Datum vypracování: 2004-10-21
Databáze universitních knihoven universit Oxford, Cambridge a Institutu neurologie university Londýn
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1. Proteomics Techniques ••• Royal Free Campus Departments Home About us Facilities Microscopy Techniques Administration Costs... ...Bioinformatics Sample Preparation Optimal sample preparation is essential for good 2D results. The ideal process will cause complete solubilization,... ...Bioinformatics Data from the mass spectrometer consists of a series of molecular ion m/z (mass/charge) values that are translated into to a... 60% Tue, 07 Sep 2004 10:08:56 GMT http://www.rfc.ucl.ac.uk/departments/Biomedical-... Introduction This facility has been set up to help researchers establish and run proteomic projects and identify biological molecules of interest – particularly proteins. The facility is currently equipped with Amersham Biosciences 2D gel electrophoresis equipment, a Waters/Micromass capillary HPLC system coupled to a qToF-µ electrospray mass spectrometer. 2 Once a protein has been isolated and digested, the mass spectrometer is a suitable tool for asking, “What is this protein?”, “Which residues are modified?”, and “What is the modification?” As there are many practical considerations in setting up these types of experiments it is strongly advised that you contact the facility staff about the design of the study prior to the preparation of samples. The following guidelines provide a brief introduction for those interested in proteomics: Sample Preparation 
Optimal sample preparation is essential for good 2D results. The ideal process will cause complete solubilization, disaggregation and denaturation of the proteins in the sample. However, as samples vary in their constituent properties, optimal procedures must be determined empirically for each type of sample. The development of the sample preparation must take into account the object of the investigation: increasing the range and number of detectable proteins can sometimes only be obtained at the expense of clarity and reproducibility. Close collaboration between investigators and facility staff should produce a sample preparation protocol tailored to the needs of the investigation. Protecting Samples against Proteolysis Disrupting tissue or cultured cells can liberate or activate endogenous proteases. As proteolytic degradation of proteins greatly complicates 2D electrophoresis, measures should be included to avoid this problem. Proteases can be inactivated by immediate snap-freezing or denaturing samples for example with 10% TCA, 8M urea or 2% SDS. However, the addition of a cocktail of protease inhibitors has often proved adequate. Facility staff will be pleased to advise collaborators on suitable strategies. Gel Electrophoresis
2D gel electrophoresis is the main technique used to separate proteins in the facility. It is suitable for analyzing the proteins present in complicated biological samples, but can be supplemented with a variety of biochemical techniques in order to concentrate proteins of interest. 2D electrophoresis is often suitable for separating highly complex protein mixtures, however some proteins will be masked by other more abundant species and many proteins will remain beyond the limit of detection. The pH range for the 1st dimension needs to be optimized, and this may consume considerable amounts of time and sample. Image Analysis Computer-aided comparison of 2D gels facilitates the identification of changes in protein mobility and abundance. The facility has two workstations equipped with PDQuest, a standard software package in proteomics. Gels can be stained with silver or Deep Purple using protocols that allow subsequent analysis by mass spectrometry. It is hoped to develop comparative fluorescence-based techniques at a later stage. Note that there is inherently some gel to gel variation in staining and mobililty; consequently changes are significant if they are reproducible and generally greater than 2 to 3-fold in intensity. Excision and Digestion
Spots from 2D gels that reproducibly differ between experimental and control samples are manually removed for digestion, usually with trypsin. As such it is not practical to excise large numbers of proteins from multiple gels. It is vital up to this stage that samples are not contaminated with environmental proteins (typically keratins). Proteolytic fragments can then be desalted and analysed directly on the mass spectrometer or separated using the CapLC HPLC system and automatically loaded onto the mass spectrometer. Mass Spectrometry The Micromass qToF-µ electrospray mass spectrometer is configured for high-sensitivity analysis of small numbers of samples. It is not a high-throughput instrumen and requires expertise to operate. It is not suitable for screening vast numbers of samples. Sensitivity is highly compromised by salts and detergents in the sample. In addition to peptide analysis, the qToF-µ is suitable for studying many intact proteins (contact the facility staff for details). Facility staff will advise you on the best buffer in which to prepare samples. Bioinformatics
Data from the mass spectrometer consists of a series of molecular ion m/z (mass/charge) values that are translated into to a corresponding series of molecular weights or ”peptide fingerprint". Each fingerprint is then compared with the predicted fingerprints of all proteins in a comprehensive sequence database to identify the parent protein. In additionmolecular ions can be fragmented by collision in order to derive sest of daughter ions, from which information on amino acid sequence and sites of covalent modification can be obtained. Note that in general covalent modifications need to be stable and of high stoichiometry to be identified. The facility is equipped with a workstation running MassLynx 4.0 software. Facility staff will assist researchers with the identification of target proteins. Useful references and web pages British Mass Spectrometry Society A basic mass spectrometry tutorial Bio-Rad Proteomics Guide International Mass Spectrometry News American Society for Mass Spectrometry Informace o projektu „Blue gene“ společnosti IBM-výzkum: http://www.research.ibm.com/bluegene/ Directory: download download -> Performance Tuning Guidelines for Windows Server 2008 May 20, 2009 Abstract download -> Northern England’s set-jetting locations download -> Bbc archives: Beyond the programme download download -> Occupational health and safety download -> Dynatest 3031 lwd light Weight Deflectometer download -> Forth coming competitive exams download -> English Olympiad Tasks: 10 th form Аудіювання, 10 клас. Текст download -> Brush High School Morning Announcements Friday, September 05, 2014 all school download -> Year 9 The Arts — Music download -> Years 7 and 8 standard elaborations — Australian Curriculum: Music draft Download 0.52 Mb. Share with your friends:
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Lesk, A,M.
D gel electrophoresis is a suitable technique for asking, “Where do differences arise amongst the proteins in two similar samples?” For example, closely matched samples from diseased and healthy cells can be compared. Differences in protein abundance or covalent modification (e.g. phosphorylation, glycosylation and acylation) can provide important clues to the pathogenesis, progress and treatment of a disease.