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.
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. 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. 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. 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. 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. 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. 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. 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. 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. Creative Proteomics Presentation
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Notas del editor
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.