TY - JOUR
T1 - SDS-PAGE-free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes
AU - Sun, Liangliang
AU - Zhang, Yanyan
AU - Tao, Dingyin
AU - Zhu, Guijie
AU - Zhao, Qun
AU - Wu, Qi
AU - Liang, Zhen
AU - Yang, Ling
AU - Zhang, Lihua
AU - Zhang, Yukui
PY - 2012/12
Y1 - 2012/12
N2 - Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS-PAGE-free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1-butyl-3-methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D-SCX-RPLC-ESI-MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.
AB - Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS-PAGE-free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1-butyl-3-methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D-SCX-RPLC-ESI-MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.
KW - 1-Butyl-3-methyl imidazolium tetrafluoroborate
KW - 2D-LC-ESI-MS/MS
KW - Cytochrome P450 enzyme
KW - Protein spectral count
KW - Uridine diphosphoglucuronosyl transferase
UR - http://www.scopus.com/inward/record.url?scp=84871283943&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84871283943&partnerID=8YFLogxK
U2 - 10.1002/pmic.201200260
DO - 10.1002/pmic.201200260
M3 - Article
C2 - 23109277
AN - SCOPUS:84871283943
SN - 1615-9853
VL - 12
SP - 3464
EP - 3469
JO - Proteomics
JF - Proteomics
IS - 23-24
ER -