title: Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis.
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 12s 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.
authors: Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R & A Center, Chinese Academy of Sciences, Dalian, China.
authors: Yu
authors: Y
authors: Liang
authors: Dingyin
authors: D
authors: Tao
authors: Junfeng
authors: J
authors: Ma
authors: Liangliang
authors: L
authors: Sun
authors: Zhen
authors: Z
authors: Liang
authors: Lihua
authors: L
authors: Zhang
authors: Yukui
authors: Y
authors: Zhang
mesh_terms: D000818:Animals; D019149:Bioreactors; D056148:Chromatography, Reverse-Phase; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin