Plasma membrane(PM)proteome is one of the major subproteomes present in the cell,and is very important in liver function.In the present work,C57 mouse liver PM was purified by density-gradient centrifugation.The purif...Plasma membrane(PM)proteome is one of the major subproteomes present in the cell,and is very important in liver function.In the present work,C57 mouse liver PM was purified by density-gradient centrifugation.The purified PM was verified by electron microscope analysis and Western blotting.The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction.Proteins were separated by 2DE and 1DE,trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry.In all,547 non-redundant mouse liver PM proteins were identified,of which 34%contributed to plasma membrane or plasma membrane-related proteins.This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.展开更多
Hainantoxin-Ⅳ(HNTX-Ⅳ)was isolated from the Chinese bird spider Ornithoctorcs hainana and identified as a novel antagonist of tetrodotoxin-sensitive(TTX-S)sodium channels.As revealed by the solution structure of HNTX...Hainantoxin-Ⅳ(HNTX-Ⅳ)was isolated from the Chinese bird spider Ornithoctorcs hainana and identified as a novel antagonist of tetrodotoxin-sensitive(TTX-S)sodium channels.As revealed by the solution structure of HNTX-Ⅳ solved by two-dimensional nuclear magnetic resonance(2D-NMR),HNTX-Ⅳ adopts an inhibitor cystine knot motif.To check the role of basic residues during HNTX-Ⅳ’s interaction with TTX-S sodium channels,R26A and K27A mutants of HNTX-Ⅳ were constructed by solid-phase chemical synthesis.The synthesized peptides were purified and refolded under optimized oxidation conditions.Correct synthesis and folding were confirmed by MALDI-TOF mass spectrometry and NMR spectroscopy,respectively.Using the whole-cell patch-clamp technique,Lys27 but not Arg26 was identified as a key residue for HNTX-Ⅳ’s bioactivity against TTX-S sodium channels,because R26A-HNTX-Ⅳ showed slightly reduced activity and K27A-HNTX-Ⅳ showed almost no inhibition.展开更多
基金Supported by China National Haman Liver Proteome Project(Grant No.2004 BA711A18)Hunan Provincial Education Department and Hunan Science and Technology Project(Grant Nos.05FJ2002 and 05FJ4018)
文摘Plasma membrane(PM)proteome is one of the major subproteomes present in the cell,and is very important in liver function.In the present work,C57 mouse liver PM was purified by density-gradient centrifugation.The purified PM was verified by electron microscope analysis and Western blotting.The results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization fraction.Proteins were separated by 2DE and 1DE,trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass spectrometry.In all,547 non-redundant mouse liver PM proteins were identified,of which 34%contributed to plasma membrane or plasma membrane-related proteins.This study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.
文摘Hainantoxin-Ⅳ(HNTX-Ⅳ)was isolated from the Chinese bird spider Ornithoctorcs hainana and identified as a novel antagonist of tetrodotoxin-sensitive(TTX-S)sodium channels.As revealed by the solution structure of HNTX-Ⅳ solved by two-dimensional nuclear magnetic resonance(2D-NMR),HNTX-Ⅳ adopts an inhibitor cystine knot motif.To check the role of basic residues during HNTX-Ⅳ’s interaction with TTX-S sodium channels,R26A and K27A mutants of HNTX-Ⅳ were constructed by solid-phase chemical synthesis.The synthesized peptides were purified and refolded under optimized oxidation conditions.Correct synthesis and folding were confirmed by MALDI-TOF mass spectrometry and NMR spectroscopy,respectively.Using the whole-cell patch-clamp technique,Lys27 but not Arg26 was identified as a key residue for HNTX-Ⅳ’s bioactivity against TTX-S sodium channels,because R26A-HNTX-Ⅳ showed slightly reduced activity and K27A-HNTX-Ⅳ showed almost no inhibition.
