Proteomics 2 d gel, mass spectrometry, maldi tof

proteomics

1. Proteomics 2D Gel, Mass Spectrometry, MALDI TOF 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 1

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3. Term originated in 1995 – refers to the proteins expressed by a genome Study of the proteome where the proteome is the total protein content of a genome and the genome is a complete set of the genetic material of a cell for an organism 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 3

4. Functional Proteomics – Study of change in protein expression within the proteome Structural Proteomics – Study of the primary, secondary, and tertiary structure of the proteins in a proteome, functional predictions from primary structure; high throughput crystallography 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 4

5. Proteomics branches 1. Proteomic analysis (analytical protein chemistry) - Characterization of proteins and their post-translational modifications 2. Expression proteomics (differential display proteomics) - Profiling of expressed proteins using quantitative methods 3. Cell-mapping proteomics (cataloging of protein-protein interactions) -Identification of protein complexes Approaches used to study proteomics : Gel-based proteomics Mass spectrometry driven proteomics Protein arrays, Yeast two-hybrid arrays,... 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 5

6. How to look at proteome? 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 6

7. One genome expresses many proteins 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 7

8. DNA mRNA Proteins Metabolyte Genome “Genomics” Proteome “Proteomics” Omics Transcriptome “ Transcriptomics ” Metabolome “Metabolomics” 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 8

9. Generalized proteomics scheme Yarmush & Jayaraman, 2002 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 9

10. Definitions of Proteomics 10 Be defined as the large-scale characterization of the entire protein complement of a cell line, tissue, or organism. Goal: -To obtain a more global and integrated view of biology by studying all the proteins of a cell rather than each one individually. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY

11. Why is Proteomics necessary? 11 Having complete sequences of genome is not sufficient to elucidate biological function . A cell is normally dependent upon multitude of metabolic and regulatory pathways for its survival Modifications of proteins can be determined only by proteomic methodologies It is necessary to determine the protein expression level The localization of gene products can be determined experimentally Protein-protein interactions Proteins are direct drug targets . 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY

12. Types of Proteomics and Their Applications to Biology 12 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY

13. The Major Techniques in Current Proteomics 13 Two-dimensional electrophoresis IEF strip separation SDS-PAGE gel separation Mass Spectrometry Protein sequencing Peptide mapping Post-translational Modification Others ICAT Yeast two hybrid assay Protein chips 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY

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15. INTRODUCTION: Gel Electrophoresis is used to separate proteins from the mixture of different proteins of different molecular size. 2D Gel Electrophoresis separates the proteins of same molecular size. It was given by O’Farrell and Klose in 1975. Firstly, the Serum proteins were separated by using this method. It separates the proteins based on molecular size as well as charges. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 15

16. TWO-DIMENSIONAL ELECTROPHORESIS This technique combines the technique IEF (first dimension), which separates proteins in a mixture according to charge (PI), with the size separation technique of SDS-PAGE second dimension). The combination of these two technique to give two-dimension(2-D)PAGE provides a highly sophisticated analytical method for analyzing protein mixtures. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 16

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18. The goal of two-dimensional electrophoresis is to separate and display all gene products present. It is the only method currently available which is capable of simultaneously separating thousands of proteins. The two dimensions of 2D PAGE are – -iso electric point -size Therefore, it has unique capability to capture detailed info about protein expression, isoforms, complex formation and post translational modification. e.g.- chemotherapy to cancer cells, occupational benzene exposure to blood cells. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 18

19. STEPS INVOLVED 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 19

20. 1. SAMPLE PREPARATION 2. RUN FIRST DIMENSION –IEF 3. RUN SECOND DIMENSION –SDS-PAGE 4. VISUALIZE AND ANALYZE How to perform 2D PAGE??? 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 20

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24. Detect separated proteins by staining or immuno detection after blotting onto a membrane. Powerful tools and techniques are used to compare the samples & identify the protein of interest. 4. Visualize And Analyze 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 24

25. Image Analysis 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 25

26. STAINING Sensitive, quantitative and MS-compatible. Comassie brilliant blue R250 staining (30-100 ng ) Colloidal comassie blue G250 staining (30-100 ng ) Diamine silver stain (1-10 ng ) : gluteraldehyde Silver nitrate stain (1-10 ng ) Sypro Ruby fluorescent stain Flamingo fluorescent gel stain. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 26

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28. IDENTIFICATION OF SPOTS: Comparison with a reference gel. Extraction of spots and biochemical analysis. The protein is detected as a strip of interconnected spots with different sizes and shapes. The spots change their shape and size with stimulation, as well as increase/decrease in number. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 28

29. IMAGING SOFTWARE: Amersham Pharmacia Biotech Typhoon BIO-RAD Molecular Imager FX 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 29

30. Imaging 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 30

31. OTHER SOFTWARES ARE : BioNumerics   2D Delta 2D ImageMaster Melanie PDQuest Progenesis and REDFIN 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 31

32. Useful 2D-gel websites GELBANK: http://www.gelbank.anl.gov GelScape: http://www.gelscape.ualberta.ca:8080/htm/index.html NCI Flicker: http://www.lecb.ncifcrf.gov/flicker/ World-2D PAGE repository: http://world-2dpage.expasy.org/repository/

