The document discusses the field of proteomics, which is the large-scale study of proteins, including their functions and structures. It defines proteomics and describes several areas within it, such as functional proteomics, expressional proteomics, and structural proteomics. It outlines typical proteomics experiments and some key methods used, including two-dimensional electrophoresis, mass spectrometry, and protein-protein interaction prediction methods like phylogenetic profiling.

1. PROTEOMICSMs.ruchiyadavlectureramity institute of biotechnologyamity universitylucknow(up)

2. introduction –what is proteomics?Definition:-The identification, characterization and quantification of all proteins involved in a particular pathway, organelle, cell, tissue, organ or organism that can be studied in concert to provide accurate and comprehensive data about that system.”

3. PROTEOMICSThe study of the expression, location, interaction, function and structure of all the proteins in a given cell or organismFunctional ProteomicsExpressional ProteomicsStructural Proteomics

4. PROTEOMICSFunctional proteomics: What is the function of each product of the 30,000 human geneswhere do protein localize,what do they interact with.( Interactomics)how are they modified (PTM)Structural proteomics:What is the 3D structure of proteins and multi-protein machines?Expression proteomics: What differences in protein expression levels accompany certain , Aspects of a cell’s physiology? (normal or pathological)

5. PROTEOME

6. Typical Proteome Experiment

7. Methods used in proteomics

8. Fundamental methods used in proteomics

9. Methods forProteinidentification

10. The identities of proteinsSize: molecular weight (utilized in 2-DE)Charge: pI (utilized in 2-DE)Hydrophobicity

11. Major techniques in modern proteomics Two dimensional electrophoresis, 2-DE Mass spectrometry

12. Global profiling proteomicsIdentification of protein markers from patient samples

13. Find the different protein spots on 2-D gels

14. how is MS used in proteomics ?

15. Perspective for MS based Proteomics

16. Principle of mass spectrometry in proteomics

17. Mass spectrometry:principleMasses of Amino Acid Residues

18. Peptide FragmentationCollision Induced DissociationH+H...-HN-CH-CO . . .NH-CH-CO-NH-CH-CO-…OHRi-1RiRi+1Prefix FragmentSuffix FragmentPeptides tend to fragment along the backbone . Fragments can also loose neutral chemical groups like NH3 and H2O.

19. The types of fragment ions observed in an MS/MS spectrum depend on many factors including primary sequence, the amount of internal energy, how the energy was introduced, charge state, etc.Peptide FragmentationFragments will only be detected if they carry at least one charge. If this charge is retained on the N terminal fragment, the ion is classed as either a, b or c.If the charge is retained on the C terminal, the ion type is either x, y or z. A subscript indicates the number of residues in the fragment.

21. Breaking Protein into Peptides and Peptides into Fragment Ions(ms/ms)Proteases, e.g. trypsin, break protein into peptides.A Tandem Mass Spectrometer further breaks the peptides down into fragment ions and measures the mass of each piece.Mass Spectrometer accelerates the fragmented ions; heavier ions accelerate slower than lighter ones.Mass Spectrometer measure mass/chargeratio of an ion.

22. N- and C-terminal PeptidesPAGNFAPGNFANPGFC-terminal peptidesN-terminal peptidesANFPGPANFG

23. Terminal peptides and ion typesPeptideH2OPNGFMass (D) 57 + 97 + 147 + 114 = 415withoutGFN PeptideH2OPMass (D) 57 + 97 + 147 + 114 – 18 = 397

24. Peptide FragmentationPeptide: S-G-F-L-E-E-D-E-L-K24

25. GVDLKL57 Da = ‘G’K DVG 99 Da = ‘V’H2ODMass Spectramass0The peaks in the mass spectrum:

26. Prefix and Suffix Fragments.

27. Fragments with neutral losses(-H2O, -NH3)

28. Noise and missing peaks.GVDLKPeptide Identification: IntensitymassMS/MS0mass0Protein Identification with MS/MS

29. Commonly used Mass Spectrometer in ProteomicsMALDI-TOFMatrix Assisted Laser Desorption Ionization Time Of FlightESI tandem MS (with HPLC, LC tandem MS or LC MS/MS)Electro Spray Ionization Mass Spectrometry

