proteomics lecture 2b.ppt
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Proteomics studies the proteome through several key areas: mass spectrometer-based proteomics to identify and quantify expressed proteins; array-based proteomics to define protein function and interactions; informatics to manage proteomics data; and clinical applications. Mass spectrometry techniques separate proteins using methods like 2D gel electrophoresis then analyze proteins/peptides by mass and charge to identify sequences and infer protein identity. This provides insights into protein functions, modifications, locations, and concentrations across diverse expression levels in the cell.
proteomics lecture 2b.ppt
- 1. Proteomics • The proteome is larger than the genome due to alternative splicing and protein modification. As we have said before we need to know • All protein-protein interactions. • One protein or peptide may have multiple functions depending on context. • Regulation of protein function. • Modification • Location • Detection and quantitation – The concentration of all proteins changes by 10 orders of magnitude within the cell. Currently there are no easy methods for determining the concentrations over this large of a concentration range.
- 2. What areas are being studied in Proteomics? • Mass spectrometer based proteomics – This area is most commonly associated with proteomics and is a method to determine which proteins are expressed and the amounts of those proteins. • Array based proteomics – This method uses various arrays to try and define the function of the proteins, regulation levels and interacting partners within the cell. • Informatics – This is trying to define what information will be needed, stored, accessed and how it can be used to study the proteome of the cell. • Clinical • Orthagonalomics
- 3. MASS SPECTROMETER BASED PROTEOMICS • Principles – Mass spectrometry is based on the fact that ions of differing charge and mass will move differently in a magnetic field. In proteomics the proteins are first separated by some means and then analyzed with a mass spectrometer
- 4. Separating the Proteome • The protein genome is separated by several different methods. • Many researchers are first separating portions of the genome, such as isolating organelles, and then analyzing that portion. • This is because often proteins of interest, regulatory proteins are in low abundance. • The most commonly used method is 2- dimensional gel electrophoresis. – Consists of using isoelectric focusing with SDS polyacrylamide gel electrophoresis
- 5. Isoelectric focusing • This separates proteins based on isoelectric point • The isoelectric point is the pH at which the protein has no net charge. • pH gradients may be large 2-10 or small 6-7 • Typically this is done with an immobilized pH gradient gel strip or with a tube gel containing a low concentration of polyacrylamide. • Ampholytes are added to create a pH gradient in an electric field and the proteins are loaded. • The IEF gel is placed in an electrophoresis system for up to 24 hours and the proteins form tight bands at their isoelectric point. • The IEFgels are now ready for the second method.
- 6. Figure is from “Principles of Biochemistry” Lehninger, Fourth Edition
- 7. SDS Polyacrylamide Gel Electrophoresis • The second dimension separates the proteins based on size. • There are two parts, the stacking gel which concentrates the sample and the running gel that is used to separate the proteins. • The IEF gel is soaked in a solution containing chemical to denature the proteins including sodium dodecyl sulfate a detergent which gives the proteins a net negative charge. This means that all proteins will move in one direction. • The IEF gel is then put in the one long well in the stacking gel, sealed in place with agarose, and the proteins subjected to an electric field to separate. • The larger proteins are found at the top and the smaller ones are found at the bottom of the gel.
- 8. 2-Dimensional Gel Electrophoresis • In a 2D gel the proteins appear as spots on the gel rather than bands. These spots can then be further processed or used for mass spectrometry directly. • Further processing usually includes spot excision, trypsin digestion, and mass spectromety • Analysis may also include differential 2D gel electrophoresis – In this case a control and sample are separately labeled with a fluorescent molecule. – The samples are mixed and electrophoresed in the same gels. – A laser scanner is used to identify each spot and a program puts the two images together.
- 11. 2-D Gel (non-denaturing) pH 4 pH 10
- 12. Alternate Separation Methods • The first dimension is run in larger agarose tube gels with ampholytes. – This has less resolution than polyacrylamide gels. The tubes are sliced and the proteins are allowed to diffuse out. • Gel regions are cut, proteins eluted and the proteins are then separated by capillary electrophoresis. • Capillary electrophoresis has a much greater resolution for the proteins mass. • Proteins are eluted from the capillary in the process and can be collected. They are readily available for mass spectrometry.
