1. Lecture 6: Peptides –MS-MS –Schiff base for ninhydrin –Protein purification

2. Figure 7-7The amino acid sequence of a polypeptide chain.

3. To make trypsin even more versatile you can modify side chains of amino acids Lys specific reaction to hide basicity See p. 170 in your book-especially the reactions with citraconic anhydride so trypsin won’t cleave at Lys residues. Also on p. 170 conversion of Cys side group with 2- bromoethylamine to make a basic group to cleave at Cys with trypsin.

4. Lysine reactions + C - OOC H CH 2 H3NH3N + NH 3 Lysine aldehyde + R’ H2OH2O C H O CH 2 + C - OOC H CH 2 H3NH3N + N C H O H+H+ + Schiff base

5. Lysine reactions + C - OOC H CH 2 H3NH3N + NH 3 Lysine Succinic anhydride O CH 2 + C - OOC H CH 2 H3NH3N + N C O-O- O 2H + + C O H2CH2C C O H2CH2C C CH 2 O

6. Determining primary structure of polypeptides Deduce the amino acid sequence of a simple polypeptide from the following results: A.Acid hydrolysis: (Ala 2, Arg, Lys 2, Met, Phe, Ser 2 ) B.Carboxypeptidase A: (Ala) C.Trypsin: (Ala,Arg), (Lys,Phe,Ser), (Lys), (Ala, Met, Ser) D.CNBr: (Ala, Arg, Lys 2, Met, Phe, Ser), (Ala, Ser) E.Thermolysin: (Ala, Arg, Ser), (Ala, Lys 2, Met, Phe, Ser) Where do we start? First, from A. (acid hydrolysis) we know how many amino acids are in the polypeptide: 9 Second from B. (carboxypeptidase A), we know the last amino acid is one of the Ala.

7. We know trypsin cleaves at the carboxy side of basic aas (Lys and Arg) Trypsin: (Ala,Arg), (Lys,Phe,Ser), (Lys), (Ala, Met, Ser), so we can rearrange the amino acids as follows: Ala-Arg, either Phe-Ser-Lys or Ser-Phe-Lys, Arg-Lys or Lys-Lys, and either Lys-(Ala, Met, Ser) or Arg-(Ala, Met, Ser). For CNBr, we got two fragments (Ala, Arg, Lys 2, Met, Phe, Ser) and (Ala, Ser). We know that cleavage occurs on the carboxy side of Met. So we know that Met-(Ser-Ala) or Met-(Ala-Ser). 1 - 2 - 3 - 4 - 5 - 6 - 7 - 8 -Ala

8. For thermolysin, we know it cleaves N-terminal to Ile, Met, Phe, Trp, Tyr, Val. So (Ala, Arg, Ser) are before Met From trypsin: Ala-Arg, Phe-Ser-Lys or Ser-Phe-Lys, Arg- Lys or Lys-Lys, and either Lys-(Ala, Met, Ser) or Arg- (Ala, Met, Ser). WE know that one Ala is the carboxy terminal amino acid, so Ala-Arg cannot be the carboxy terminus. Therefore, the only other possibility is the last sequence (Ala, Met, Ser) where Ala is the carboxy terminal amino acid. So the order at the carboxy terminus is basic aa-Met-Ser- Ala or basic aa-Ser-Met-Ala 1 - 2 - 3 - 4 - 5 - 6 - 7 - 8 -Ala

9. For CNBr, we know that cleavage occurs on the carboxy side of Met. So, combined with the trypsin result we get basic aa-Met (Ser-Ala). For thermolysin, we know it cleaves N-terminal to Ile, Met, Phe, Trp, Tyr, Val. So (Ala, Arg, Ser) are before Met or Phe. We know from the CNBr cleavage that the Met must be before Ser-Ala, so for the (Ala, Lys 2, Met, Phe, Ser) Phe must be the 1st aa in this sequence. We also know that a basic aa precedes Met from the trypsin experiment. Since the only basic aas in this fragment are Lys, the order must be : Phe-Lys-Lys-Met-Ser-Ala 1 - 2 - 3 - 4 - 5 - basic aa - Met - Ser -Ala

