Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis

Yu Liang, Dingyin Tao, Junfeng Ma, Liangliang Sun, Zhen Liang, Lihua Zhang, Yukui Zhang

Research output: Contribution to journalArticlepeer-review

Abstract

A novel kind of hydrophilic monolith based immobilized enzyme reactors (IMERs) was prepared both in UV-transparent capillaries and on glass microchips by the photopolymerization of N-acryloxysuccinimide and poly(ethylene glycol)diacrylate, followed by trypsin immobilization. The performance of capillary IMERs for protein digestion was evaluated by the digestion of myoglobin with the residential time from 12. s to 71. s. With μRPLC-ESI-MS/MS analysis, the obtained sequence coverages were all over 80%, comparable to that obtained by in-solution digestion for 12. h. The nonspecific absorption of BSA on monolithic support was evaluated, and no obvious protein residue was observed by a fluorescence assay. Moreover, no carry-over of the digests on the capillary IMER was found after the digestion of myoglobin (24 μg) and BSA (9 μg), which further demonstrated the good hydrophilicity of such matrix. In addition, an integrated microchip-based system involving on-line protein digestion by microchip-based IMER, peptides separation by nanoRPLC and identification by ESI-MS/MS was established, by which a mixture of standard proteins and one RPLC fraction of Escherichia coli extract were successfully identified, indicating that the hydrophilic monolith based IMER might provide a promising tool for high-throughput proteomic analysis.

Original languageEnglish (US)
Pages (from-to)2898-2905
Number of pages8
JournalJournal of Chromatography A
Volume1218
Issue number20
DOIs
StatePublished - May 20 2011

Keywords

  • Capillary
  • Hydrophilic monolith
  • Immobilized enzyme reactor
  • Microchip
  • Proteomic analysis

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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