- 13. Alternative Separation Methods • Whole proteome is analyzed at once. • Proteome is digested with protease (trypsin) • Digested proteome is injected to HPLC with 2 columns in series (mixed bed ion exchange and reverse phase) • Peptides are eluted from ion exchange onto reverse phase and then separated on reverse phase column. • Peptides then enter ESI-MS-MS
- 14. Mass Spectrometery • Separates ions based on mass to charge ratio. – Charges are placed on the protein or the peptide by ionization. • Two most common types of ionization are: • Matrix-Assisted Laser Desorption Ionization. – MALDI causes fragmentation of the protein during ionization. Can be used to get more information about the fragments. Easier to do than ESI. • Electrospray ionization (ESI) – ESI can give whole protein masses as well as complex masses. If the proteins is first separated by reverse phase HPLC before injection only the subunits masses will be known.
- 15. Matrix-Assisted Laser Desorption Ionization (MALDI) • MALDI causes fragmentation of the protein during ionization. Can be used to get more information about the fragments. Easier to do than ESI. • Requires sample to be placed in matrix that absorbs appropriate wavelength light. • Matrix generates heat and forms ions of matrix and what is around it.
- 17. Mass Analyzers • Important parameters – Sensitivity • How few ions can be detected. – Resolution • How well different masses can be determined. – mass accuracy • How reproducible and correct are the masses.
- 18. Mass Analyzers (MS) • Quadrapole • High Sensitivity, acceptable mass accuracy and resolution • Easily coupled to chromatography • Time of Flight • High Sensitivity, high mass accuracy, high resolution • Limited to small m/z ratios • Not easily coupled to chromatography • Easily coupled to MALDI
- 19. Mass Analyzers (MS) • Ion Trap • High Sensitivity • Low mass accuracy and resolution • Fourier Transform ion cyclotron • High sensitivity, mass accuracy, resolution, dynamic range • Expensive, difficult to operate, low fragmentation efficiency
- 20. Mass Spectrometers • Instruments are often coupled – MS/MS • ESI - quadrapole MS -TOF-MS – Collider • Is essentially a Quadrapole MS with collision gas included • Forms collision induced ions – Gives collision induced spectra (CID)
- 23. Protein identification and Quatitation • To quantitate – add stable isotopes – post separation modification • SH, NH2, N-linked carbohydrates • Incorporation of isotopes in culture
- 24. MS Quantitation
- 25. Protein Identification • Use collision induced spectra – provides sequence information – provides unique m/z spectra for each peptide • Problem is large number of CID & large amount of information. – Need methods for searching – Filtering has been tried (limited success) – Most successful with human intervention. – Improving • Use unique mass of peptides to identify sequence and multiple ions to identify proteins
- 26. Amino Acid Masses Amino acid Mass(avg) Amino acid Mass(avg) G 57.0520 D 115.0886 A 71.0788 Q 128.1308 S 87.0782 K 128.1742 P 97.1167 E 129.1155 V 99.1326 M 131.1986 T 101.1051 H 137.1412 C 103.1448 F 147.1766 I 113.1595 R 156.1876 L 113.1595 Y 163.1760 N 114.1039 W 186.2133
- 27. Peptide Fragmentation 100 0 250 500 750 1000 m/z % Intensity K 1166 L 1020 E 907 D 778 E 663 E 534 L 405 F 292 G 145 S 88 b ions 147 260 389 504 633 762 875 1022 1080 1166 y ions
- 28. Mass Analyzers (MS) Quadrapole • Sensitive, acceptable mass accuracy and resolution • Easily coupled to chromatography. – Time of Flight • Sensitive, high mass accuracy, high resolution • Limited to small m/z ratios • Not easily coupled to chromatography – Ion Trap • Sensitive • Low mass accuracy – Fourier Transform ion cyclotron • High sensitivity, mass accuracy, resolution, dynamic range • Expensive, difficult to operate, low fragmentation efficiency
- 30. Proteomics • The proteome is larger than the genome due to alternative splicing and protein modification. As we have said before we need to know • All protein-protein interactions. • Function – One protein or peptide may have multiple functions depending on context. • Regulation of protein function. • Modification • Location – Location will help us to understand the proteins role in the cell, what its function is, and what controls its function. • Detection and quantitation – The concentration of all proteins changes by 10 orders of magnitude within the cell. Currently there are no easy methods for determining the concentrations