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. PROTEOMICS The study of the expression, location, interaction, function and structure of all the proteins in a given cell or organism Functional Proteomics Expressional Proteomics Structural Proteomics
4. PROTEOMICS Functional proteomics: What is the function of each product of the 30,000 human genes where 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)
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 Peptides P A G N F A P G N F A N P G F C-terminal peptides N-terminal peptides A N F P G P A N F G
23. Terminal peptides and ion types Peptide H2O P N G F Mass (D) 57 + 97 + 147 + 114 = 415 without G F N Peptide H2O P Mass (D) 57 + 97 + 147 + 114 – 18 = 397
29. Commonly used Mass Spectrometer in Proteomics MALDI-TOF Matrix Assisted Laser Desorption Ionization Time Of Flight ESI tandem MS (with HPLC, LC tandem MS or LC MS/MS) Electro Spray Ionization Mass Spectrometry
45. Mass spectrum mapping Top spectrum depicts a theoretical mass spectrum, as might be generated by a sequence search algorithm, matching an actual peptide MS/MS spectrum (bottom).
52. Expression proteomics Separation, quantification and identification of large numbers of proteins from biological specimens 2D gel electophoresis Mass Spec analysis
53. Ms/ms for post translational modification PTM-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.
59. Genomic Context Phylogenetic profiles Conservation of gene neighborhood Gene Fusion Similarity of phylogenetic trees Correlated mutations
60. 1.Phylogenetic Profile Based on the pattern of the presence or absence of a given gene in a set of genomes A profile is constructed for each protein (Prot a–Prot d), recording its presence (1) or absence (0) in a set of organisms Pairs 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 FUSION Rosetta 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.
66. 5.Correlated mutations A 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 method Five 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).
70. types of protein microarrays Three types of protein microarrays are currently used to study the biochemical activities of proteins: Analytical microarrays, Functional microarrays, and Reverse phase microarrays
71. Analytical microarrays Different 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 microarrays Native 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
74. Functional protein microarrays Functional 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. RPA RPAs 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