| 1 Biotin labeling and receptor analysis of Huwentoxin-â… The Chinese bird spider Selenocosmia(=ornnithoctonus) huwena, distributed in the hilly areas of Yunnan and Guangxi in the south of China,is a recently identified new species.The crude venom of O.huwena contains a mixture of compounds with different types of biological activities,of which neuropeptide toxins have been widely and deeply studied.Huwentoxin-â… (HWTX-â… ),a neurotoxic peptide,is the most abundant toxic component in the crude vernon of the spider.It contains 33 amino acid residues,including six cysteine residues that form three pairs of disulfide bonds.Being a blocker of N-type Ca2+ channel, suggested by patch-clamp experiments,the toxin can block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation with its action site located on the foresynaptic membrane.In order to purify and characterize the receptor of HWTX-â… in plasma membrane and obtain more direct proofs about the action mechanism of HWTX-â… ,in the present work,we first optimized the experimental conditions on biotin labeling of HWTX-â… and analyzed the effects of biotinylation on the bioactivity of HWTX-â… ,and then purified and characterized the receptor proteins of HWTX-â… from the plasma membrane in rat phrenic nerve diaphragm synapses,employing biotin/avidin affinity chromatrography and proteomics-based strategies. The labeling experiments demonstrated that when the mole ratio of biotinylation reagent and HWTX-â… was 2:1,most HWTX-â… molecules were labeled by only one biotin group,with the labeling rate of 42%. Mass spectrometry and electrophysiological experiments indicated that biotin labeling occurred on Lys13 of HWTX-â… and this biotinylation did not affect the bioactivity of HWTX-â… .After the protein fraction obtained by affinity chromatography was separated by SDS-PAGE,digested by trypsin and identified by mass spectrometry,several proteins were found that might interact with HWTX-â… .2 Application of performic acid oxidation-assisted digestion in the plasma membrane proteomicsMembrane proteins play critical roles in many biological process and are often the molecular targets for drug action.The study of plasma membrane proteins has become one of the hotspot of proteomics.The development of this field can offer new targets for the medication development and new markers for disease diagnose.Exploring the function of integral membrane proteins is very important for us to understand their roles in physiological and pathological processes. However,the low abundance and the hydrophobic nature of their transmembrane domains(TMDs) make them difficult to analyze.During proteomic analysis,hydrophobic peptides or transmembrane domains are often under-represented,which reduce the total sequence coverage and the reliability of protein identification.Here we developed a new on-membrane digestion strategy for membrane protein identification,which involved the use of mild performic acid oxidation treatment(PAOT).PAOT was helpful to the analysis of highly hydrophobic proteins because performic acid can oxidize the Cys and Met residues into cysteic acid and methionine sulfone,respectively,which significantly decreased the hydrophobicity of the proteins and thus increased their cleavage and detection efficiency. Taking human CNE1 cells as experimental materials,we made a comparative study on the identification of integral membrane proteins based on PAOT-assisted digestion and other two conventional digestion methods:trypsin/chymotrypsin digestion and methanol-modified digestion. The results showed that the total numbers of integral membrane proteins identified based on PAOT,trypsin/chymotrypsin and methanol-modified digestions were 101,59 and 72,which included 67,40 and 31 proteins with GRAVY＞0,respectively.So,compared with other two methods, PAOT-assisted digestion could lead to more integral membrane proteins with high hydrophobicity to be identified,and was more suitable for membrane proteome analysis.
|
PDF Full Text Request