33. 1 . Spot number: 10,000-150,000 gene products in a cell. PTM makes it difficult to predict real number. It’s impossible to display all proteins in one single gel . 2.Molecular weights : Protein >250kDa do not enter 2 nd SDS – PAGE properly . 3.Reproducibility: Variation most comes from sample preparation . 4.Less sensitivity (lower resolution) Challenges for 2DE 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 33

34. Solves the Gel to gel variations problem in 2D PAGE, by enabling the multiple samples in single gels. Sensitivity improved due to use of fluorescent dye and laser. Advantages 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 34

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36. Mass spectrometry (MS) is an analytical technique that produces spectra (singular spectrum) of the masses of the atoms or molecules comprising a sample of material. Mass spectrometry is an extremely valuable analytical technique in which the molecules in a test sample are converted into gaseous ions that are subsequently separated in a mass spectrometer according to their mass-to-charge ratio (m/z) and detected . 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 36

37. SPECTROPHOTOMETER 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 37

38. History Mass spectroscopy was first performed at the cambridge university, in 1912 by J.J Thomson (1856-1940) when he obtained the mass spectra of O2, N2, CO Mass spectroscopy took off in 1930s and advance technology resulted in the development of double focusing Mass spectrometers capable of accurate determination. The modern techniques of mass spectrometry were devised by Arthur Jeffrey Dempster and F.W. Aston in 1918 and 1919 respectively. In 1989, half of the Nobel Prize in Physics was awarded to Hans Dehmelt and Wolfgang Paul for the development of the ion trap technique in the 1950s and 1960s. In 2002, the Nobel Prize in Chemistry was awarded to John Bennett Fenn for the development of electrospray ionization (ESI ) 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 38

39. Technique can be used for Qualitative and quantitative analysis Providing information about the mass of atoms and molecules Molecular structure determination (organic & inorganic) Identification and characterization of materials 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 39

40. COMPONENTS OF A MASS SPECTROMETER - Sample input system - Ionization source - Mass analyzer - Detector - Vacuum pumps - Computer based data acquisition and processing system 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 40

41. The mass spectrum is a plot of a relative abundance of the ions at each m/z ratio. In most cases , the nascent molecular ions of the analyte produce fragment ions by cleavage of the bond and the resulting fragmentation pattern constitute the mass spectrum . 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 41

42. PRINCIPLE AND INSTRUMENTATION 1.) IONISATION Ionization is a process of charging a molecule. The sample molecule must be charged in order to measure them using a mass spectrometer The atom is ionized by knocking one or more electrons off to give a positive ion. The particles in the sample (atoms or molecules) are bombarded with a stream of electrons to knock one or more electrons out of the sample particles to make positive ions. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 42

43. PRINCIPLE AND INSTRUMENTATION 2.) ACCELERATION The ions are accelerated so that they all have the same kinetic energy Ionisation chamber at +1000v The positive ions are repelled away from the positive Ionisation chamber and pass through 3 slits with voltage in the decreasing order. All the ions are accelerated into a freely focused beam . Ion beam 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 43

44. PRINCIPLE AND INSTRUMENTATION 3.) DEFLECTION The ions in the deflected by a magnetic field according to their masses Different ions are deflected by the magnetic field by different amounts. Amount of deflection depends upon : 1) The mass of the ion 2) The charge on the ion 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 44

45. PRINCIPLE AND INSTRUMENTATION 4.) DETECTION The beam of ions passing through the machine is detected electrically. Ions leaves space in metal by neutralizing it & the electrons in the wire shuffle along to fill it. The flow of electrons in the wire is detected as an electric current which can be amplified & recorded 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 45

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48. THE TWO MOST COMMON METHOD OF MASS SPECTROMRTRY FOR PROTEIN ANALYSIS 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 48

49. MALDI-TOF MS The protein sample is mixed with a chemical matrix that includes a light absorbing substance excitable by a laser. The laser pulse is used to excite the chemical matrix, creating a microplasma that transfers a energy to protein molecule substance excitable by a laser. Molecule in the sample ionizing them and ejecting them into the gas phase. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 49

50. ELECTROSPARY IONISATION MASS SPECTROMETRY Electrospray is an especially soft ionisation method , capable of generating molecular ions (without fragmentation) from biological macromolecules present in aqueous solution 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 50

51. esI -MS A solution of macromolecules is sprayed in the form of fine droplets from a glass capillary under the influence of a strong electric field The droplets pick up the positive charge as they exit the capillary Evaporation of the solvent leaves multiply charged molecules 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 51

52. Applications of mass spectrometry to study proteins Identifying unknown proteins Protein sequencing (peptides are sequenced by generating multiple sets of proteins Identification of chemical modification (PTM in proteins after synthesis) Identification of organisms (identifying bacteria by finger printing proteins) 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 52

53. Applications Mass spectrometry is also used to determine the isotopic composition of elements within a sample. Differences in mass among isotopes of an element are very small, and the less abundant isotopes of an element are typically very rare. Mass spectrometry is an important method for the characterization and sequencing of proteins. The two primary methods for ionization of whole proteins are ESI and MALDI. Mass spectrometry (MS), with its low sample requirement and high sensitivity, has been predominantly used in glycobiology for characterization and elucidation of glycan structures. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 53

