Two dimensional liquid chromatography;    Monolith column;    Protein separation;    Intact protein;   

DOI  :  10.1016/j.talanta.2018.01.072

来源: Elsevier

【 摘 要 】

A comprehensive two-dimensional liquid chromatography (LCxLC) system consisting of twelve columns in the second dimension was developed for comprehensive analysis of intact proteins in complex biological samples. The system consisted of an ion-exchange column in the first dimension and the twelve reverse-phase columns in the second dimension; all thirteen columns were monolithic and prepared inside 250 mu m i.d. capillaries. These columns were assembled together through the use of three valves and an innovative configuration. The effluent from the first dimension was continuously fractionated and sequentially transferred into the twelve second dimension columns, while the second-dimension separations were canied out in a series of batches (six columns per batch). This LCxLC system was tested first using standard proteins followed by real-world samples from E. coli. Baseline separation was observed for eleven standard proteins and hundreds of peaks were observed for the real-world sample analysis. Two-dimensional liquid chromatography, often considered as an effective tool for mapping proteins, is seen as laborious and time-consuming when configured offline. Our online LCxLC system with increased second-dimension columns promises to provide a solution to overcome these hindrances.

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