A.a sequence analysis 112070804002


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A.a sequence analysis 112070804002

  1. 1. Prepared By: PARTH Guided By: Rajesh ParmarApmc college of pharmaceutical education and research. 1
  2. 2.  Introduction Amino acid analysis General techniques for analysis of amino acids Method used for amino acid analysis Reference. 2
  3. 3.  Definition: “Amino acids are organic compounds containing an amine group and a carboxylic acid group, a side chain that varies between different amino acids.” 3
  4. 4.  One amino acid molecule can react with another and become joined through an amide linkage. This polymerization of amino acids creates proteins, this condensation reaction yields the newly formed peptide bond and a molecule of water. 4
  5. 5.  20 amino acid are encoded by genetic code. Several amino acids are called "essential" for humans because they cannot be created from other compounds by the human body, and so must be taken in as food. Non essential amino acids which the body can synthesized, Alanine, Proline, Asparagine, Aspartic acid, Cysteine, Tyrosine, Serine, Glycine, Glutamine and Glutamic acid. Essential amino acids which the body cant synthesize are, Isoleucine, leucine, Lysine, Methionine, Valine, Phenyl alanine, Threonine, Tryptophan, Histidne and Arginine. 5
  6. 6.  Amino acid analysis refers to the methodology used to determine the amino acid composition or content of proteins, peptides, and other pharmaceutical preparations. Proteins and peptides are macromolecules consisting of covalently bonded amino acid residues organized as a linear polymer. The sequence of the amino acids in a protein or peptide determines the properties of the molecule. 6
  7. 7.  Amino acid analysis can be used to Quantify protein and peptides, Determine the identity of proteins or peptides based on their amino acid composition, Support protein and peptide structure analysis, Evaluate fragmentation strategies for peptide mapping, Detect atypical amino acids that might be present in a protein or peptide. It is necessary to hydrolyze a protein/peptide to its individual amino acid constituents before amino acid analysis. 7
  8. 8.  General techniques for analysis of amino acids are:1. Determining amino acid composition Hydrolysis Separation Quantitative analysis2. N-terminal amino acid analysis3. C-terminal amino acid analysis 8
  9. 9. 4. Edman degradation5. Mass spectrometry6. Predicting protein sequence from DNA/RNA sequences  The two major direct methods of protein sequencing are mass spectrometry and the Edman degradation reaction 9
  10. 10. 1.) Determination of amino acid composition Hydrolysis Hydrolysis is done by heating a sample of the protein in 6M hydrochloric acid to 100-110 oC for 24 hr or longer. Proteins with many bulky hydrophobic groups may require longer heating periods. 10
  11. 11.  However, these conditions are so vigorous that some amino acids (serine, threonine, tyrosine, tryptophan, glutamine and cystine) are degraded. Rastall suggests a variety of reagents to prevent or reduce degradation - thiol reagents or phenol to protect tryptophan and tyrosine from attack by chlorine, and pre- oxidising cysteine. 11
  12. 12. H O H H O H H O H H O H H O H H O H 3N C C N C C N C C N C C N C C N C C O C H3 C H 2O H C H2 C H(C H 3 ) 2 H C H 2C H 2S C H 3 A S F V G M H 3 O , h eat (tot al hydr olysis) H O H O H O H O H O H OH 3N C C O + H 3N C C O + H 3N C C O + H 3N C C O + H3 N C C O + H 3N C C O C H3 C H 2O H C H2 C H(C H 3 ) 2 H C H 2C H 2S C H 3 A S F V G M (equ imolar mixture of A, S , F , V , G , an d M ) Hydrolysis: conversion of a peptide into a mixture of its component amino acids 12
  13. 13.  Separation The amino acids can be separated by ion-exchange chromatography or hydrophobic interaction chromatography. Amino acids will be eluted when the pH reaches their respective isoelectric points. The latter technique may be employed through the use of reversed phase chromatography. Many commercially available C8 and C18 silica columns have demonstrated successful separation of amino acids in solution in less than 40 minutes through the use of an optimised elution gradient. 13
  14. 14.  Quantitative analysis Once the amino acids have been separated, their respective quantities are determined by adding a reagent that will form a coloured derivative. If the amounts of amino acids are in excess of 10 nmol, ninhydrin can be used for this - it gives a yellow colour when reacted with proline, and gives a purple colour. The concentration of amino acid is proportional to the absorbance of the resulting solution. 14
  15. 15.  Sangers method of peptide end-group analysis:A. derivatization of N-terminal end with Sangers reagent 2-4 dinitroflorobenzen(DNFB),B. Total acid hydrolysis of the dinitrophenyl peptide 15
  16. 16. H O H H O H H O H H O H H O H H O H 3N C C N C C N C C N C C N C C N C C O C H3 C H 2O H C H2 C H (C H 3 ) 2 H C H 2C H 2S C H 3 A S F V G M S angers R eagent (2,4- dinitrof luor obe nzene) H H O H H O H H O H H O H H O H H OO 2N N C C N C C N C C N C C N C C N C C OH C H3 C H 2O H C H2 C H (C H 3 ) 2 H C H 2C H 2S C H 3 NO2 16