54. Disadvantages This often fails to distinguish between optical and geometrical isomers and the positions of substituent in o-, m- and p- positions in an aromatic ring. Also , its scope is limited in identifying hydrocarbons that produce similar fragmented ions. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 54

55. Introduction Matrix-assisted laser desorption/ionization (MALDI)  is a soft ionization technique used in mass spectroscopy, allowing the analysis of biomolecules (biopolymers  such as DNA, Proteins, peptide   and sugars) and organic molecules  (such as polymers, dendrimers  and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods . Method where a laser is used to generate ions of high molecular weight samples, such as proteins and polymers . 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 55

56. Mechanism The mechanism remains uncertain. It may involve absorption of light by the matrix. Transfer of this energy to the analyte - which then ionizes into the gas phase as a result of the relatively large amount of energy absorbed. To accelerate the resulting ions into a flight-tube in the mass spectrometer they are subjected to a high electrical field 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 56

57. MATRIX ASSISTED LASER DESORPTION IONIZATION-TIME OF FLIGHT It is three steps method. The sample is mixed with a suitable matrix & applied to a metal plate. A pulsed laser irradiate a sample triggering desorption of matrix material. Ionization of analyte molecules 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 57

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59. PARTS OF MALDI TOF Sample plate Matrix Laser Variable voltage grid Vaccum system Flight tube Time mass detectors 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 59

60. Matrix The analyte incorporation in to a suitable matrix is the first step of the MALDI process, and is an important feature of the MALDI method A typical sample preparation involves using 10 -6 M solution of the analyte mixed with 0.1 M solution of the matrix The solvents are then evaporated in a vacuum of the MS, and the matrix crystallizes with the analyte incorporated 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 60

61. According to Sigma Aldrich, the matrix must meet the following properties and requirements : Be able to embed and isolate analyte (e.g. by co-crystallization) Be soluble in solvents compatible with analyte Be vacuum stable Absorb the laser wavelength Cause co-desorption of the analyte upon laser irradiation Promote analyte ionization 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 61

62. MALDI-TOF Properties of a good Matrix They are of a fairly low molecular weight They are often acidic, therefore act as a proton source to encourage ionization of the analyte . Basic matrices have also been reported. They have a strong optical absorption in either the UV or IR range, so that they rapidly and efficiently absorb the laser irradiation. This efficiency is commonly associated with chemical structures incorporating several conjugated double bonds, as seen in the structure of cinnamic acid. They are functionalized with polar groups, allowing their use in aqueous solutions. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 62

63. MALDI-TOF 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 63

64. Structure of Matrix 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 64

65. Laser Numerous gas and solid state lasers have been developed for use in MALDI. Most MALDI devices use a pulsed UV laser N 2 source at 337 nm. Neodymium-yttrium aluminum garnet ( Nd:YAG ) Emits at 355 nm and gives a longer pulse time IR lasers are also used The most common IR laser is the erbium doped-yttrium aluminum garnet ( Er:YAG ). Emits at 2.94 micrometer. It is “softer” than the UV, which is useful for certain biomolecules . 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 65

66. The MALDI method uses a pulse laser Laser fires in intervals Pulsed laser produces individual group of ions 1 st pulse=1 st group of ions 2 nd pulse= 2 nd group of ions, etc. Each group of ions generated are detected With continuous pulsing, the signal resolution increases 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 66

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68. Time Mass Detector The typical detector used with MALDI is the time of flight mass detector (TOF-MS) TOF is a method where the ions are accelerated by an electric field, resulting in ions of the same strength to have the same kinetic energy. The time it takes for each ion to traverse the flight tube and arrive at the detector is based on its mass-to-charge ratio; therefore the heavier ions have shorter arrival times compared to lighter ions. 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 68

69. MALDI-TOF Spectrometer used for MALDI The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due to its large mass range. The TOF measurement procedure is also ideally suited to the MALDI ionization process 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 69

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71. Advantages o f MALDI TOF MS Soft ionization - analyze intact biomolecules and synthetic polymers Broad mass range - analyze a wide variety of biomolecules Simple mixtures are okay Relatively tolerant of buffers and salts Fast data acquisition Easy to use and maintain, no water or gas hook ups required High sensitivity, superior mass resolution and accuracy 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 71

72. APPLICATIONS Proteomics Pharmaceutical Analysis To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural source, peptides & their amino acid sequences Bioavailability studies Drug metabolism studies, pharmacokinetics Characterization of potential drugs Drug degradation product analysis Screening of drug candidates Identifying drug targets 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 72

73. APPLICATIONS Microbiology It is used for the identification of microorganisms. Species diagnosis by this procedure is much faster, more accurate & cheaper than other procedures based on biochemical tests. Forensic analysis Environmental analysis Pesticides on foods Soil and groundwater contamination 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 73

74. THANK YOU…!!! 3/14/2018 DEPT. OF PLANT BIOTECHNOLOGY 74