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Proteomics

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Refers to the creation and advancement of algorithms ... Translations - putative proteins resulting from modifying (i.e. intron splicing) ... – PowerPoint PPT presentation

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Title: Proteomics


1
Proteomics
  • Dr. Caroline Clower
  • Assistant Professor of Chemistry
  • Department of Natural Sciences
  • Clayton State University
  • ITFN 4800
  • April 23, 2009

2
Bioinformatics
Statistics
Evolutionary Biology
Genetics
Mathematics
Biochemistry/ Molecular Biology
Bioinformatics
Computer Science
Chemistry
Medicine
Physics
3
Bioinformatics vs. Computational Biology
  • Used interchangeably
  • Actually different terms
  • Distinction made by National Institutes of Health
  • Bioinformatics
  • Refers to the creation and advancement of
    algorithms
  • Computational and statistical techniques to solve
    problems arising from management and analysis of
    biological data
  • Computational Biology
  • Refers to hypothesis-driven investigation of a
    specific problem using computers, using
    experimental or simulated data to advance
    scientific knowledge

4
Questions and Answers
  • Fundamental questions in science and medicine
  • What are the evolutionary origins of this
    protein?
  • What gene does this DNA sequence code for?
  • What does this gene do?
  • How does this enzyme work and what does it look
    like?
  • When is this gene expressed?
  • What genes are expressed before the onset of
    cancer?
  • What drugs can be used to treat this disease?
  • What mutations are responsible for this genetic
    disorder?
  • Analytical tools
  • Experimental (electrophoresis, spectroscopy,
    etc.)
  • Computational (programs, databases, internet,
    etc.)
  • No single comprehensive database exists for
    accessing all the information needed to manage
    data

5
Applications
  • Locate mutations responsible for genetic diseases
    and disorders
  • Aids in treatment and diagnosis
  • Pharmacogenomics
  • Designer drugs and therapies
  • Biotechnology
  • Discover and exploit new enzymes
  • Environmental clean-up
  • Antibiotics and other chemotherapeutic agents
  • Useful products

6
The Omics
  • Genomics
  • DNA Sequence homology locations of genes and
    functional sites phylogeny mapping infer
    function
  • Transcriptomics
  • mRNA sequence and structure
  • Metabolomics
  • Proteins and enzymatic pathways involved in cell
    metabolism
  • Glycomics
  • Carbohydrates of a cell
  • Interactomics
  • Complex interactions of protein networks in a
    cell
  • Nutrigenomics
  • Interactions between diet and genes
  • Proteomics

7
Introduction to Proteomics
  • Proteome
  • Sum total of an organisms proteins
  • Essential to understanding how organisms work
    (more so than genomics)
  • Characterization difficult
  • Protein structure is complicated
  • Chemical modification can occur
  • Proteins must be isolated and purified
  • Proteins must be crystallized
  • Structure predictions (bioinformatics) are
    difficult and often inaccurate

8
Proteomics
  • Large scale analysis of proteins
  • Amino acid sequence and protein structure
  • Named in 1995 development began in 1960s
  • Macromolecules carry information
  • Availability of computers
  • Development of algorithms
  • Information
  • Protein sequences
  • Primary sequence (SWISS-PROT and PIR databases)
  • Direct submissions - protein sequencing
  • Secondary sequence (GenPept and TrEMBL databases)
  • Translations - putative proteins resulting from
    modifying (i.e. intron splicing) nucleic acid
    sequence
  • Predicted structure (many algorithms)
  • Solved structure (Protein Data Bank)

9
Protein Data Bank
  • Archive of 3D structural data of biological
    macromolecules
  • Based on experimental data
  • Managed by the Research Collaboratory for
    Structural Bioinformatics (RCSB)
  • Rutgers, UCSD, UW-Madison
  • As of March 31, 2009 contained 56751 structures

10
Applications of Information
  • Structure reveals function
  • Porins
  • Enzymes
  • Knowledge of structure and function allow other
    applications
  • Pharmacotherapy, etc.