10. Remember for thermolysin, we know it cleaves N-terminal to Ile, Met, Phe, Trp, Tyr, Val. So (Ala, Arg, Ser) are before Met or Phe. We know from the trypsin digest that Ala-Arg are in a specified order so the final sequence must be Ala-Arg- Ser 1 - 2 - 3 - Phe - Lys - Lys - Met - Ser -Ala Ala - Arg - Ser - Phe - Lys - Lys - Met - Ser -Ala

11. Peptide characterization by mass spectrometry Mass spectrometry (MS) is an important technique for characterizing and sequencing polypeptides. MS measure the mass-to-charge (m/z) ratio for ions in the gas phase where m is the mass of th ion and z is the charge. Prior to 1985 MS could not be used for the analysis of proteins or nucleic acids because the gas phase ions produced destroyed the molecules. Three techniques developed to solve this problem: –Electrospray ionization (ESI) –Matix-assisted laser desorption/ionization (MALDI) –Fast atom bombardment (FAB).

12. Figure 7-8aThe generation of the gas phase ions required for the mass spectrometric analysis of proteins. (a) By electrospray ionization (ESI). Page 172

13. Figure 7-8bThe generation of the gas phase ions required for the mass spectrometric analysis of proteins. (b) By matrix-assisted laser desorption/ionization (MALDI). Page 172

14. Figure 7-8cThe generation of the gas phase ions required for the mass spectrometric analysis of proteins. (c) By fast atom bombardment (FAB). Page 172

15. Figure 7-9The ESI-MS spectrum of the 16,951-D horse heart protein apomyoglobin. Page 173 p 1 = M + z 1 z1z1 p 2 = M + z 1 -1 z 1 -1 Z1 = 25 for peak 1 P1 = 678.6 The mass of = 678.6 (25)-25=16940 1884.7(9)-9=1695.33

16. Peptide characterization by mass spectrometry Gas phase macromolecular ions are detected by MS which measures their m/z values with high accuracy >0.01%. If the ion’s charge value can be determined, it is the most accurate method. ESI-MS has several advantages over other forms of MS because of the high ionic charges of the ions produced allows analysis of compounds with greater molecular mass >100 k. ESI-MS can be configured with other purification equipment (HPLC for example) so that it can be used to analyze a tryptic digest of a protein by determining the molecular masses of the components. Tandem mass spectrometers (MS-MS) can be used to directly sequence short polypeptides (<25 aa) but cannot distinguish Ile and Leu (same mass) and is unreliable for Gln and Lys (differ by only 0.036 D)

17. Figure 7-10The use of a tandem mass spectrometer (MS/MS) in amino acid sequencing. Page 174

18. Carboxyl and amino terminal reactions are important for chemical synthesis of peptides Polypeptides can be chemically synthesized by covalently linking (coupling) amino acids, one at a time, to the terminus of a growing polypeptide chain. In the chemical synthesis of polypeptides, the polypeptide chain is being synthesized from the C-terminus to the N-terminus. The amino acid being added to the chain must already have its own  - amino group chemically protected (blocked) to prevent it from reacting with other similar molecules as well as with the N-terminal amino group of the chain. Once the new amino acid is coupled, the now N-terminal amino acid must be deprotected (deblocked) so that the next peptide bond can be formed.

19. Figure 7-34Flow diagram for polypeptide synthesis by the solid phase method.

20. Amino group reactions H C R COO - NH 2 Amino acid + H C R N C-R’ HCl Substituted amide Cl C R’ O COO - O H+H+ + H

21. Protection of the amino group t-Butyloxycarbonyl chloride (Boc) + H2NH2N C R HCl Cl C (CH 3 ) 3 C-O O COO - H C (CH 3 ) 3 C-O O NH C R COO - H Boc-amino acid  -amino acid

22. Figure 7-35A selection of amino acids with benzyl-protected side chains and a Boc-protected  -amino acid group. Page 206

23. Carboxyl group reactions H CR COOH NH 3 + Amino acid NH 3 + H CR C NH 2 O H3NH3N + H2OH2O Amide H CR COOH NH 3 + Amino acid R’-NH 2 + H CR C N-R’ O H3NH3N + H2OH2O Substituted amide H

24. Carboxyl group reactions H CR COOH NH 3 + Amino acid R’-OH + H CR C O-R’ O H3NH3N + H2OH2O Ester

25. Protection of the carboxyl groups H CR COOH NH 3 + Amino acid NH 3 + H CR C NH 2 O H3NH3N + H2OH2O Amide H CR COOH NH 3 + Amino acid R’-NH 2 + H CR C N-R’ O H3NH3N + H2OH2O Substituted amide H

