proteomics lecture 2b.ppt protein structure determination
- 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