1. 1 OUTCOME 1: KEY ASPECTS OF PROTEOMICS Proteomics is “the large scale study of proteins, particularly their structures and functions”.

2. Two Dimensional Gel Electrophoresis Separates proteins in a mixture based on their charge and mass. Separation in the first dimension relies upon differences in CHARGE and utilises the technique of isoelectric focusing. A complex protein mixture can be loaded onto a polyacrylamide gel which incorporates a pH gradient. 2

3. 3 Isoelectric focusing

4. 4 Proteins migrate through the matrix until they reach a point in the pH gradient where their charge is the same as the surrounding pH. They stop at this point in the gel: their isoelectric point.

5. SDS PAGE 2 nd stage relies upon differences in MASS. Isoelectric gel is soaked in a denaturing solution containing SDS: –the proteins unfold, same in terms of shape. –the SDS charge (negative) swamps any inherent charge on the proteins. Proteins migrate based solely on differences in mass. Smaller proteins travel further than larger ones. 5

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7. Large proteins will travel slower and less far Second dimension: SDS-PAGE First dimension: Isoelectric focussing (IEF) 2D gel electrophoresis Gel with inbuilt pH gradient Low (acidic) pH High (basic) pH neutral pH Sample of mixed proteins apply electric current Proteins with overall positive charge will move towards negative electrode Proteins with overall negative charge will move towards positive electrode As surrounding pH changes, charge on individual amino acids changes, causing overall charge on protein to change. When overall charge is zero, protein stops moving. Thus IEF represents CHARGE-BASED SEPARATION Gel is treated with SDS. This saturates all proteins with negative charges Apply an electric current. As all proteins are negatively charged, all will move towards positive electrode Acrylamide acts as a seive. Small proteins will travel faster and further Thus SDS PAGE is SIZE-BASED SEPARATION

8. 8 Successive application of both techniques in two dimensions provides maximum separation and allows thousands of proteins to be resolved in a single experiment.

9. Interpreting 2D gels not an easy task. technique yields enough protein to enable structural analysis to be carried out. each ‘spot’ can be analysed in more detail to reveal its identity (see later) 9

10. Interpreting 2D gels measure MWt and Isoelectric point of each protein in the gel. Compare results to predicted sequences number of software programmes are available to perform the analysis. differences due to:  errors in DNA sequencing  post-translational modifications to protein  proteolysis 10

11. Limitations Between 5-10,000 different proteins in a cell, so 2D-PAGE not sensitive enough to detect rare proteins Split original sample into different fractions reduce complexity of protein mixture. Technique is then used in combination with mass spectrometry to identify all major proteins present in a sample. 11

12. 2D gel electrophoresis is useful when comparing related samples (different expression) such as occurs in normal v. disease tissue. Can indicate altered proteins or expression of novel proteins in disease tissue Information may be useful as the relevant proteins could represent diagnostic markers and/or targets for drugs. 12

13. 13 Here Gel 3 is being compared against Gel 1 (see image 4 for overlay) and against gel 2 (see image 5 for overlay). Comparison shows spot A to be present in only Gel 1 but spot B to be present in all Gels.

14. Peptide Mass Fingerprinting PMF relies on the use of a proteolytic enzyme (protease) to digest the target protein into a number of small peptides. The collection of peptides produced by this digestion will be unique to the protein in question and so, once separated by mass spectroscopy, will act as a fingerprint for identification purposes. 14

15. Peptide Mass Fingerprinting Suitable protease (eg trypsin) Digestion must go to completion: –the peptide fragments of varying masses will be characteristic of the protein. –The samples must survive the MS procedure without fragmenting too much: –MALDI-TOF and ESI-TOF are the currently favoured MS techniques. 15

16. Peptide Mass Fingerprinting There must be information against which to compare the fingerprint: –this relies upon the ability to search databases. –If there is no relevant information in a database then PMF is of no use. –Databases commonly searched are Genbank and Swissprot 16

17. 17 OVERVIEW OF PMF Peptide (spot from SDS-PAGE gel) Tryptic cleavage (complete) Peptide fragments Mass Spectrometry “ Fingerprint ”

18. From 2D gel to MS The spot is cut from the gel and protease is added to digest in-gel. The sample then has to be prepared for entry to the mass spectrometer. MS works on different formats but essentially all involve movement of ions, through a vacuum, towards a detector. The detector records mass/charge ratios. 18

19. Ionising Peptides The 2 main methods for ionising peptides are MALDI (matrix assisted laser desorption ionization) and ESI (electrospray ionisation): In MALDI a laser is used to create the ionized peptides and in ESI high voltage (3 or 4 kv) is used. MALDI can cope with a higher number of samples (high throughput). 19

20. Ionising Peptides ESI is known as a ‘soft’ ionisation method as it ionises the sample without causing any of it to fragment. Similarly with MALDI the ionisation process doesn’t cause excessive decomposition of the sample. Both versions of mass spectrometry are suitable for use with peptides which are relatively fragile molecules. 20

