Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Proteome and Gene Expression Analysis Chapter 15 & 16 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. 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. 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. Getting the Data 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. 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. 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 Extracting the Proteins • First, the proteins are extracted from the cells using lysis. – This involves a detergent that destroys the membranes of the cell. 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. QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see thi s picture. Using the 2D Gel • Staining makes the spots containing the individual (we hope) proteins visible. segmenting – 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. QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture. dust “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… QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture. Protein Identification Using Mass Spectrometry QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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. Spectrum: Protein Fingerprint QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Not part of this subject, but cool… Protein Arrays for Measuring Protein-Protein Interactions • You can synthesize proteins from DNA directly on a substrate. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. – 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. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Next time: Analyzing Gene and Protein Expression Data QuickTime™ and a TIFF (U ncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (U ncompres sed) decompressor are needed to see this picture. Gene expression clustering Protein Expression Clustering