TY - JOUR
T1 - Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis
AU - Liang, Yu
AU - Tao, Dingyin
AU - Ma, Junfeng
AU - Sun, Liangliang
AU - Liang, Zhen
AU - Zhang, Lihua
AU - Zhang, Yukui
N1 - Funding Information:
The authors are grateful for the financial support from National Natural Science Foundation ( 20935004 ), National Basic Research Program of China ( 2007CB714503 and 2007CB914100 ), Creative Research Group Project by NSFC (No. 21021004 ), National Key Technology R. & D. Program ( 2008BAK41B02 and 2009BAK59B02 ), and Knowledge Innovation Program of Chinese Academy of Sciences ( KJCX2YW.H09 ).
PY - 2011/5/20
Y1 - 2011/5/20
N2 - 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.
AB - 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.
KW - Capillary
KW - Hydrophilic monolith
KW - Immobilized enzyme reactor
KW - Microchip
KW - Proteomic analysis
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U2 - 10.1016/j.chroma.2011.02.073
DO - 10.1016/j.chroma.2011.02.073
M3 - Article
C2 - 21450299
AN - SCOPUS:79955080967
VL - 1218
SP - 2898
EP - 2905
JO - Journal of Chromatography A
JF - Journal of Chromatography A
SN - 0021-9673
IS - 20
ER -