21. TOF-MS The analyser/detector part of the mass spectrometer is a ‘time-of-flight’. TOF-MS times how long it takes fragments to travel from one end of a tube to the detector at the other end. Lighter fragments move faster down the tube than heavier ones. On this basis it’s possible to separate and analyse peptides based on their differing masses. 21

22. TOF-MS The horizontal axis shows time it took the fragments to travel to the detector. Since flight time depends on mass the horizontal axis also represents mass. The vertical axis represents intensity. 22

23. Searching Databases The initial MS spectrum can often provide information to search a database. Software programs translate genomes into proteins, theoretically cut the proteins using your protease and then calculate the mass of each peptide fragment. A comparison is then made of the peptide fragments from the ‘unknown’ protein and the theoretical fragments encoded by the genome. After some statistical analysis the best matches are highlighted. The advantage of this technique is that only the masses of the peptides, rather than their sequences, has to be known, but the big disadvantage is that there has to be relevant information in the database. 23

24. Searching Databases After some statistical analysis the best matches are highlighted. The advantage of this technique is that only the masses of the peptides, rather than their sequences, has to be known, but the big disadvantage is that there has to be relevant information in the database. 24

25. TRYPTIC DIGESTION and TOF-MS (time of flight mass spectroscopy)

26. metphecysalaleulyshisleuargphecyshislysgluphe Lets imagine this protein is an enzyme called Xase It could be identified by determining its entire amino acid sequence, but this is a time-consuming process. There is a quicker way!

27. And because trypsin will always cut Xase in exactly the same places, tryptic digestion of Xase will always produce the same four fragments. This result, 4 fragments of these exact sizes, can be described as the tryptic digestion fingerprint of the protein Xase. metphecysalaleuhisleuphecyshislysarglysgluphe The proteolytic enzyme trypsin cuts polypeptide chains after lysine (lys) and arginine (arg) residues. Therefore trypsin would cut Xase at these three points Therefore tryptic digestion of Xase will produce four fragments. metphecysalaleu lys phecyshislys gluphe arghisleu m.w = 693 m.w = 406 m.w = 515 m.w = 275 Adding this result to an international database will allow other researchers to identify Xase in their samples.

28. A researcher finds an unknown protein is being produced by a species of cell they are studying. They analyse the protein as follows: They perform a tryptic digestion of the protein 693 406 515 They ionise the fragments by MALDI or ESI They subject the fragments to TIME OF FLIGHT MASS SPECTROSCOPY Ionised fragments are fired through a vacuum towards a detector Smallest fragments travel fastest, largest fragments travel slowest. 275 681 1196 1889 TIME 275

29. metphecysalaleuhisleuphecyshislysarglysgluphe 693 406 515 275 On checking a database they find a protein with an identical fingerprint to their unknown protein: Xase metphecysalaleuhisleuphecyshislysarglysgluphe 1099 790 Incomplete digestion

30. Obtaining Further Information If the database search is not fruitful then further information is required. This is usually done by tandem mass spectroscopy or by Edman degradation. 30

31. Tandem Mass Spectroscopy MS-MS Most popular experimental method for identifying proteins: –employs more than one analyser (up to 4?), –requires very little sample ( ≤40 ρmoles). Can carry out structural and sequencing studies and determine the amino-acid sequences of the individual proteolytic peptides contained in the digest mixture 31

32. Principle of MS-MS First MS ionises the peptides. Next MS causes specific peptide to fragment further. Resultant spectrum can provide sequence information not available by single MS. Once the amino acid sequence information has been obtained further database searching can be carried out to identify the protein. 32

33. Sequence Determination Each peak on the graph represents one peptide fragment. The peak at the extreme right will represent the entire peptide (i.e. Molecular weight, MWt). 33

34. 34 Each peak actually represents millions of fragments, each with the same mass, and therefore the same amino acid sequence

35. Using basic arithmetic the mass of each amino acid in the peptide is determined. e.g.842 – 743 = 99 = amino acid 1 743 – 672 = 71 = amino acid 2 All but 2 amino acids have unique masses: valine, for example, is 99Da (Daltons), whilst alanine is 71 Da. It is therefore possible to identify each amino acid based solely on mass. Having worked out the sequence, a computer is used to identify the protein, i.e. compare to databases. 35

36. Edman Degradation This technique to sequence peptides involves removal of one amino acid at a time from the N-terminus of a peptide. Process involves the following steps: –Coupling of PITC (phenyl isothio cyanate) to free N-terminus of peptide. –Cleavage of N-terminal amino acid –Conversion of the cleaved amino acid to the stable PTH amino acid which is then analysed by HPLC. 36

37. Step 1 –Free N terminal amino group reacts with PITC in alkaline medium. This gives the PTC derivative. Step 2 –The PTC – peptide is then treated with trifluoroacetic acid to hydrolyse the first peptide bond whilst the other peptide bonds remain intact. 37

