TY - JOUR
T1 - Serially coupled microcolumn reversed phase liquid chromatography for shotgun proteomic analysis
AU - Tao, Dingyin
AU - Zhu, Guijie
AU - Sun, Liangliang
AU - Ma, Junfeng
AU - Liang, Zhen
AU - Zhang, Weibing
AU - Zhang, Lihua
AU - Zhang, Yukui
PY - 2009/4
Y1 - 2009/4
N2 - Microcolumn RPLC (μRPLC) is one of the optimum separation modes for shotgun proteomic analysis. To identify as many proteins as possible by MS/MS, the improvement on separation efficiency and peak capacity of μRPLC is indispensable. Although the increase in column length is one of the effective solutions, the preparation of a long microcolumn is rather difficult due to the high backpressure generated during the packing procedure. In our recent work, through connecting microcolumns of 5, 10, and 15 cm length via unions with minimal dead volume, long microcolumns with length up to 30 cm were obtained, with which 318 proteins were identified from proteins extracted from Escherichia coli by μRPLC-ESI MS/MS, and similar distributions of Mw and pI were found with single and various coupled microcolumns. Furthermore, by using MS/MS with improved sensitivity, with such a serially coupled 30 cm long microcolumn, 1692 proteins were identified within 7 h from rat brain tissue, with false positive rate (FPR) <1%. All these results demonstrated that serially couple microcolumns might be of great promising to improve the separation capacity of μRPLC in shotgun proteomic analysis.
AB - Microcolumn RPLC (μRPLC) is one of the optimum separation modes for shotgun proteomic analysis. To identify as many proteins as possible by MS/MS, the improvement on separation efficiency and peak capacity of μRPLC is indispensable. Although the increase in column length is one of the effective solutions, the preparation of a long microcolumn is rather difficult due to the high backpressure generated during the packing procedure. In our recent work, through connecting microcolumns of 5, 10, and 15 cm length via unions with minimal dead volume, long microcolumns with length up to 30 cm were obtained, with which 318 proteins were identified from proteins extracted from Escherichia coli by μRPLC-ESI MS/MS, and similar distributions of Mw and pI were found with single and various coupled microcolumns. Furthermore, by using MS/MS with improved sensitivity, with such a serially coupled 30 cm long microcolumn, 1692 proteins were identified within 7 h from rat brain tissue, with false positive rate (FPR) <1%. All these results demonstrated that serially couple microcolumns might be of great promising to improve the separation capacity of μRPLC in shotgun proteomic analysis.
KW - Serially coupled microcolumn
KW - Shotgun proteome analysis
KW - μRPLC MS/MS
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U2 - 10.1002/pmic.200800649
DO - 10.1002/pmic.200800649
M3 - Article
C2 - 19294701
AN - SCOPUS:65349188080
SN - 1615-9853
VL - 9
SP - 2029
EP - 2036
JO - Proteomics
JF - Proteomics
IS - 7
ER -