1.Purification And Identification Of HWTX-I Receptors From Rat Diaphragm Muscle Cell Membrane 2.Gel-embedment-based Preparation Of Membrane Proteins And Its Mass Spectrometry Analysis

1 Purification and identification of HWTX-Ⅰreceptors from rat diaphragm muscle cell membrane 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-Ⅰ(HWTX-Ⅰ), a neurotoxic peptide, is the most abundant toxic component in the crude vemon 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 reversibly 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 receptors of HWTX-Ⅰon rat diaphragm muscle cell membrane and obtain more direct evidence of the action mechanism of HWTX-Ⅰ, in the present work, we first labeled HWTX-Ⅰwith biotin, and then purified and characterized the receptor proteins of HWTX-Ⅰfrom the plasma membrane in rat phrenic nerve diaphragm synapses, employing biotin/avidin affinity chromatrography and chemical crosslinking. After the protein fraction obtained by affinity chromatography was desalted by ultrafiltration, digested by trypsin in solution and analyzed by mass spectrometry, several proteins were found that might interact with HWTX-Ⅰ.In addition, by using the chemical crosslinking and Western blotting methods, we detected the receptors qualitatively in the molecular weight range of 50-80 kD. After the proteins in this range were digested and analyzed by mass spectrometry, several proteins that might interact with HWTX-Ⅰwere also identified.2 Gel-embedment-based preparation of membrane proteins and its mass spectrometry analysisMembrane proteins are important biological and pharmacological targets involved in intercellular communication, vesicle trafficking, ion transport, protein translocation/integration, and propagation of signaling cascades. Contemporary genomic analyses indicate that 20-30% of all open reading frames (ORFs) encode for membrane proteins. Comprehensive and high throughput analysis of membrane proteins especially integral membrane proteins (IMPs) would greatly facilitate our understanding of their roles in cell life. However, their analysis presents a special challenge due to their hydrophobic nature and low abundance. To enhance the solubility of membrane proteins, the buffer containing some organic solvents or detergents was usually employed to extract these proteins. Nevertheless, although these additives could improve the solubility and thus the extraction efficiency, most of them decrease protease activity in the subsequent enzymolysis and interfere with MS analysis when used at a higher concentration. Thus how to remove the additives from the membrane protein lysate obtained by extraction is a subject worthy of investigation.In the present study, a new method involving heat gel-embedment and improved in-gel digestion was developed to prepare membrane proteins for its analysis by mass spectrometry. In this method, membrane protein lysate containing high concentration of SDS was mixed with an acrylamide solution and the proteins were embedded and immobilized in gel matrix when the gel polymerized. For investigating the effect of temperature on protein embedment, it was made at different temperatures (25,35 or 45℃). After removal of SDS from the gel matrix by repeated washing, the in-gel digestions were performed in the presence of 0.1% RapiGest reagent (ALS),0.1% sodium deoxycholate and 10% ACN, respectively. The resultant peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry. The results showed that, compared with that at 25℃, gel-embedment at 45℃improved the protein embedment and thus protein identification, with the identified IMPs increased by 27%.0.1% sodium deoxycholate was more efficient than 0.1% ALS and 10% ACN in terms of improving the digestion and tryptic digest recovery of the gel-embedded proteins particularly the hydrophobic IMPs. Out of the 326 IMPs identified by heat gel-embedment combined with improved in-gel digestion strategies, 149 (46%) proteins had at least two mapped transmembrane domains. These results indicate that our newly developed protocol could facilitate the high throughput analysis of membrane proteins particularly the integral membrane proteins.