26. Amino group reactions H C R COO - NH 2 Amino acid + H C R N C-R’ H2OH2O Schiff base H C R’ O COO - H H+H+ + Primary amine imine H C R COO - NH 2 Amino acid + H C R N C-R’ HCl Substituted amide Cl C R’ O COO - O H+H+ + H

27. Example of the Schiff base reaction using Ninhydrin O O OH Ninhydrin H2OH2O O O O  H2NH2N C COO - H R + O O O-O- NH 2 COO - + R O O OH NH COO - R Proton transfer

28. Ninhydrin reaction O O OH NH COO - R H2OH2O O O  N C-O - R O O H H : + CO 2 O O N R - O O N R : : O H H + - O H H

29. Ninhydrin reaction O O N R : : O H H + - O H H O O N R : OH H - O H H O O N R H - H + O O NH 2 R OH - H + + R H + R O H

30. Ninhydrin reaction O O NH 2 - O O O O N H O O O O-O- N Two resonance forms of Ruheman’s Purple can be detected at 570 nm O O O  + Imine formation followed by same mechanism

31. Summary of reactions of amino acids Peptide bond formation –Activation –Protection Schiff’s base formation Sanger’s reagent Dansylation Edman degradation Peptide hydrolysis Hydrolysis of Gln, Asn Modification of Lys Reactions of Cys (  -mercaptoethanol, DTT, iodoacetate) Met/CNBr Hydrazine Cleavage

32. Protein purification Protein purification can be thought of as a series of fractionation steps designed to: –Get the protein of interest almost exclusively in one fraction –Get a significant amount of contaminants in a different fraction Objective: To separate the protein of interest from a complex mixture of proteins (from tissues or recombinant organism) while maintaining biological function. Biological function can be maintained by controlling the pH, the temperature, and ionic strength (e.g. salt concentration) of the buffers used during purification. Similar techniques are used to isolate all proteins but the less starting material we have the more work it is to isolate it.

33. Outline of steps for protein purification Choose your protein source. Methods of solubilization A unique assay (a test) for the protein of interest. Assay for total protein A series of fractionation steps adapted to the protein Assays for purity If the protein you want to purify is an enzyme, it’s biological activity can be used as a unique assay for its presence throughout the purification.

34. Starting materials for protein purification Proteins constitute a major fraction of the mass of all living things Sometimes proteins are available in abundance (hemoglobin) but they may also be very scarce (lac repressor in wild type E. coli cells). Therefore, the choice of where proteins are obtained is important. If more of the protein is present in the source, the easier it is to purify. Proteins may be obtained from an original source (tissues from specific organism, yeast, etc.) Proteins may also be obtained from recombinant organisms (clones) that overexpress the gene encoding the protein of interest. In E. coli and yeast recombinant protein may constitute up to 30% of the total protein.

35. Methods of solubilization 1st step is to get the protein into solution If the protein is located in the cytosol, we need to break open the cell. For animal cells this can be accomplished with osmotic lysis. Put the cells in hypotonic solution; less solutes than inside the cell. For cells with a cell wall (bacteria, plants) we have to use other methods. For bacteria, lysozyme is often effective - Selectively degrades bacterial cell wall. Can also use detergents or organic solvents, although these may also denature the protein.

36. Mechanical processes to break open the cell High speed blender Homogenzier French press Sonicator. After the cells have been broken, the crude lysate, may be filtered or centrifuged to remove the particulate cell debris. The protein of interest is in the supernatant.

37. For proteins that are components of membranes or subcellular assembly Remove the assembly from the rest of the cellular material (mitochondria for example). Can be done by differential centrifugation-cell lysate is centrifuged at a speed that removes only the cell components denser than the desired organelle followed by a centrifugation at speed that spins down the organelle.

38. Stabilization of proteins pH Temperature Inhibition of proteases Retardation of microbes that can destroy proteins –Sodium azide is often used.

39. Assay of proteins Done throughout the purification process to make sure that your protein of interest is there. If the protein of interest is an enzyme, using a reaction for which that enzyme is a catalyst is a good way to monitor protein recovery. Monitor the increase of the product of the enzymatic reaction –Fluorescence –Generation of acid to be monitored by titration –Coupled enzymatic reaction - couple with another enzyme to make an observable substance.