Proteomics

Proteomics

1. genotype

   • Genome is the blueprint of what could be made.

2. phenotype

   • Phenotype is what is made. – mRNA expression. – Non-coding RNA. – Proteins and proteoforms. – Lipids. – Metabolites.

3. different genes expressed in different cells

   • We identified a total of 11,731 proteins, and on average 10,361 ± 120 proteins in each cell line. This very high proteome coverage enabled analysis of a broad range of processes and functions. Despite the distinct origins of the cell lines, our quantitative results showed surprisingly high similarity in terms of expressed proteins.
   • lobal similarity of the proteomes did not imply equal expression levels of individual proteins across the 11 cell lines, as we found significant differences in expression levels for an estimated two-third of them. The variability in cellular expression levels was similar for low and high abundance proteins, and even many of the most highly expressed proteins with household roles showed significant differences between cells. Metabolic pathways, which have high redundancy, exhibited variable expression, whereas basic cellular functions such as the basal transcription machinery varied much less.

4. genome --> transcriptome

   • Whole genomes sequenced in less than a wee
   • Multiple forms of mRNA from one gene. – Multiple forms of primary protein sequence. – Proteoform. • Transcriptome: Abundance of different mRNA’s in a cell type
   • multiple forms of mRNA from one gene
   • transcriptome

5. more proteins than genes

   • post-translational cleavage
   • estimation of proteoforms

6. fractionation

   • uantity of a mixture (gas, solid, liquid, enzymes, suspension, or isotope) is divided during a phase transition, into a number of smaller quantities (fractions) in which the composition varies according to a gradient.a process which a certain 
   • Fractionation is necessary to reduce sample complexity and dynamic range of protein concentration
   • Use chemical properties to reduce sample complexity
   • size
   • charge
   • hydrophobicity

7. same genome, different proteome

8. whats the proteome?

   • The Proteome is ALL of the proteins expressed by a cell, organism or tissue under a certain set of environmental conditions. • Genomics measures Genotype. • Proteomics is a measure of Phenotype. – Phenotype also defined by metabolites (metabolome), lipids (lipidome). • A cell is NOT just what proteins are expressed, but: – When is the protein expressed and how much? – How is it modified post-translation? – What is the protein interacting with?
   • different from genomics
   • what does proteomics study?
     • Obtain a global view of cell or organism at the protein level under a certain environmental condition. – Measure quantitative differences in amount of protein expressed. • Includes analysis of the modifications possible on proteins, including cleavage, phosphorylation, glycosylation, lipidation, etc. • The challenge of proteomics is to quantify the changes in proteins when the system they are part of changes.
   • not just what proteins are expressed

9. what is it?

   • challenges
   • why study it?
     • • To determine phenotypic changes in a system. • To ascertain the function and interactions that proteins have in normal, diseased and infected cells. • Determine mechanisms of cellular function. • How do protein interactions and complexes alter in disease? • Find targets for therapeutic intervention. • Find “Biomarkers” for early diagnosis of disease.
     • PCR
       • Genomics/Transcriptomics – can ‘amplify’ signal using PCR. – Targeted amplification of low abundance mRNA. • Proteins – no equivalent of PCR. • Want to detect these, start with more. • But more of these, overwhelming signal. – Back where you started. – And if you can’t get more? 

10. Analysis of proteome

    • How is the Proteome analysed? • Proteomics uses well established methodologies to fractionate and separate proteins from one another. – Electrophoresis. – Chromatography. • Mass spectrometry (MS) is used to produce data that reflects protein/peptide amino acid sequence. • MS data is “matched” using algorithms to known or predicted protein sequences.
    • electrophoresis
      • 2D gel
      • isoelectric focusing
    • Chromatography
      • output meaning
    • Mass Spec
      • intact spectra
      • fragmented spectra
        • fragmentation
      • searching/matching MS data
    • smashing cells open
      • Smash everything open. • Divide the protein into manageable groups • Cut it up to peptides. • Measure peptide mass in the MS and smash the peptide to measure the fragments. • Search databases for matches. • Interpret what it all means biologically.
      • A. Do you want to catalogue everything? B. Do you want to find out what proteins are interacting? • Often you can’t have both. – To analyse a protein, first you have to get to it and second you have to solubilise it.
    • how to interpret data

11. technologies

12. Workflow

    • heterogenity
      • Heterogeneity of proteins means each workflow is tailored to the particulars of the sample and the subset of proteins needing analysis.
      • Solubilise
        • proteins so they can be fractionated
        • Fractionate
          • Fractionate proteins by their differing physical properties.
          • Analyse
            • Analysing unfractionated samples displays only the highest abundance proteins. – 2-300 proteins seen of 23,000+ proteins and 500,000+ proteoforms. identify and quantify. 

13. if unfractionated?

14. problems

    • theoretical peptide mass=measured mass
    • if sequence not in database
    • peptide for difinition not detected

15. peptide vs. protein

    • how many proteins expressed

16. quantitating changes in proteome

    • ID
    • amount
    • measure
    • mass spec
    • 2D-PAGE

17. examples

    • comprehensive ms based proteome quantification of haploid versus diploid yeast
    • plasmodium falciparum proteome changes in reponse to treatment