38. Step 3 –The process completes with the liberated cyclic derivative of the N-terminal residue rearranging to give the PTH derivative. Identification of the original N-terminal residue is by HPLC The shortened peptide chain goes through a second cycle of the same procedure. This is repeated until all amino acids are identified: –sequence is determined from N  C terminus. 38

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40. N C C O R4R4 HH H R3R3 N C C O HH H N C C O R2R2 HH H EDMAN DEGRADATION N C C O R1R1 H H H PITC Add strong acid. eg. TFA (trifluroacetic acid) peptide bond breaks Add strong acid. eg. TFA (trifluroacetic acid) peptide bond breaks Add strong acid. eg. TFA (trifluroacetic acid) peptide bond breaks

41. Protein-Protein Interactions Protein-protein interactions are critical to all cellular processes. Antibody-antigen, receptor-ligand,..etc involve specific interactions of two or more proteins 41

42. Protein-Protein Interactions Some protein-protein interactions are long term interactions: –triple helix formed by collagen can form for weeks or even years without dissociating other protein-protein interactions are more transient: –kinase enzyme binding a protein substrate in order to phosphorylate it may dissociate after less than one microsecond. 42

43. Protein-Protein Interactions Interactions occur at defined domains and most interfaces are flat, circular areas on the protein surface. Water is usually excluded. Interactions mediated by non-covalent bonds such as hydrogen bonds, ionic bonds, Van der Waals forces and hydrophobic interactions 43

44. In the human body there may be 100,000 protein – protein interactions, ranging from a combination of 2 identical proteins to complexes involving 50 to 100 different protein subunits. Flattened circular binding domains Non-covalent bonding (hydrophobic interactions, ionic bonding, Van der Waals forces, hydrogen bonds)

45. ANALYSIS OF PROTEIN COMPLEXES – MASS SPECTROSCOPY Traditional Mass Spec preparation techniques were too harsh for non-covalently-bound proteins to survive. Soft ionisation methods (ESI or MALDI) are more gentle; complexes survive the process.

46. ANALYSIS OF PROTEIN COMPLEXES – MASS SPECTROSCOPY Separation of subunits prior to MS allows individual identification. Then cross linking of subunits allows the oligomeric complex to be analysed by MS This can reveal whether stoichiometry of complex is: dimer trimer tetramer

47. ANALYSIS OF PROTEIN COMPLEXES – ANTIBODY TRAPPING Purify the protein and raise antibodies against it. Attach antibodies to beads and perform affinity chromatography: Protein mix from cell is passed through column Protein (including other proteins complexed with it) are trapped by the antibody Protein complex removed from beads and analysed by 2D electrophoresis and MS

48. Cellular proteins are run through affinity column ANALYSIS OF PROTEIN COMPLEXES – AFFINITY TAG SEPARATION Protein is cloned and expressed carrying an AFFINITY TAG such as protein A Protein is expressed in cell and contents extracted. Tagged protein and proteins complexed with it attach to bead in column Tagged protein and proteins complexed with it are released and eluted from column Proteins are analysed by 2D electrophoresis and mass spec. However: Complexes may not survive release and elution membrane-bound complexes difficult to isolate tags may prevent complex forming

49. FRET is fluorescence resonance energy transfer. ANALYSIS OF PROTEIN COMPLEXES – FRET There are two variations of this technique The protein with which it is thought to complex is labelled with a complementary fluorescent molecule that can absorb the fluorescence of the first. In both methods, a protein suspected of forming a complex is fluorescently labelled and emits light of a specific wavelength. If the two fluorescent molecule are brought very close together (ie. if the proteins form a complex), the wavelength emitted changes.

50. FRET is fluorescence resonance energy transfer. ANALYSIS OF PROTEIN COMPLEXES – FRET This time, the protein with which it is thought to complex is labelled with a fluorescent quencher molecule that absorbs the fluorescence of the first and stops fluorescent emittance. In the second version, the protein suspected of forming a complex is again fluorescently labelled and emits light of a specific wavelength. Therefore if the two fluorescent molecule are brought very close together (ie. if the proteins form a complex), the fluorescence stops. Q

51. For transcription to occur, a TRANSCRIPTIONAL ACTIVATOR (TA) protein is required. This protein has TWO DOMAINS: BD – a DNA binding domain AD – the activation domain Only if both domains are present will transcription of DNA take place. ANALYSIS OF PROTEIN COMPLEXES – YEAST TWO HYBRID ASSAY

52. In YEAST TWO HYBRID ANALYSIS, the two domains are separated onto proteins suspected of forming a complex. Fusion proteins are constructed (from the DNA sequence) that will each carry one of the domains. ANALYSIS OF PROTEIN COMPLEXES – YEAST TWO HYBRID ASSAY One carries the BD domain The other carries the AD domain Only if the proteins form a complex can a REPORTER GENE be transcribed and produce a quantifiable, coloured product

53. Other Techniques Bioinformatics - predicting protein interactions by comparing mRNA expression, analysis of genome sequence Structural approaches such as X-ray crystallography and NMR analyse the contact areas between proteins Molecular approaches such as site- directed mutagenesis allows specific modifications. 53