Proteome and Gene Expression Analysis

1. Proteome and Gene Expression Analysis Chapter 15 & 16

2. The Goals • Functional Genomics: • To know when, where and how much genes are expressed. • To know when, where, what kind and how much of each protein is present. • Systems Biology: • To understand the transcriptional and translational regulation of RNA and proteins in the cell.

3. Genes and Proteins • First, we’ll talk about how to find out what genes are being transcribed in the cell. • This is often referred (somewhat misleadingly) to gene “expression”. • Second, we’ll look at measuring the levels of proteins in the cell. • The real “expression” of protein coding genes… • Third, we’ll talk about how we process and analyze the raw data using bioinformatics.

4. Review: Gene Arrays • Put a bunch of different, short single-stranded DNA sequences at predefined positions on a substrate. • Let the unknown mixture of tagged DNA or RNA molecules hybridize to the DNAs. • Measure the amount of hybridized material.

5. Getting the Data

6. Getting Protein Expression Data • To be able to understand protein expression, we need the concentrations of all proteins (the “proteome”) in difference cell and tissue types under varying conditions. • Large scale identification of proteins is much more limited than for RNA. • Nothing really equivalent to RNA expression microarrays or high-throughput sequencing exists yet. • Relatively low-throughput technologies are all that we have right now.

7. Measuring Protein Expression • In order to measure all the types of protein in a cell we must • Extract the proteins • Purify the proteins • Identify the individual proteins • How do we accomplish purification and identification of proteins.

8. The Technologies:Protein Expression • Low-throughput • 2D Gel Electrophoresis + Mass Spectrometry • Liquid chromatograph + Mass Spectrometry • Protein microarrays • Limited in application at this point • Can be used for things other than protein expression like protein-protein interactions

9. Extracting the Proteins • First, the proteins are extracted from the cells using lysis. • This involves a detergent that destroys the membranes of the cell.

10. Separating the Proteins:2D Gel Electrophoresis • First step: pI/pH • Proteins are introduced to a gel with an imobilized pH gradient. • A charge is applied. • Proteins migrate until the pH causes them to lose their charge (isoelectric point) and then stop. • Second step: mass • First gel transferred to second gel • SDS (detergent) breaks structure and charges the proteins proportional to their mass.

11. Using the 2D Gel • Staining makes the spots containing the individual (we hope) proteins visible. • The gel is photographed. • Protein level (concentration) can be estimated by image processing. • Individual, stained spots can be cut out for evaluation by Mass Spectrometry. segmenting dust

12. “Two channel” 2D Gels • Low signal-to-noise is a problem with protein gels, as it is with RNA expression arrays. • A similar trick of putting two cell lysates (samples) on one gel can help. • Registration problems and sample-dependent effects are thereby minimized. • However, 1-channel gels allow comparing more than two samples…

13. Protein Identification Using Mass Spectrometry

14. Steps of Mass Spectrometry • Digest: • Sample (spot) is digested with a proteolytic enzyme • Spectrum: • Peaks correspond to the mass-charge ratio of protein fragments • These provide a fingerprint • Identify: • Compare fingerprint to theoretical fingerprints • Post-translational modifications screw things up.

15. Spectrum: Protein Fingerprint

16. Tricks: Protein “chips” • If you had an antibody to every possible protein and could put it on a chip, and you could label the proteins in your sample, you would have something equivalent to an RNA expression microarray. • Getting reliable antibodies is difficult and expensive. • Arrays with 500 to 2000 proteins are available commercially; Clontech, Eurogentec, Arrayit etc.

17. Not part of this subject, but cool…

18. Protein Arrays for Measuring Protein-Protein Interactions • You can synthesize proteins from DNA directly on a substrate. • Nano-well approach • “Printing” approach: DAPA (DNA to Protein Array) • These can be used for measuring binding between proteins, but not for identification of proteins.

19. Next time:Analyzing Gene and Protein Expression Data Protein Expression Clustering Gene expression clustering