Establishment of a PF2D-MS/MS platform for rapid profiling and semiquantitative analysis of membrane protein biomarkers.
Current proteome profiling techniques have identified relatively few mammalian membrane proteins despite their numerous important functions. To establish a standard throughput-potential profiling platform for membrane proteins, Triton X-100-solubilized rat liver microsomal proteins were separated on a 2-D separation system (2-D liquid phase fractionation (PF2D)) in two different pH ranges (4.0-8.5 and 7.0-10.5). This system produced 182 proteins with more than two transmembrane domain (TMD), including 16 TMDs with high confidence. Comparative 2-D liquid maps with high resolution and reproducibility have been constructed for liver microsome from the phenobarbital (PB) treated rats. PF2D was also found to be useful for the semiquantification of some representative cytochrome P450 family proteins (e.g., cytochrome P450 2B2) that were induced by PB treatment compared with untreated controls. Thus, the combination of both high-detection capacity and rapid preliminary semiquantification in a PF2D platform could become a standard system for the routine analysis of membrane proteins.
Lee
Hyoung-Joo
HJ
Department of Biochemistry, Yonsei Proteome Research Center and Biomedical Proteome Research Center, Yonsei University, Sudaemoon-Ku, Seoul, Korea.
Kwon
Min-Seok
MS
Lee
Eun-Young
EY
Cho
Sang Yun
SY
Paik
Young-Ki
YK
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry; D015415:Biomarkers / Q000737:chemistry*; D000072467:Cytochrome P450 Family 2; D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation* / Q000379:methods; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D008862:Microsomes, Liver / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010634:Phenobarbital / Q000737:chemistry; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D051381:Rats; D014357:Trypsin / Q000737:chemistry
D015415:Biomarkers; D003902:Detergents; D008565:Membrane Proteins; D017830:Octoxynol; D001189:Aryl Hydrocarbon Hydroxylases; C089142:Cyp2b3 protein, rat; D000072467:Cytochrome P450 Family 2; D014357:Trypsin; D010634:Phenobarbital