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Membrane protein identification by shotgun proteomics

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Membrane proteins play important roles in various cellular processes, such as cell adhesion, immune response, metabolism and signal transduction. They are popular targets for proteomics research and the common candidates for drug development. Shotgun proteomics methods are available for the identification of membrane proteins.

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Membrane protein identification by shotgun proteomics

  1. 1. Creative Proteomics Presentation Membrane Protein Identification by Shotgun Proteomics LOGO
  2. 2. Creative Proteomics Presentation Introduction of Membrane Proteins Integral Membrane Proteins Peripheral Membrane Protein Lipid- anchored Protein Reference: Lodish H. Molecular cell biology. Macmillan, 2008.
  3. 3. Creative Proteomics Presentation Introduction of Membrane Proteins The integral membrane proteins • Span across the lipid bilayer and are amphipathic. • Their hydrophilic regions protrude into the cytoplasm or the extracellular environment for interaction with soluble proteins and molecules whereas the hydrophobic regions work for the embedding of the proteins into the lipid bilayer Peripheral membrane proteins • Peripheral membrane proteins are attached to one side of the membrane in several ways, like an in- plane a-helix Lipid-anchored membrane proteins • attached to one side of the membrane through covalent bonds to lipid groups.
  4. 4. Creative Proteomics Presentation Play important roles in various cellular processes, such as cell adhesion, immune response, metabolism and signal transduction. Popular targets for proteomics research and the common candidates for drug development. It is reported that about 60% of approved drugs target membrane proteins 1 2 3 Introduction of Membrane Proteins 4 Low abundance, limited solubility, restricted enzyme accessibility are main issues in limiting the amount of information obtained in the study of membrane proteins. Shotgun proteomics methods have relieved some difficulty in the identification of membrane proteins.
  5. 5. Creative Proteomics Presentation 1 2 4 3 Enrichment of Membrane Protein Processes for membrane protein identification Membrane Protein Separation Protease Digestion Mass spectrometry and computational analysis
  6. 6. Creative Proteomics Presentation Enrichment of Membrane Protein Gilmore J M, Washburn M P. Advances in shotgun proteomics and the analysis of membrane proteomes. Journal of proteomics, 2010, 73(11): 2078-2091.
  7. 7. Creative Proteomics Presentation Membrane Protein Separation Membrane Protein Separation Gel Based Solution Based • Blue-Native Electrophoresis (BNE) • Clear-Native Electrophoresis (CNE) • High Resolution Clear- Native Electrophoresis (hrCNE) • Multidimensional Protein Identification Technology (MuPIT) • Immobilized pH Gradient Isoelectric Focusing (IPG-IEF) • Mass Spectrometry- Based (GeLC-MS/MS)
  8. 8. Creative Proteomics Presentation Protease Digestion Trypsin: typical, some membrane proteins tend to lack trypsin cleavage sites, and it leads to large peptide fragments which keep up their hydrophobic nature and are not detected by the mass spectrometer Proteinase K: a non-specific protease can be used in membrane proteins. But peptides generated by non-specific proteases are difficult to predict because of the random location of positive charges Elastase and pepsin has been well characterized for the analysis of membrane proteins.
  9. 9. Creative Proteomics Presentation Mass spectrometry and computational analysis • Tandem mass spectrometry analysis equipped with a MALDI or ESI ion source is often used for membrane protein identification • Peptides are analyzed by mass spectrometry and obtain mass spectra • Subsequently, the bioinformatics tools and software are used for membrane protein identification
  10. 10. Creative Proteomics Presentation Our Membrane Protein Identification Service Buffer and detergent screening for solubilization and purification Purification from cytoplasm, periplasm, or cell culture supernatants Further purification steps for crystallization-ready, homogeneous protein fractions Affinity purification using His, Rho1D4, GST, or strep tags Our service
  11. 11. Creative Proteomics Presentation THANK YOU FOR LISTENING LOGO Please contact us for more information Web: Email: www.creative-proteomics.com info@creative-proteomics.com

