A Novel Method For Identification Rice Root Plasma Membrane By Ultrasonic Assisted Methanol Extraction NanoRPLC-LTQ-Orbitrap Detection And Its Application

The metal ions in soil root system visa transmembrane transporter system on the Plasma membraneto achieve the root cells. Plastids and apoplastic is the two ways for metal ions enter the vascular systemto achieve leaves and seeds. These functions implemented are in large part of the total cell proteinmembrane protein. Shotgun method based on liquid phase which provides powerful means for theanalysis of membrane proteins, However, due to the membrane protein of low abundance, stronghydrophobicity and exist most of the plasma membrane protein glycosylation phosphorylationmodification after translation of micro heterogeneity of membrane proteins, So for high sensitivity,high-throughput to analysis of the proteomics exist a bigger challenge. In these theses, our purpose is tosolve the membrane protein solvent problem base on the shotgun method of proteomics. our choose themost suitable Solvent by comparing the different strength of the soluble liquid and optimizing massspectrometry conditions, caparisoning three types of collision modes. To establish an effective protocolfor the identification of these highly hydrophobic membrane proteins that is ideally suited for bothdiscovery and qualitative proteomic analysis using nano-liquid reversed-phase chromatography-LTQ-Orbitrap and applicant the method in detection Cadmium-related proteins in rice root plasmamembrane.. Cadmium（Cd）pollution is a major problem in the quality and safety of rice, on the levelof proteomics research on cadmium treatment differences can provides the necessary technical means toreveal the mechanism of cadmium transport mechanism and to probe into rice inherent nature of lifeactivities. The main results as follows:1、Using BSA and membrane protein of rice as the sample, grope for CID, PQD, HCD collisionmodes distinction, the response value with BSA protein, peptides matching rate and stability, thenumber of membrane protein and stability as judgment, finally established the spray voltage2.0kv formass spectrometry, CID mode for the collision mode and nanoliter liquid reversed-phasechromatography separation methods.2、Through comparison and optimization get large number of membrane proteins of ultrasonicassisted methanol extraction method of rice root plasma membrane protein than low concentration ofurea system, the system of high concentration of urea and guanidine hydrochloride system, SDS system,methanol with SDS system. This experiment established methanol-ultrasonic assisted method to extractroot membrane proteins based on two-phase aqueous systems。By the method,758kinds of membraneproteins including324membrane proteins identified with nanoliter liquid reversed-phasechromatography combined with high resolution mass spectrometry (nanoLC-LTQ-Orbitrap) were42.7%,which accounted for the total proteins. During324membrane proteins, there were38kinds oftransmembrane proteins,9kinds of inner membrane proteins。3、It was found that cadmium accumulation in rice root and leaf increased as the cadmiumtreatment time and concentration of rice tillering and rice seed, Cadmium accumulation of root is muchlarger than the leaf. Description of rice under Cd treatment in vivo with rapid absorption of cadmiumion transport system, and after a week of cadmium in rice seedling treatment has made significant changes Compared with the photographic, indicating that there are cadmium stress physiologies in Cd.The method of Ultrasonic assisted methanol to extract root membrane proteins used in identifying theroot plasma membrane proteins under Cd treatment, could achieve high-throughput and simultaneousanalysis of a large number of root plasma membrane proteins of cadmium-related also. The methodprovided theoretical and technical support for the transport and coercion mechanism of cadmium in rice.180kinds of Cd-related proteins Classified by function can include36kinds of cadmium stress protein,39kinds of metal ion binding protein,64kinds of defense related proteins,11kinds of ATP or GTPenzyme related proteins and functional overlapping protein made28species were identified in oneexperiment. The protein Q9FE01was only found under high cadmium concentration and longtime ofprocessing, which defined as ion binding protein and stress proteins, with bivalent ion binding sites, andcalcium, iron, potassium ions and bivalent iron ions but did not report with cadmium ion, thecombination of this experiment guess it may for cadmium ion binding protein.