30. Mass spectrometry used to sequence short stretches of polypeptide

31. Single Stage MSMS

32. Tandem Mass Spectrometry(MS/MS)Precursor selection30

33. Tandem Mass Spectrometry (MS/MS)Precursor selection + collision induced dissociation(CID)MS/MS

34. Tandem mass spectrometry

35. MS FOR PEPTIDE MASS AND SEQUENCE DETECTIONMALDI-TOF

36. Typical result from MALDI-Tof (spectrum)

37. High throughput technique:2D electrophoresis + Mass spectrometrySeparationidentification

38. Basic concept for routine protein analysis

39. Peptide mass fingerprinting (PMF)

40. Peptide mass fingerprinting (PMF)

41. Principles of Fingerprinting*SequenceMass (M+H)Tryptic Fragmentsacedfhsakdfgeasdfpkivtmeeewendadnfekgwfeacekdfhsadfgeasdfpkivtmeeewenkdadnfeqwfeacedfhsadfgekasdfpkivtmeeewendakdnfegwfe>Protein 1acedfhsakdfqeasdfpkivtmeeewendadnfekqwfe>Protein 2acekdfhsadfqeasdfpkivtmeeewenkdadnfeqwfe>Protein 3acedfhsadfqekasdfpkivtmeeewendakdnfeqwfe4842.054842.054842.05

42. Principles of FingerprintingSequenceMass (M+H)Mass Spectrum>Protein 1acedfhsakdfqeasdfpkivtmeeewendadnfekqwfe>Protein 2acekdfhsadfqeasdfpkivtmeeewenkdadnfeqwfe>Protein 3acedfhsadfqekasdfpkivtmeeewendakdnfeqwfe4842.054842.054842.05

43. Data analysis of ms/ms method

44. Peptide mass fingerprinting (PMF) or mapping

45. Mass spectrum mappingTop spectrum depicts a theoretical mass spectrum, as might be generated by a sequence search algorithm, matching an actual peptide MS/MS spectrum (bottom).

46. Computer prog. search databases that contain information

47. Peptide analysis

48. Pmf on webMascotwww.matrixscience.comProFoundhttp://129.85.19.192/profound_bin/WebProFound.exeMOWSEhttp://srs.hgmp.mrc.ac.uk/cgi-bin/mowsePeptideSearchhttp://www.narrador.embl-heidelberg.de/GroupPages/Homepage.htmlPeptIdenthttp://us.expasy.org/tools/peptident.html

49. mascot

50. mascot

51. Ptm identification

52. Expression proteomicsSeparation, quantification and identification of large numbers of proteins from biological specimens2D gel electophoresisMass Spec analysis

53. Ms/ms for post translational modificationPTM-specific mass increments of peptides and amino acid residues or diagnostic fragment ions in mass spectra reveal the presence of PTMs.The MS spectrum is acquired to determine the molecular mass of the peptides.Next, peptides are in turn selected for MS/MS. Fragmentation of the peptide amide bond produces a set of fragment ions that generate a l readout of the sequence in the tandem mass spectrum.The presence of a PTM will change the mass of the modified amino acid residue and of the peptide. MS/ MS often reveals the mass of the PTM and the identity and position of the modified amino acid residue.