  17. 17.  The number of methods available for C-terminal amino acid analysis is much smaller than the number of available methods of N-terminal analysis. The most common method is to add carboxypeptidases to a solution of the protein, take samples at regular intervals, and determine the terminal amino acid by analysing a plot of amino acid concentrations against time. 17
  18. 18. Carboxypeptidase: C-terminal AA Analysis H O H H O H H O H H O H H O H H OH 3N C C N C C N C C N C C N C C N C C O C H3 C H 2O H C H2 C H (C H 3 ) 2 H C H 2C H 2S C H 3 A S F V G M C a rbox ype pt ida se H O H H O H H O H H O H H O H OH 3N C C N C C N C C N C C N C CO + H 3N C CO C H3 C H 2O H C H2 C H (C H 3 ) 2 H C H 2C H 2S C H 3 M C a rbox ype pt ida se H O H H O H H O H H O H OH 3N C C N C C N C C N C CO + H3 N C CO C H3 C H 2O H C H2 C H (C H 3 ) 2 H G 18
  19. 19.  Edman degradation, developed by Pehr Edman, is a very important reaction for protein sequencing, because it allows the ordered amino acid composition of a protein to be discovered. Now a day, Automated Edman sequencers are used, and are able to sequence peptides up to approximately 50 amino acids long. 19
  20. 20.  A reaction scheme for sequencing a protein by the Edman degradation follows - some of the steps are describe below.1. Break any disulfide bridges in the protein with an oxidising agent like per formic acid or reducing agent like 2- mercaptoethanol.2. Separate and purify the individual chains of the protein complex, if there are more than one. Determine the amino acid composition of each chain.3. Determine the terminal amino acids of each chain. 20
  21. 21. 4. Break each chain into fragments under 50 amino acids5. Separate and purify the fragments.6. Determine the sequence of each fragment.7. Repeat with a different pattern of cleavage.8. Construct the sequence of the overall protein. 21
  22. 22.  Phenylisothiocyanate is reacted with an uncharged terminal amino group, under mildly alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative. Then, under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. 22
  23. 23. 23
  24. 24. 24
  25. 25.  The amino acid sequence of a protein can also be determined indirectly from  the mRNA or,  the DNA that codes for the protein. (e.g. prokaryotes). 25
  26. 26.  This can then be used to isolate the mRNA coding for the protein, which can then be replicated in a polymerase chain reaction to yield a significant amount of DNA, which can then be sequenced relatively easily. 26
  27. 27.  Gel electrophoresis Electrophoresis is a separation technique based on the movement of charged ions under the influence of an electrical field. This technique is primarily used for the separation of amino acids and peptides on the basis of their charge. In this experiment of separating amino acids, a phosphate buffer (pH 6) will be used as the conducting liquid and cellulose as the supporting medium. 27
  28. 28.  Polyacrylamide Gel Electrophoresis (PAGE) PAGE (Polyacrylamide Gel Electrophoresis) SDS(sodium dodecyl sulfste) is the most widely used analytical method to resolve separate components of a protein mixture based on their size H O H O H OM ixture of : H 3N C C O + H 3N C CO + H3 N C CObu ffered a t pH 6.0 C H 2C O 2 C H3 C H 2 C H 2 C H 2 C H2 N H 3 D ( pI= 2.8) A (pI= 6.0) K ( pI= 9.7) 28
  29. 29.  Before current is turned on: K A D After current is turned on: K A D 29
  30. 30.  The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence The polymerase chain reaction (PCR) is an in vitro technique which allows the amplification of a specific deoxyribonucleic acid (DNA) region that lies between two regions of known DNA sequences.. 30
  31. 31.  This helix comprises of two single strands of DNA running antiparallel to each other and held together non-covalently by hydrogen bonds. The hydrogen bonds form between the complementary bases, i.e., adenine (A) with thymine (T) and guanine (G) with cytosine (C). 31
  32. 32.  X-ray crystallography is a technique which is widely used to determine structures of proteins. It exploits the fact that X-rays are scattered or diffracted in a predictable manner when they pass through a protein crystal. X-rays are diffracted when they encounter electrons, so the nature of the scattering depends on the number of electrons that are present in each atom and the organization of the atoms in space. 32
  33. 33.  In principle X-ray analysis of very large and complex organic molecules like proteins is possible but the mathematical analysis of the diffraction patterns is very complex because large number of atoms in the molecule may yield thousands of diffraction spots. X-ray crystallography is the primary method used to determine protein structures 33
  34. 34.  Has allowed determination of structures as large as viruses and ribosomes to be completed X-ray methods are fast and depend on computers and robots X-ray structures are generally more accurate than NMR structures. 34
  35. 35.  Isoelectric focusing takes place in a pH gradient and is limited to molecules which can be either positively or negatively charged (amphoteric molecules), like proteins, enzymes and peptides. Each amino acid has an isoelectric point(pI) numerically equal to the pH at which the zwitterion concentration is at a maximum. O OH O O O O C C C H OH H 3N H H 3N H H 2N H R R R @ pH < pI @ pH = pI @ pH > pI 35