11
Protein Structure and Proteomics
12
Levels of Protein Structure
  • Primary
  • Linear sequence of amino acids
  • Secondary
  • Local structure certain motifs are common
  • Tertiary
  • Complete 3D shape
  • Quaternary
  • gt1 peptide chain

13
Primary Structure
  • Linear sequence of amino acids
  • Ala-Glu-Val-Thr-Asp-Pro-Gly or AEVTDPG
  • Cannot be predicted by any algorithm
  • Experimental
  • Protein sequencing
  • First protein insulin
  • Approach
  • Denature protein
  • Break into small segments
  • Determine sequences of segments
  • Animation

14
Example
Margaret Dayhoffs FORTRAN programs
15
Secondary Structure
  • Regular repeating structure
  • Helices (coil)
  • Sheets (extended zig-zag)
  • Turns (short loops)
  • Rotation of backbone
  • Only some allowed angles
  • Ramachandran diagram

16
Classification of Proteins by Secondary Structure
  • Fibrous
  • High composition of single secondary structure
  • Strong and flexible
  • Collagen (connective tissue, skin, tendons,
    cartilage)
  • Triple helix
  • Silk fibroin
  • b-sheet
  • a-Keratin (hair, wool, skin, nails)
  • Coiled coil
  • b-Keratin (scales, feathers)
  • b-sheet

17
Classification of Proteins by Secondary Structure
  • Globular
  • Majority of all proteins
  • Contain several types of secondary structure
  • Percentage of protein (on average)
  • 31 a-helix
  • 28 b-sheet
  • 13 turns/bends
  • 28 loops and random coil
  • Compact spherical shapes

18
Secondary Structure Prediction
  • Based on observed frequencies in known structures
  • Chou-Fasman algorithm
  • P (a), P (b), P (turn)
  • Probability of an AA participating in various
    secondary structures
  • Uses a window of 6 residues

19
Secondary Structure Prediction
  • PELE Results from many different algorithms
  • H a helix
  • E b strand
  • T b turn
  • C random coil
  • Algorithms listed to the right (initials indicate
    authors)
  • JOI Joint prediction
  • Assigns the structure using a "winner takes all"
    procedure

20
Tertiary Structure
  • 3D structure (overall shape) of an entire
    polypeptide
  • Interactions between secondary structural
    components
  • No algorithm predicts the 3D shape with high
    accuracy
  • Can predict cellular location and sequence motifs
  • Experimentally determined by
  • X-ray crystallography (85 of structures in the
    PDB)
  • 2D Nuclear Magnetic Resonance (15)
  • Computational modeling/Homology modeling (lt1)

21
X-ray crystallography
  • Precise positions of atoms
  • Mathematical analysis of film
  • Fourier transform
  • Creates electron density map
  • Combine with principles of chemical bonding to
    create structure

22
2D-NMR NOESY
  • Graphically displays atoms in close proximity
  • Calculates structure

23
Computational Modeling
  • Sequence alignment to find homologous protein
  • BLAST algorithm
  • Searches for maximal local alignments
  • Inserts gaps to optimize alignment
  • Breaks sequence down into subsequences (words)
  • Searches for those words in database
  • Extends sequence on either side of word to look
    for certain score (threshold)

24
Scoring Alignments
  • Scoring matrices based on similarity and
    probability of substitutions/mutations
  • Example
  • Alignment of mouse and crayfish trypsin
  • Raw score 30
  • Evaluate alignment with
  • Alignment score (S)
  • E score expected number of sequences that have
    scores S that would be found randomly
  • Low value for E indicates search result is not
    random and sequences are likely to be related

Mouse I V G G Y N C E E N S V P Y
Q 5 4 5 5 -3 2 -2 2 3 0 0 -1 6
10 4 Crayfish I V G G T D A V L G E
F P Y Q
25
Protein Modeling Programs
  • Investigate and manipulate structure
  • Structure overlays and alignment
  • Pharmacophores
  • Drug design from protein structure

26
Websites of Interest
  • ExPASy proteomics tools (http//us.expasy.org/tool
    s/)
  • NCBI (http//www.ncbi.nlm.nih.gov/)
  • PDB (www.pdb.org)
  • Protein Explorer (www.proteinexplorer.org)
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