54. Tandem mass spectrometry (MS/MS) for mapping posttranslational modifications

55. List of modification and their properties

56. Ptm identification tools

57. Protein –protein interaction

58. Ppi methods

59. Genomic ContextPhylogenetic profilesConservation of gene neighborhoodGene FusionSimilarity of phylogenetic treesCorrelated mutations

60. 1.Phylogenetic ProfileBased on the pattern of the presence or absence of a given gene in a set of genomesA profile is constructed for each protein (Prot a–Prot d), recording its presence (1) or absence (0) in a set of organismsPairs of proteins with identical (or similar) phylogenetic profiles are predicted to interact (Prot a and Prot c in this case)

61. 2.Gene cluster and gene neighborhood methods,Proteins whose genes are physically close in the genomes of various organisms are predicted to interact.B

62. 3.GENE FUSIONRosetta Stone method: The Rosetta Stone approach infers protein interactions from protein sequences in different genomes . It is based on the observation that some interacting proteins/domains have homologs in other genomes that are fused into one protein chain, a so-called Rosetta Stone protein

63. REDUCED MSA(4,5)To obtain a quantitative indicator of the interaction between two proteins (Prot a and Prot b), the MSAs of both proteins are reduced to the set of organisms common to the two proteins (Org 1–Org 5). Each of the reduced alignments is used to construct the corresponding intersequence distance matrix.

64. 4. Similarity of phylogenetic trees (Mirrortree)Matrices are commonly used to construct the corresponding phylogenetic trees. Linear correlation between these distance matrices is calculated. High correlation values are interpreted as indicative of the similarity between phylogenetic trees and hence are taken as predicted interactions.

65. 5.Correlated mutations

66. 5.Correlated mutationsA correlation coefficient is calculated for every pair of residues. The pairs are divided into three sets: two for the intraprotein pairs (Caa and Cbb; pairs of positions within Prot a and within Prot b) and one for the interprotein pairs (Cab; one position from Prot a and one from Prot b).The distributions of correlation values are recorded for these three sets. The ‘interaction index’ is calculated by comparing the distribution of interprotein correlations with the two distributions of intraprotein correlations

67. 6. Classification methods/RDF methodFive different features/domains are used Each interacting protein pair is encoded as a string of 0, 1, and 2. The decision trees are constructed based on the training set of interacting protein pairs .Decisions are made if proteins under the question interact or not (‘‘yes’’ for interacting, ‘‘no’’ for non-interacting).

68. PPI DATABASE

69. Protein microarray

70. types of protein microarraysThree types of protein microarrays are currently used to study the biochemical activities of proteins: Analytical microarrays, Functional microarrays, and Reverse phase microarrays

71. Analytical microarraysDifferent types of ligands, including antibodies, antigens, DNA or RNA aptamers, carbohydrates or small molecules, with high affinity and specificity, are spotted down onto a derivatized surface. Protein samples from two biological states to be compared are separately labelled with red or green fluorescent dyes, mixed, and incubated with the chips. Spots in red or green colour identify an excess of proteins from one state over the other.These types of microarrays can be used to monitor differential expression profiles and for clinical diagnostics. Examples include profiling responses to environmental stress and healthy versus disease tissues

72. Functional protein microarraysNative proteins or peptides are individually purified or synthesized using high-throughput approaches and arrayed onto a suitable surface to form the functional protein microarrays. These chips are used to analyse protein activities, binding properties and post-translational modifications. functional protein microarrays can be used to identify the substrates of enzymes of interest. This class of chips is particularly useful in drug and drug-target identification and in building biological networks

73. Analytical versus functional protein microarrays.

74. Functional protein microarraysFunctional protein microarrays differ from analytical arrays in that functional protein arrays are composed of arrays containing full-length functional proteins or protein domains.These protein chips are used to study the biochemical activities of an entire proteome in a single experiment. They are used to study numerous protein interactions, such as protein-protein, protein-DNA, protein-RNA, protein-phospholipid, and protein-small molecule interactions

75. reverse phase protein microarray (RPA)In RPA, cells are isolated from various tissues of interest and are lysed. The lysate is arrayed onto a nitrocellulose slide using a contact pin microarrayer. The slides are then probed with antibodies against the target protein of interest, and the antibodies are typically detected with chemiluminescent, fluorescent, or colorimetric assays.

76. RPARPAs allow for the determination of the presence of altered proteins that may be the result of disease. Specifically, post-translational modifications, which are typically altered as a result of disease, can be detected using RPAs

78. PROTEIN CHIPS

79. Protein microarray