  36. 36. Equipment for Isoelectric focusing (IEF):(IEF System) Protein IEF CellAmersham Pharmacia Biotech Inc. Bio-Rad Laboratories 36
  37. 37.  This is a technique for detecting atoms that have nuclei that possess a magnetic moment. These are atoms in which either the protons or the neutrons or both are odd in number. 1H atom (one proton), 13C and 14N are some such isotopes of hydrogen, carbon and nitrogen respectively In principle, it is possible to obtain a unique signal for each hydrogen atom except in case of those which are chemically equivalent. However, this problem has been bypassed by using 2D NMR spectroscopy. 37
  38. 38.  A COSY (correlated spectroscopy) experiment gives peaks between hydrogen atoms that are covalently connected through one or two other atoms, for example, the hydrogen atoms connected to nitrogen or carbon atoms within the same amino acid residue. A NOE (nuclear Overhauser spectroscopy) spectrum, on the other hand gives peaks between pairs of hydrogen atoms that are close together in space even if they are from amino acid residues that are quite distant in the primary sequence. 38
  39. 39.  Mass spectroscopy This process, known as matrix-assisted laser desorption/ionization mass spectrometry, or MALDI MS, has been successfully used to measure the mass of a wide range of macromolecules. A solution of analytes is passed through a charged needle that is kept at a high electrical potential, dispersing the solution into a fine charged microdroplets. 39
  40. 40.  This creates a spectrum of species with different mass-to-charge ratios. Each successive peak corresponds to a species that differs from that of its neighboring peak by a charge difference of 1 and a mass difference of 1 (1 proton). The mass of the protein can be determined from any two neighboring peaks. The measured m/z of one peak is 40
  41. 41. where M is the mass of the protein, n2 is the number of charges, and X is the mass of the added groups (protons in this case). 41
  42. 42. LC/MS 42
  43. 43. A protein solution is dispersed into highly charged droplets by passagethrough a needle under the influence of a high-voltage electric field. Thedroplets evaporate, and the ions (with added protons in this case) enter themass spectrometer for m/z measurement 43
  44. 44. Amino acid sequence analysis by MS - an exampleThe spectrum generated (b) is a family of peaks, with each successivepeak (from right to left) corresponding to a charged species increased by1 in both mass and charge. 44
  45. 45.  Chromatographic method have been used to determination of the purity of small organic molecules and protein the result depend on size and /or shape , charge, and hydrophobicity of the protein RP-HPLC reveres phase high performance liquid chromatography) IEC (ion exchange chromatography) SEC (size exclusion chromatography) HIC (hydrophobic interaction chromatography) 45
  46. 46. Principle: A portion of the material to be separated will be found in the mobile phase A portion will be adsorbed to the solid adsorbent particles. 46
  47. 47. Calculate the Rf value Distance of the amino acid from the originRf = Distance of the solvent front from the origin D1 Rf= D2 47
  48. 48.  Negatively charged proteins will bind to positively charged column materials (anion exchangers), while positively charged or neutral proteins will flow through the column. The more negatively charged, the tighter it will bind and the higher the salt it will take to elute it from the column Some highly charged proteins, where the charge is 48 not evenly distributed, can bind to both anion and
  49. 49. 49
  50. 50. SO 3 K (stron gly r eta ine d)sulfon ate d A (slightly re ta ine d, & SO 3pol ystyre ne D (unre ta ine d) SO 3 D A K 50
  51. 51. REVERSE PHASE & HYDROPHOBICINTERACTION separation of hydrophobic proteins stationary phase is non polar liquid (hydrophobic) polar mobile phase polarity of mobile phase reduced to proteins. hydrophobic interaction chromatography phenyl sephadex and octal sephadex is used with weak interaction to prevent denaturation 51
  52. 52. The PTC-amino acids are separated on a reversephase C18 silica column and the PTC chromophoreis detected at 254 nm. All of the amino acids willelute in approximately 25 minutes.Many commercially available C8 and C18 silicacolumns have demonstrated successful separation ofamino acids in solution in less than 40 minutes throughthe use of an optimised elution gradient. 52
  53. 53. 53
  54. 54. 1. Lehninger Principles of Biochemistry, 4th Edition Chapter 9.2. Henry Jakubowski. Biochemistry Online, chapter 2 B.[1]3. Hanno Steen & Matthias Mann. The abcs (and xyzs) of peptide sequencing. Nature Reviews Molecular Cell Biology, 5:699-711, 2004.4. Sergio Marceline Michael W. King. Analysis of protein.[2]5. R A Rastall. Investigating protein structure and function.[3]6. Alberts Bray Johnson Lewis Raff Roberts & Walter. 1998. Essential Cell Biology: An Introduction to the Molecular Biology of the Cell. Garland Publishing, New York.7. http://www.pafko.com/history/h_intro.html
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