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Introduction to proteomics

Concha Gil, ProteoRed, Universidad Complutense de Madrid

Thursday, August 3.

Proteome: Protein complement expressed by a genome in certain environmental conditions at a given moment.

Genome Proteome
Static, it has the information Dynamic, it carries out functions

From genes to cellular functions

DNA -(transcription)-> RNA -(splicing)-> mRNA -(translation)-> Protein --> Function

Complexity:

  • Diverse protein species per gene
  • RNA processing
  • Post-translational modifications (PTMs)
  • Proteins have different physico-chemical properties
  • Different extraction methods required
  • Different sub-cellular locations
  • Study of subproteomes

Systematic (large scale) study of protein properties to obtain a global, integrated view of disease processes, cellular processes and networks at the protein level:

  • Expression levels
  • Macromolecular interactions
  • Post-translational modifications

Proteomic technology:

  • Separation techniques (protein and peptides complex mixtures)
    • Mono- & Bi-dimensional gel electrophoresis (2-DE)
    • Multidimensional Chromatography (2DLC)
  • Identification techniques (protein and peptide characterization)
    • Mass spectrometry (MS)
  • Bioinformatics
    • Softwares and proteomics servers
    • DNA and protein databases

A mass spectrometer generates gas-phase ions from a sample, separating them according to their mass-to-charge ratio (m/z) and produces a record of their abundances.

  • Protein mass it not enough for protein identification
  • Proteins must be digested by proteases
  • The exact mass of the peptides or fragments will be measured

Identification methods

  • Peptide mass fingerprinting (PMF)
  • Trypsin digestion

Experimental design: Non-gel proteomics

  1. Sample preparation
  2. Digestion
  3. Peptide separation (2DLC)
  4. Identification (MS/MS)

Expression proteomics

  • Detection of changes in the proteome in different environmental conditions
  • Comparison and relative quantitation of the amount of protein- Different labeling can be used.

Proteomics types

  • Quantitative proteomics: Analysis of protein expression at larger scale.
  • Functional proteomics: Interaction proteomics and protein networks.