Today’s Proteomics Session II 台大生技教改暑期課程
What’s “proteomics” ? "The analysis of the entire protein complement expressed by a genome, or by a cell or tissue type.“ Wasinger VC et al Progress with gene-product mapping of the mollicutes: Mycoplasma genitalium. Electrophoresis 16 (1995) 1090-1094 • Two MOST related technologies: • 2-D electrophoresis: separation of complex protein mixtures • Mass spectrometry: Identification and structure analysis
Today’s topics • Introduction to Proteomics • Technology of Proteomics • Applications of Proteomics
Move over Genome…on to Proteomics • If the genome is the blueprint of an organism---who reads it? • At this point no computer algorithm can solve this • A computer can decode all 6 reading frames of an organism • A computer can compare these.. But then what?
Genomic DNA • Structure • Regulation • Information • Computers cannot determine which of these 3 roles DNA play solely based on sequence… (although we would all like to believe they can) Those are what we need to know about protein
Definitions of Proteomics Classical - restricted to large scale analysis of gene products involving only proteins Inclusive - combination of protein studies with analyses that have genetic components such as mRNA, genomics, and yeast two-hybrid • Don’t forget that the proteome is dynamic, changing to reflect the environment that the cell is in
1 gene = 1protein? • 1 gene is no longer equal to one protein • In fact, the definition of a gene is debatable..(ORF, promoter, pseudogene, gene product, etc) • 1 gene=how many proteins?
Differential protein expression Stimulus Stimulus RNA Protein DNA Transcription Translation x4 x1 Scenario 1: can be analyzed by microarray technology RNA Protein DNA Transcription Translation x3 Scenario 2: can be solved by proteomics technology RNA Protein DNA Transcription Translation x3
Co- and Post-translational modification Co-translational modified Post-translational modified
Why Proteomics? (summary) • Annotation of genomes, i.e. functional annotation • Genome + proteome = annotation • Protein Function • Protein Post-Translational Modification • Protein Localization and Compartmentalization • Protein-Protein Interactions • Protein Expression Studies • Differential gene expression is not the answer
Types of Proteomics • Protein Expression • Quantitative study of protein expression between samples that differ by some variable • Structural Proteomics • Goal is to map out the 3-D structure of proteins and protein complexes • Functional Proteomics • To study protein-protein interaction, 3-D structures, cellular localization and PTMS in order to understand the physiological function of the whole set of proteome.
Technology of Proteomics • Separation of proteins • 1DE (convention) • 2DE (modern) • Multi-dimensional HPLC (modern) • Analysis of proteins • Edman Sequencing (convention) • Mass Spectrometry (modern) • Database utilization
Isolated RNA Electrophoresis Blotting Labelling on probes !! Developing Probing Traditional RNA technique : Northern blotting 1. Estimated time to get results: 2-3days 2. Expressed Gene (mRNA) checked: 1-8 species 3. Accuracy: Low to moderate
Clustered experiments Clustered genes High-throughput method: Microarray Labelling on sample mRNA as probe cDNA or oligonucleotide spotted on chips data analysis 1. Estimated time to get results: 5-7 days 2. Expressed Gene (mRNA) checked: thousands 3. Accuracy: moderate to high
Traditional Protein technique: peptide sequencing Cut desired band Database searching for homolog Peptide N terminal sequencing • Protein purification: necessary • Protein idetified: 1 per purified sample
High throughput technique: 2D electrophoresis + Mass spectrometry • Protein purification: not necessary • Protein idetified: up to thousands per unpurified sample
In our course, we will focus much on A. 2-DE B. Mass spectrometry
Major technique in proteomic research:2-D electrophoresis (separation) • First dimension: • denaturing isoelectric focusing • separation according to the pI • 2. Second dimension: • SDS electrophoresis (SDS-PAGE) • Separation according to the MW Interested spot MS analysis Digest to peptide fragment
The principle of IEF The IEF is a very high resolution separation method, and the pI of a protein can be measured.
Immobilized pH gradient strips (IPG strips) • Introduced by Gorg. A. • Ref: Gorg. A (1994), Westermeier (2001) • Dried gel strips can be stored at -20 to -80 from months to years.
2-DE instruments, 1st dimension Amersham Biosciences Bio-Rad
2-DE instruments, 2nd dimension Amersham Biosciences 23 x 20 cm 8 x 10 cm 16 x 16 cm
2-DE instruments, 2nd dimension Bio-Rad
Examples of 2-DE results D Healthy control Patient MS analysis Digest to peptide fragment
Major technique in proteomic research:Mass Spectrometry (analysis) Ion source Ion separator detector Ion source: substance to ion gas Mass analysis: according to mass/charge (m/z) Detection: femtomole –attomole (10-15 – 10-18 mole)
Commonly used Mass Spectrometer in Proteomics MALDI-TOF Matrix Assisted Laser Desorption Ionization Time Of Flight ESItandem MS (with HPLC, LC tandem MS or LC MS/MS) • Electro Spray Ionization MS Quadrupole
Commercial available MALDI-Tof Microflex ™, Bruker Voyager DE-PRO™, ABI MALDI micro™, Micromass
Peptide fingerprinting with MALDI-TOF Gel Database Protein ? 1 2 3 tryptic stored data or theoretical? peptides digestion mass spectrometry compare: ?? is identical to ??
ESI Quadrupole MS • Nano electrospray: >30 min spray time for 1 mL sample • Highly charged molecules are selected by ac modulation of transverse fields