Notas

  • Hello, welcome to watch the creative proteomics videos about protein identification,. Today, we are going to learn some basic knowledge Membrane Protein Identification by Shotgun Proteomics
  • Membrane proteins are a class of proteins that interact with or are part of, biological membranes. Membrane proteins can be classified into three parts based on their location and interactions with membranes: integral (membrane penetrating); peripheral (attached via non-covalent bonds); or lipid-anchored (attached through covalent bonds).
  • The integral membrane proteins span across the lipid bilayer and are amphipathic. Their hydrophilic regions protrude into the cytoplasm or the extracellular environment for interaction with soluble proteins and molecules whereas the hydrophobic regions work for the embedding of the proteins into the lipid bilayer. Peripheral membrane proteins are attached to one side of the membrane in several ways, like an in-plane a-helix. And the lipid-anchored membrane proteins are attached to one side of the membrane through covalent bonds to lipid groups.
  • Membrane proteins play important roles in various cellular processes, such as cell adhesion, immune response, metabolism and signal transduction. They can act as transporters, receptors and structures proteins. Therefore, membrane proteins are popular targets for proteomics research and the common candidates for drug development. It is reported that about 60% of approved drugs target membrane proteins. However, low abundance, limited solubility, restricted enzyme accessibility are main issues in limiting the amount of information obtained in the study of membrane proteins.
    Shotgun proteomics methods have relieved some difficulty in the identification of membrane proteins. However, the major difficulty of membrane proteome analysis still lies in the preparation of membrane proteins.
  • Next, the main processes for membrane protein identification will be introduction, including Enrichment of Membrane Protein, Membrane Protein Separation, Protease Digestion, Mass spectrometry and computational analysis.
  • Because the integral membrane proteins are low abundant nature, enrichment of membrane protein is essential for proteomic analysis. There are couples of strategies, including: subcellular fractionation (separated by increasing speeds using a glycerol or sorbitol gradient), delipidation (Solubilization of the membrane in the presence of detergent is performed followed by delipidation using a chloroform/methanol solution to extract and solubilize membrane proteins from the lipid bilayers), affinity purification (using biotinylation), and removal of non-membrane proteins (use of high salt and high pH has been successful in removing cytosolic and membrane-associated proteins).
  • The membrane protein separation is often divided into gel-based separation and solution-based separation. An earlier gel-based separation is SDS-PAGE prior to mass spectrometry, which is now known as GeLC. In this approach, the gel is cut into slices and digested with trypsin. And the peptides were extracted from gel slices and analyzed by MS/MS. At last, the peptides are identified with databases. There are some other gel-based separation methods, such as blue-native electrophoresis (BNE), clear-native electrophoresis, and high resolution clear-native electrophoresis.
    Speaking to solution-based separation, multidimensional protein identification technology is a 2D chromatographic approach to separating proteins, in which proteins are digested into peptides and then the peptides are separated by using strong cation exchange and reverse phase chromatography. It is reported that another approach to separating peptides or proteins is immobilized pH gradient isoelectric focusing. In this method, digested membrane proteins can be separated by IPG-IEF in the presence of 60% methanol.
  • For effective digestion, the backbone of the proteins must be accessible for the proteolytic enzymes. However, access to certain parts of membrane proteins is often blocked by sugars or lipids. Trypsin digestion is a typical way to digestion. However, some membrane proteins tend to lack trypsin cleavage sites, and it leads to large peptide fragments which keep up their hydrophobic nature and are not detected by the mass spectrometer. Proteinase K which is a non-specific protease can be used in membrane proteins. But peptides generated by non-specific proteases are difficult to predict because of the random location of positive charges. Elastase and pepsin has been well characterized for the analysis of membrane proteins.
  • Tandem mass spectrometry analysis equipped with a MALDI or ESI ion source is often used for membrane protein identification. Peptides were analyzed by mass spectrometry and obtain mass spectra. Subsequently, the bioinformatics tools and software are used for membrane protein identification.
  • Our membrane protein service offering includes: Purification from cytoplasm, periplasm, or cell culture supernatants; Buffer and detergent screening for solubilization and purification; Affinity purification using His, Rho1D4, GST, or strep tags; and Further purification steps for crystallization-ready, homogeneous protein fractions
  • Thanks for watching our video. At creative proteomics, we provide the most reliable services for Membrane Protein Identification by Shotgun Proteomics. If you have any questions or specific requirements. Please do not hesitate to contact us. We are very glad to cooperate with you.

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