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Mass spectrometry and proteomics. Mass spectrometry and proteomics. Eva Dimitrova and Jessica Connor. Genomics. DNA (Gene). Transcription. Transcriptomics. RNA. Translation. Functional Genomics. PROTEIN. Proteomics. Enzymatic reaction. METABOLITE. Metabolomics.
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Mass spectrometry and proteomics Mass spectrometry and proteomics Eva Dimitrova and Jessica Connor
Genomics DNA (Gene) Transcription Transcriptomics RNA Translation Functional Genomics PROTEIN Proteomics Enzymatic reaction METABOLITE Metabolomics The “omics” nomenclature…
Proteomics definition “Proteomics is a science that focuses on the study of proteins : their roles, their structures, their localization, their interactions, and other factors.” www.lexicon-biology.com
3 Kinds of Proteomics • Functional Proteomics The identification of protein functions, activities or interactions at a global or organismwide scale • Expressional Proteomics The analysis of global or organismwide changes in protein expression • Structural Proteomics The high throughput, or high volume expression and structure determination of proteins by Xray, NMR or computerbased methods
Components of Expressional Proteomics Protein Separation Mass Spectroscopy Bioinformatics
Pathway Step 1: Sample prep Step 2: Separation Step 3: Mass spectrometry
General overview Aebersold, R & Mann, M. (2003, March). Mass spectrometry based proteomics. Nature. 422, 198-207
Sample preparation Sample preparation involves everything that lies between the sample and 1st dimension of the 2D SDS gel • Cells and cell cultures – multiply • Homogenation and protein isolation • Contaminant removal/ cleanup • Fractionation
Cleanup and fractionation • General Purpose Cleanup • Improve Resolution • Improve Reproducibility • Fractionation • Reduce Complexity • Improve Range of Detection • Enrich low-abundance proteins www.expressionproteomics.com
2D-SDS PAGE gel The first dimension (separation by isoelectric focusing) - gel with an immobilised pH gradient - electric current causes charged proteins to move until it reaches the isoelectric point The second dimension (separation by mass) -pH gel strip is loaded onto a SDS gel -SDS denatures the protein (to make movement solely dependent on mass, not shape) and eliminates charge. Can Resolve: ~1500-2500 proteins
Staining Technology • Staining • Silver • Coomassie blue • Fluorescent dyes • Sypro Ruby-$$$ • Radioisotopic labeling
Trypsin digestion Trypsin • Serine protease • Claves at the carboxyl end of lysine and arginine (except when either is followed by proline)
Ionization method MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF Quadrapole MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif. How does a mass spectrometer work? Create ions Separate ions Detect ions • Mass spectrum • Database analysis
Definitions ESI- Electron Spray Ionization is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized MALDI- Matrix-assisted laser desorption/ionization is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules and large organic molecules, which tend to be fragile and fragment when ionized by more conventional ionization methods.
Mass analyser • TOF – time of flight • Ion trap • Quadropole • Fourier transform ion cyclotron
Mass Spec Principles Sample + _ Detector Ionizer Mass Analyzer
Typical Mass Spectrum Relative Abundance aspirin m/z ratio: Molecular weight divided by the charge on this protein 120 m/z-for singly charged ion this is the mass
ESI and MALDI Aebersold, R & Mann, M. (2003, March). Mass spectrometry based proteomics. Nature. 422, 198-207
Stable isotope protein labeling Aebersold, R & Mann, M. (2003, March). Mass spectrometry based proteomics. Nature. 422, 198-207
Artificially trypsinated Fragmented using trypsin Artificial spectra built Spot removed from gel Peptide Mass Identification Spectrum of fragments generated MATCH Library Database of sequences (i.e. SwissProt)
How MS sequencing works • Peptide mass and database matching • Further f ragmentation of the peptides occur in a predictable fashion, mainly at the peptide bonds • The resulting daughter ions have masses that are consistent with KNOWN molecular weights of di-peptides, tri-peptides, tetra-peptides… Ser-Glu-Leu-Ile-Arg-Trp Collision Cell Ser-Glu-Leu-Ile-Arg Ser-Glu-Leu-Ile Ser-Glu-Leu Etc…
Data Analysis Limitations -You are dependent on well annotated genome databases -Data is noisy. The spectra are not always perfect. Often requires manual determination. -Database searches only give scores. So if you have a false positive, you will have to manually validate them
Why Proteomics? • Proteins are the active biological agents in cells • DNA sequences don’t show how proteins function or how biological processes occur • Proteins undergo post transcriptional modifications • 3D structures affect protein function • Alternative splicing
The Human Proteome Initiative. (2007) http://ca.expasy.org/sprot/hpi/hpi_desc.html Retrieved March 24, 2009.
Challenges • Analyses of complex mixtures are not comprehensive • Difficult to prepare a pure sample • Protein expression is very sensitive to environmental conditions • Difficult to use ion currents to determine peptide abundance
Protein Profiling • Generate large scale proteome maps • Annotate and correct genomic sequences • Analyze protein expression as a function of cellular state
Analysis of Plasmodium falciparum(malaria parasite)proteome Figure 1: Proteins identified in each stage are plotted as a function of their broad functional classification. To avoid redundancy, only one class was assigned per protein. Florens, L. et al. (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature. 419, 520-526.
Analysis of Myconcogene proteome Fig. 3. Summary of functionally related expression changes in Myc(+) cells. The proteins reduced or induced in Myc(+) cells are shown in green or red, respectively. The numbers denote fold expression change. The arrows denote activation and the blocked lines denote inhibition. Shiio, Y. et al. (2002) Quantitative proteomic analysis of Myc oncoprotein function. EMBO J. 21, 5088–5096.
Protein Interactions • When analyzing a new protein, first question to ask is – to what proteins does it bind? • Method: Use new protein as an affinity agent to isolate its binding partners • Will not detect low affinity, transient, or cellular environment specific interactions
Protein Interaction Experiments • Steps 1. Bait presentation using endogenous proteins 2. Affinity purification of complex 3. Analysis of bound proteins
Studies of Large Protein Complexes • Spliceosome in yeast and human cells • Nuclear pore complex in yeast • Nucleolus in human cells • Largest organelle mapped • Found over 400 nucleolar proteins • Still not complete
Analyzing Protein Modifications • Finding all modifications on a single protein • Identified by examining the measured mass and fragmentation spectra • Proteome wide scanning of modifications • Not complete
Additional Challenges • Experimental design • Large amounts of data, absence of hypotheses • Must take advantage of statistical methods • Data collection • High throughput collection • High quality data • Data analysis, visualization, and storage • Data Publication
Future Directions • Influence on clinical diagnostics and therapy • Analysis of whole proteins • Tissue imaging • Using mass tags for high throughput protein identification
Other Applications of Mass Spectrometry • Isotope dating and tracking • Trace gas analysis • Mapping the location of individual atoms • Pharmacokinetics • Space exploration • Respiratory gas analysis
Conclusion Proteomics is extremely valuable for understanding biological processes and advancing the field of systems biology. “The ultimate goal of systems biology is the integration of data from these observations into models that might, eventually, represent and simulate the physiology of the cell.”
